WorldWideScience

Sample records for multimeric heme complexation

  1. Identification of the Mitochondrial Heme Metabolism Complex.

    Science.gov (United States)

    Medlock, Amy E; Shiferaw, Mesafint T; Marcero, Jason R; Vashisht, Ajay A; Wohlschlegel, James A; Phillips, John D; Dailey, Harry A

    2015-01-01

    Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.

  2. Methylation-regulated decommissioning of multimeric PP2A complexes

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li; Rowse, Michael; Stanevich, Vitali; Chen, Hui; Li, Yitong; Satyshur, Kenneth A.; Johnson, Benjamin; Gu, Ting-Jia; Liu, Zuojia; Xing, Yongna

    2017-12-01

    Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.

  3. Aggregate complexes of HIV-1 induced by multimeric antibodies.

    Science.gov (United States)

    Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

    2014-10-02

    Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

  4. Gas-phase spectroscopy of ferric heme-NO complexes

    DEFF Research Database (Denmark)

    Wyer, J.A.; Jørgensen, Anders; Pedersen, Bjarke

    2013-01-01

    and significantly blue-shifted compared to ferric heme nitrosyl proteins (maxima between 408 and 422 nm). This is in stark contrast to the Q-band absorption where the protein microenvironment is nearly innocent in perturbing the electronic structure of the porphyrin macrocycle. Photodissociation is primarily...... maxima of heme and its complexes with amino acids and NO. Not so innocent: Weakly bound complexes between ferric heme and NO were synthesised in the gas phase, and their absorption measured from photodissociation yields. Opposite absorption trends in the Soret-band are seen upon NO addition to heme ions...

  5. Fitting multimeric protein complexes into electron microscopy maps using 3D Zernike descriptors.

    Science.gov (United States)

    Esquivel-Rodríguez, Juan; Kihara, Daisuke

    2012-06-14

    A novel computational method for fitting high-resolution structures of multiple proteins into a cryoelectron microscopy map is presented. The method named EMLZerD generates a pool of candidate multiple protein docking conformations of component proteins, which are later compared with a provided electron microscopy (EM) density map to select the ones that fit well into the EM map. The comparison of docking conformations and the EM map is performed using the 3D Zernike descriptor (3DZD), a mathematical series expansion of three-dimensional functions. The 3DZD provides a unified representation of the surface shape of multimeric protein complex models and EM maps, which allows a convenient, fast quantitative comparison of the three-dimensional structural data. Out of 19 multimeric complexes tested, near native complex structures with a root-mean-square deviation of less than 2.5 Å were obtained for 14 cases while medium range resolution structures with correct topology were computed for the additional 5 cases.

  6. Moessbauer spectroscopic study of polymer-bound heme complexes

    International Nuclear Information System (INIS)

    Tsuchida, Eishun; Nishide, Hiroyuki; Yokoyama, Hiroyuki; Inoue, Hidenari; Shirai, Tsuneo.

    1984-01-01

    Moessbauer spectra were measured on the heme complexes of poly(1-vinyl- and 1-vinyl-2-methylimidazole)(PVI and PMI) and heme derivatives with covalently bound imidazoleligand (IH) and 2-methylimidazole-ligand (MIH) embedded in poly(1-vinyl-2-pyrrolidone) film. Quadrupole splitting (ΔE sub(Q)) for the carbon monoxide adduct of PMI-heme indicated large electronic field gradient at the iron nucleus, probably due to steric hindrance of the polymer chain, and this behavior agreed with its low affinity with carbon monoxide. PMI-heme formed an oxygen adduct and its isomer shift and ΔE sub(Q) values were obtained. (author)

  7. Identification of the receptor scavenging hemopexin-heme complexes

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Maniecki, Maciej B; Jacobsen, Christian

    2005-01-01

    and is suggested to facilitate cellular heme metabolism. Using a ligand-affinity approach, we purified the human hemopexin-heme receptor and identified it as the low-density lipoprotein receptor-related protein (LRP)/CD91, a receptor expressed in several cell types including macrophages, hepatocytes, neurons......, and syncytiotrophoblasts. Binding experiments, including Biacore analysis, showed that hemopexin-heme complex formation elicits the high receptor affinity. Uptake studies of radio-labeled hemopexin-heme complex in LRP/CD91-expressing COS cells and confocal microscopy of the cellular processing of fluorescent hemopexin......-heme complexes are removed by a receptor-mediated pathway showing striking similarities to the CD163-mediated haptoglobin-hemoglobin clearance in macrophages. Furthermore, the data indicate a hitherto unknown role of LRP/CD91 in inflammation....

  8. Total adiponectin and adiponectin multimeric complexes in relation to weight loss-induced improvements in insulin sensitivity in obese women

    DEFF Research Database (Denmark)

    Polak, J.; Kovacova, Z.; Holst, C.

    2008-01-01

    , and LMW). The HMW form was suggested to be closely associated with insulin sensitivity. This study investigated whether diet-induced changes in insulin sensitivity were associated with changes in adiponectin multimeric complexes. SUBJECTS: Twenty obese women with highest and twenty obese women with lowest...... diet induced changes in insulin sensitivity (responders and non-responders respectively), matched for weight loss (body mass index (BMI)=34.5 (s.d. 2.9) resp. 36.5 kg/m(2) (s.d. 4.0) for responders and non-responders), were selected from 292 women who underwent a 10-week low-caloric diet (LCD; 600 kcal...

  9. Photochemical organic oxidations and dechlorinations with a mu-oxo bridged heme/non-heme diiron complex.

    Science.gov (United States)

    Wasser, Ian M; Fry, H Christopher; Hoertz, Paul G; Meyer, Gerald J; Karlin, Kenneth D

    2004-12-27

    Steady state and laser flash photolysis studies of the heme/non-heme mu-oxo diiron complex [((6)L)Fe(III)-O-Fe(III)-Cl](+) (1) have been undertaken. The anaerobic photolysis of benzene solutions of 1 did not result in the buildup of any photoproduct. However, the addition of excess triphenylphosphine resulted in the quantitative photoreduction of 1 to [((6)L)Fe(II)...Fe(II)-Cl](+) (2), with concomitant production by oxo-transfer of 1 equiv of triphenylphosphine oxide. Under aerobic conditions and excess triphenylphosphine, the reaction produces multiple turnovers (approximately 28) before the diiron complex is degraded. The anaerobic photolysis of tetrahydrofuran (THF) or toluene solutions of 1 likewise results in the buildup of 2. The oxidation products from these reactions included gamma-butyrolactone (approximately 15%) for the reaction in THF and benzaldehyde (approximately 23%) from the reaction in toluene. In either case, the O-atom which is incorporated into the carbonyl product is derived from dioxygen present under workup or under aerobic photolysis conditions. Transient absorption measurements of low-temperature THF solutions of 1 revealed the presence of an (P)Fe(II)-like [P = tetraaryl porphyrinate dianion] species suggesting that the reactive species is a formal (heme)Fe(II)/Fe(IV)=O(non-heme) pair. The non-heme Fe(IV)=O is thus most likely responsible for C-H bond cleavage and subsequent radical chemistry. The photolysis of 1 in chlorobenzene or 1,2-dichlorobenzene resulted in C-Cl cleavage reactions and the formation of [[((6)L)Fe(III)-Cl...Fe(III)-Cl](2)O](2+) (3), with chloride ligands that are derived from solvent dehalogenation chemistry. The resulting organic products are biphenyl trichlorides or biphenyl monochlorides, derived from dichlorobenzene and chlorobenzene, respectively. Similarly, product 3 is obtained by the photolysis of benzene-benzyl chloride solutions of 1; the organic product is benzaldehyde (approximately 70%). A brief

  10. Heme transport and erythropoiesis

    Science.gov (United States)

    Yuan, Xiaojing; Fleming, Mark D.; Hamza, Iqbal

    2013-01-01

    In humans, systemic heme homeostasis is achieved via coordinated regulation of heme synthesis, transport and degradation. Although the heme biosynthesis and degradation pathways have been well characterized, the pathways for heme trafficking and incorporation into hemoproteins remains poorly understood. In the past few years, researchers have exploited genetic, cellular and biochemical tools, to identify heme transporters and, in the process, reveal unexpected functions for this elusive group of proteins. However, given the complexity of heme trafficking pathways, current knowledge of heme transporters is fragmented and sometimes contradictory. This review seeks to focus on recent studies on heme transporters with specific emphasis on their functions during erythropoiesis. PMID:23415705

  11. Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

    Science.gov (United States)

    Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

    2010-03-01

    The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

  12. A Mononuclear Non-Heme Manganese(IV)-Oxo Complex Binding Redox-Inactive Metal Ions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Junying; Lee, Yong-Min; Davis, Katherine M.; Wu, Xiujuan; Seo, Mi Sook; Cho, Kyung-Bin; Yoon, Heejung; Park, Young Jun; Fukuzumi, Shunichi; Pushkar, Yulia N.; Nam, Wonwoo [Ewha; (Purdue); (Osaka)

    2013-05-29

    Redox-inactive metal ions play pivotal roles in regulating the reactivities of high-valent metal–oxo species in a variety of enzymatic and chemical reactions. A mononuclear non-heme Mn(IV)–oxo complex bearing a pentadentate N5 ligand has been synthesized and used in the synthesis of a Mn(IV)–oxo complex binding scandium ions. The Mn(IV)–oxo complexes were characterized with various spectroscopic methods. The reactivities of the Mn(IV)–oxo complex are markedly influenced by binding of Sc3+ ions in oxidation reactions, such as a ~2200-fold increase in the rate of oxidation of thioanisole (i.e., oxygen atom transfer) but a ~180-fold decrease in the rate of C–H bond activation of 1,4-cyclohexadiene (i.e., hydrogen atom transfer). The present results provide the first example of a non-heme Mn(IV)–oxo complex binding redox-inactive metal ions that shows a contrasting effect of the redox-inactive metal ions on the reactivities of metal–oxo species in the oxygen atom transfer and hydrogen atom transfer reactions.

  13. Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

    Science.gov (United States)

    Jiang, Yongying; Ortiz de Montellano, Paul R

    2008-05-05

    Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

  14. A functional mimic of natural peroxidases : synthesis and catalytic activity of a non-heme iron peptide hydroperoxide complex

    NARCIS (Netherlands)

    Choma, CT; Schudde, EP; Kellogg, RM; Robillard, GT; Feringa, BL

    1998-01-01

    Site-selective attachment of unprotected peptides to a non-heme iron complex is achieved by displacing two halides on the catalyst by peptide caesium thiolates, This coupling approach should be compatible with any peptide sequence provided there is only a single reduced cysteine. The oxidation

  15. Dinitrosyl non-heme iron complexes at the gamma radiation treatment of animals

    International Nuclear Information System (INIS)

    Aliev, D.I.; Alieva, I.N.; Abilov, Z.G.; Gurbanov, I.S.

    2003-01-01

    Full text: At the present time there are a great number investigations dedicated to revealing of mechanism formation of 2,03 complexes at the some pathologies in an organism. These complexes are represented weakly bounded form of non-heme iron, including into beside iron two nitrogen oxide molecules (NO) and two paired RS- groups of proteins or low-molecular compounds. 2,03 complexes are characterized by an axial symmetrical tensory of the g-factor with g=2,037, g=2,012 and g=2,03. In this study the data testifying 2,03 complexes formation into liver of animal treated by the fatal dose of gamma-radiation are reported. The changing of the ESR signal form was observed. It was shown that the form and intensity of the 2,03 signal in healthy and irradiated animals are differ from each other. The analysis of the 2,03 signal parameters is confirm this fact, too. The conclusion was made that 2,03 complexes ESR signal may be considered as an indicator of integrity of intracellular membranes of the gamma-irradiated animals

  16. Experimental and Computational Evidence for the Mechanism of Intradiol Catechol Dioxygenation by Non-Heme Iron(III) Complexes

    Science.gov (United States)

    Jastrzebski, Robin; Quesne, Matthew G; Weckhuysen, Bert M; de Visser, Sam P; Bruijnincx, Pieter C A

    2014-01-01

    Catechol intradiol dioxygenation is a unique reaction catalyzed by iron-dependent enzymes and non-heme iron(III) complexes. The mechanism by which these systems activate dioxygen in this important metabolic process remains controversial. Using a combination of kinetic measurements and computational modelling of multiple iron(III) catecholato complexes, we have elucidated the catechol cleavage mechanism and show that oxygen binds the iron center by partial dissociation of the substrate from the iron complex. The iron(III) superoxide complex that is formed subsequently attacks the carbon atom of the substrate by a rate-determining C=O bond formation step. PMID:25322920

  17. Mononuclear non-heme iron(III) complexes of linear and tripodal ...

    Indian Academy of Sciences (India)

    The rate of oxygenation depends on the solvent and the. Lewis acidity of iron(III) ... has been achieved by non-heme iron enzymes and their ..... oxygen atoms of nitrate ion (figure 3). ... enhanced covalency of iron-catecholate interaction and.

  18. The antimalarial activity of Ru-chloroquine complexes against resistant Plasmodium falciparum is related to lipophilicity, basicity, and heme aggregation inhibition ability near water/n-octanol interfaces.

    Science.gov (United States)

    Martínez, Alberto; Rajapakse, Chandima S K; Jalloh, Dalanda; Dautriche, Cula; Sánchez-Delgado, Roberto A

    2009-08-01

    We have measured water/n-octanol partition coefficients, pK(a) values, heme binding constants, and heme aggregation inhibition activity of a series of ruthenium-pi-arene-chloroquine (CQ) complexes recently reported to be active against CQ-resistant strains of Plasmodium falciparum. Measurements of heme aggregation inhibition activity of the metal complexes near water/n-octanol interfaces qualitatively predict their superior antiplasmodial action against resistant parasites, in relation to CQ; we conclude that this modified method may be a better predictor of antimalarial potency than standard tests in aqueous acidic buffer. Some interesting tendencies emerge from our data, indicating that the antiplasmodial activity is related to a balance of effects associated with the lipophilicity, basicity, and structural details of the compounds studied.

  19. The antimalarial activity of Ru–chloroquine complexes against resistant Plasmodium falciparum is related to lipophilicity, basicity, and heme aggregation inhibition ability near water/n-octanol interfaces

    Science.gov (United States)

    Martínez, Alberto; Rajapakse, Chandima S. K.; Jalloh, Dalanda; Dautriche, Cula

    2012-01-01

    We have measured water/n-octanol partition coefficients, pKa values, heme binding constants, and heme aggregation inhibition activity of a series of ruthenium–πarene–chloroquine (CQ) complexes recently reported to be active against CQ-resistant strains of Plasmodium falciparum. Measurements of heme aggregation inhibition activity of the metal complexes near water/n-octanol interfaces qualitatively predict their superior antiplasmodial action against resistant parasites, in relation to CQ; we conclude that this modified method may be a better predictor of antimalarial potency than standard tests in aqueous acidic buffer. Some interesting tendencies emerge from our data, indicating that the antiplasmodial activity is related to a balance of effects associated with the lipophilicity, basicity, and structural details of the compounds studied. PMID:19343380

  20. Involvement of heme oxygenase-1 in β-cyclodextrin-hemin complex-induced cucumber adventitious rooting process.

    Science.gov (United States)

    Lin, Yuting; Li, Meiyue; Huang, Liqin; Shen, Wenbiao; Ren, Yong

    2012-09-01

    Our previous results showed that β-cyclodextrin-hemin complex (CDH) exhibited a vital protective role against cadmium-induced oxidative damage and toxicity in alfalfa seedling roots by the regulation of heme oxygenase-1 (HO-1) gene expression. In this report, we further test whether CDH exhibited the hormonal-like response. The application of CDH and an inducer of HO-1, hemin, were able to induce the up-regulation of cucumber HO-1 gene (CsHO1) expression and thereafter the promotion of adventitious rooting in cucumber explants. The effect is specific for HO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blocked the above responses triggered by CDH, and the inhibitory effects were reversed further when 30% saturation of CO aqueous solution was added together. Further, molecular evidence showed that CDH triggered the increases of the HO-1-mediated target genes responsible for adventitious rooting, including one DnaJ-like gene (CsDNAJ-1) and two calcium-dependent protein kinase (CDPK) genes (CsCDPK1 and CsCDPK5), and were inhibited by ZnPP and reversed by CO. The calcium (Ca2+) chelator ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and the Ca2+ channel blocker lanthanum chloride (LaCl3) not only compromised the induction of adventitious rooting induced by CDH but also decreased the transcripts of above three target genes. However, the application of ascorbic acid (AsA), a well-known antioxidant in plants, failed to exhibit similar inducible effect on adventitious root formation. In short, above results illustrated that the response of CDH in the induction of cucumber adventitious rooting might be through HO-1-dependent mechanism and calcium signaling. Physiological, pharmacological and molecular evidence showed that β-cyclodextrin-hemin complex (CDH) was able to induce cucumber adventitious rooting through heme oxygenase-1 (HO-1)-dependent mechanism and calcium signaling.

  1. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus

    2013-01-01

    Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange...... in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads...... complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor...

  2. Oxygen Atom Exchange between H2O and Non-Heme Oxoiron(IV) Complexes: Ligand Dependence and Mechanism.

    Science.gov (United States)

    Puri, Mayank; Company, Anna; Sabenya, Gerard; Costas, Miquel; Que, Lawrence

    2016-06-20

    Detailed studies of oxygen atom exchange (OAE) between H2(18)O and synthetic non-heme oxoiron(IV) complexes supported by tetradentate and pentadentate ligands provide evidence that they proceed by a common mechanism but within two different kinetic regimes, with OAE rates that span 2 orders of magnitude. The first kinetic regime involves initial reversible water association to the Fe(IV) complex, which is evidenced by OAE rates that are linearly dependent on [H2(18)O] and H2O/D2O KIEs of 1.6, while the second kinetic regime involves a subsequent rate determining proton-transfer step between the bound aqua and oxo ligands that is associated with saturation behavior with [H2(18)O] and much larger H2O/D2O KIEs of 5-6. [Fe(IV)(O)(TMC)(MeCN)](2+) (1) and [Fe(IV)(O)(MePy2TACN)](2+) (9) are examples of complexes that exhibit kinetic behavior in the first regime, while [Fe(IV)(O)(N4Py)](2+) (3), [Fe(IV)(O)(BnTPEN)](2+) (4), [Fe(IV)(O)(1Py-BnTPEN)](2+) (5), [Fe(IV)(O)(3Py-BnTPEN)](2+) (6), and [Fe(IV)(O)(Me2Py2TACN)](2+) (8) represent complexes that fall in the second kinetic regime. Interestingly, [Fe(IV)(O)(PyTACN)(MeCN)](2+) (7) exhibits a linear [H2(18)O] dependence below 0.6 M and saturation above 0.6 M. Analysis of the temperature dependence of the OAE rates shows that most of these complexes exhibit large and negative activation entropies, consistent with the proposed mechanism. One exception is complex 9, which has a near-zero activation entropy and is proposed to undergo ligand-arm dissociation during the RDS to accommodate H2(18)O binding. These results show that the observed OAE kinetic behavior is highly dependent on the nature of the supporting ligand and are of relevance to studies of non-heme oxoiron(IV) complexes in water or acetonitrile/water mixtures for applications in photocatalysis and water oxidation chemistry.

  3. Nitrite to nitric oxide interconversion by heme FeII complex assisted by [CuI(tmpa)]+

    KAUST Repository

    Turias, Francesc; Solà , Miquel; Falivene, Laura; Cavallo, Luigi; Poater, Albert

    2015-01-01

    The present computational study complements the recent experimental efforts by Karlin and coworkers to describe the interconversion of nitrite to nitric oxide by means of an iron porphyrin complex together with a Cu chemical system, i.e., the iron(II) complex (F8TPP)FeII [F8TPP = tetrakis(2,6-difluorophenyl)porphyrinate(2−)] and a preformed copper(II)–nitrito complex [(tmpa)CuII(NO2)][B(C6F5)4] [tmpa = tris(2-pyridylmethyl)amine], being the latter an oxidized species of [(tmpa)CuI(MeCN)]+. By DFT calculations, we unravel how the reduction of nitrite to nitric oxide takes place through a μ-oxo heme-FeIII–O–CuII complex, following a mimetic path as in the cytochrome c oxidase. Mayer bond order (MBO) and energy decomposition analyses are used to analyze the bonding strength of such nitro derivatives to either copper or iron. © 2015 Springer Science+Business Media New York

  4. Nitrite to nitric oxide interconversion by heme FeII complex assisted by [CuI(tmpa)]+

    KAUST Repository

    Turias, Francesc

    2015-09-09

    The present computational study complements the recent experimental efforts by Karlin and coworkers to describe the interconversion of nitrite to nitric oxide by means of an iron porphyrin complex together with a Cu chemical system, i.e., the iron(II) complex (F8TPP)FeII [F8TPP = tetrakis(2,6-difluorophenyl)porphyrinate(2−)] and a preformed copper(II)–nitrito complex [(tmpa)CuII(NO2)][B(C6F5)4] [tmpa = tris(2-pyridylmethyl)amine], being the latter an oxidized species of [(tmpa)CuI(MeCN)]+. By DFT calculations, we unravel how the reduction of nitrite to nitric oxide takes place through a μ-oxo heme-FeIII–O–CuII complex, following a mimetic path as in the cytochrome c oxidase. Mayer bond order (MBO) and energy decomposition analyses are used to analyze the bonding strength of such nitro derivatives to either copper or iron. © 2015 Springer Science+Business Media New York

  5. Comparison of MukB homodimer versus MukBEF complex molecular architectures by electron microscopy reveals a higher-order multimerization

    International Nuclear Information System (INIS)

    Matoba, Kyoko; Yamazoe, Mitsuyoshi; Mayanagi, Kouta; Morikawa, Kosuke; Hiraga, Sota

    2005-01-01

    The complex of MukF, MukE, and MukB proteins participates in organization of sister chromosomes and partitioning into both daughter cells in Escherichia coli. We purified the MukB homodimer and the MukBEF complex and analyzed them by electron microscopy to compare both structures. A MukB homodimer shows a long rod-hinge-rod v-shape with small globular domains at both ends. The MukBEF complex shows a similar structure having larger globular domains than those of the MukB homodimer. These results suggest that MukF and MukE bind to the globular domains of a MukB homodimer. The globular domains of the MukBEF complex frequently associate with each other in an intramolecular fashion, forming a ring. In addition, MukBEF complex molecules tend to form multimers by the end-to-end joining with other MukBEF molecules in an intermolecular fashion, resulting in fibers and rosette-form structures in the absence of ATP and DNA in vitro

  6. A dual component heme biosensor that integrates heme transport and synthesis in bacteria.

    Science.gov (United States)

    Nobles, Christopher L; Clark, Justin R; Green, Sabrina I; Maresso, Anthony W

    2015-11-01

    Bacterial pathogens acquire host iron to power cellular processes and replication. Heme, an iron-containing cofactor bound to hemoglobin, is scavenged by bacterial proteins to attain iron. Methods to measure intracellular heme are laborious, involve complex chemistry, or require radioactivity. Such drawbacks limit the study of the mechanistic steps of heme transport and breakdown. Hypothesizing heme homeostasis could be measured with fluorescent methods, we coupled the conversion of heme to biliverdin IXα (a product of heme catabolism) by heme oxygenase 1 (HO1) with the production of near-infrared light upon binding this verdin by infrared fluorescent protein (IFP1.4). The resultant heme sensor, IFP-HO1, was fluorescent in pathogenic E. coli exposed to heme but not in the absence of the heme transporter ChuA and membrane coupling protein TonB, thereby validating their long-standing proposed role in heme uptake. Fluorescence was abolished in a strain lacking hemE, the central gene in the heme biosynthetic pathway, but stimulated by iron, signifying the sensor reports on intracellular heme production. Finally, an invasive strain of E. coli harboring the sensor was fluorescent during an active infection. This work will allow researchers to expand the molecular toolbox used to study heme and iron acquisition in culture and during infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Control of intracellular heme levels: Heme transporters and Heme oxygenases

    Science.gov (United States)

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology. PMID:21238504

  8. Control of intracellular heme levels: Heme transporters and heme oxygenases

    OpenAIRE

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

  9. Multimerization rules for G-quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Kolesnikova, Sofia; Hubálek, Martin; Bednárová, Lucie; Cvačka, Josef; Curtis, Edward A.

    2017-01-01

    Roč. 45, č. 15 (2017), s. 8684-8696 ISSN 0305-1048 Institutional support: RVO:61388963 Keywords : tetramolecular G-quadruplexes * RNA G-quadruplexes * circular dichroism Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 10.162, year: 2016 https://academic.oup.com/nar/article/45/15/8684/4002725/Multimerization-rules-for-Gquadruplexes

  10. Photoenhanced Oxidative DNA Cleavage with Non-Heme Iron(II) Complexes

    NARCIS (Netherlands)

    Li, Qian; Browne, Wesley R.; Roelfes, Gerard

    2010-01-01

    The DNA cleavage activity of iron(II) complexes of a series of monotopic pentadentate N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py)-derived ligands (1-5) was investigated under laser irradiation at 473, 400.8, and 355 nm in the absence of a reducing agent and compared to that under

  11. Mitochondrial Cytochrome c Oxidase Biogenesis Is Regulated by the Redox State of a Heme-Binding Translational Activator.

    Science.gov (United States)

    Soto, Iliana C; Barrientos, Antoni

    2016-02-20

    Mitochondrial cytochrome c oxidase (COX), the last enzyme of the respiratory chain, catalyzes the reduction of oxygen to water and therefore is essential for cell function and viability. COX is a multimeric complex, whose biogenesis is extensively regulated. One type of control targets cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. In this study, we investigated whether Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment. We report that Cox1 synthesis is attenuated under oxidative stress conditions and have identified one of the underlying mechanisms. We show that in vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation. H2O2-induced downregulation of mitochondrial translation has so far not been reported. We show that high H2O2 concentrations induce a global attenuation effect, but milder concentrations specifically affect COX1 mRNA processing and translation in an Mss51-dependent manner. The redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production.

  12. The hydroxyl functionality and a rigid proximal N are required for forming a novel non-covalent quinine-heme complex.

    Science.gov (United States)

    Alumasa, John N; Gorka, Alexander P; Casabianca, Leah B; Comstock, Erica; de Dios, Angel C; Roepe, Paul D

    2011-03-01

    Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this important distinction, we study three newly synthesized and several commercially available QN analogues lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin (Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN (DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid-state NMR spectroscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. A role for heme in Alzheimer's disease: Heme binds amyloid β and has altered metabolism

    OpenAIRE

    Atamna, Hani; Frey, William H.

    2004-01-01

    Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-b (P < 0.01) and 26% less heme-a (P = 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in he...

  14. The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS.

    Science.gov (United States)

    Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina; Hunter, Gregory A; Gakh, Oleksandr; Isaya, Grazia; Ferreira, Gloria C; Al-Karadaghi, Salam

    2016-05-27

    Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: Implications for the enzyme mechanism‡

    OpenAIRE

    Ogura, Hiroshi; Evans, John P.; Peng, Dungeng; Satterlee, James D.; de Montellano, Paul R. Ortiz; Mar, Gerd N. La

    2009-01-01

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase-1, (hHO) has been investigated by 1H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. 2D 1H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts, that places the lone iron π-spin in the dxz orbital, rather than the dyz orbital found in the cya...

  16. Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

    Science.gov (United States)

    Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

    2017-09-01

    Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

  17. Oxidative stress/damage induces multimerization and interaction of Fanconi anemia proteins.

    Science.gov (United States)

    Park, Su-Jung; Ciccone, Samantha L M; Beck, Brian D; Hwang, Byounghoon; Freie, Brian; Clapp, D Wade; Lee, Suk-Hee

    2004-07-16

    Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function.

  18. Solution NMR characterization of magnetic/electronic properties of azide and cyanide-inhibited substrate complexes of human heme oxygenase: implications for steric ligand tilt.

    Science.gov (United States)

    Peng, Dungeng; Ogura, Hiroshi; Ma, Li-Hua; Evans, John P; de Montellano, Paul R Ortiz; La Mar, Gerd N

    2013-04-01

    Solution 2D (1)H NMR was carried out on the azide-ligated substrate complex of human heme oxygenase, hHO, to provide information on the active site molecular structure, chromophore electronic/magnetic properties, and the distal H-bond network linked to the exogenous ligand by catalytically relevant oriented water molecules. While 2D NMR exhibited very similar patterns of two-dimensional nuclear Overhauser spectroscopy cross peaks of residues with substrate and among residues as the previously characterized cyanide complex, significant, broadly distributed chemical shift differences were observed for both labile and non-labile protons. The anisotropy and orientation of the paramagnetic susceptibility tensor, χ, were determined for both the azide and cyanide complexes. The most significant difference observed is the tilt of the major magnetic axes from the heme normal, which is only half as large for the azide than cyanide ligand, with each ligand tilted toward the catalytically cleaved α-meso position. The difference in chemical shifts is quantitatively correlated with differences in dipolar shifts in the respective complexes for all but the distal helix. The necessity of considering dipolar shifts, and hence determination of the orientation/anisotropy of χ, in comparing chemical shifts involving paramagnetic complexes, is emphasized. The analysis shows that the H-bond network cannot detect significant differences in H-bond acceptor properties of cyanide versus azide ligands. Lastly, significant retardation of distal helix labile proton exchange upon replacing cyanide with azide indicates that the dynamic stability of the distal helix is increased upon decreasing the steric interaction of the ligand with the distal helix. Copyright © 2013. Published by Elsevier Inc.

  19. Heme Sensor Proteins*

    Science.gov (United States)

    Girvan, Hazel M.; Munro, Andrew W.

    2013-01-01

    Heme is a prosthetic group best known for roles in oxygen transport, oxidative catalysis, and respiratory electron transport. Recent years have seen the roles of heme extended to sensors of gases such as O2 and NO and cell redox state, and as mediators of cellular responses to changes in intracellular levels of these gases. The importance of heme is further evident from identification of proteins that bind heme reversibly, using it as a signal, e.g. to regulate gene expression in circadian rhythm pathways and control heme synthesis itself. In this minireview, we explore the current knowledge of the diverse roles of heme sensor proteins. PMID:23539616

  20. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

    International Nuclear Information System (INIS)

    Wojtowicz, Halina; Wojaczynski, Jacek; Olczak, Mariusz; Kroliczewski, Jaroslaw; Latos-Grazynski, Lechoslaw; Olczak, Teresa

    2009-01-01

    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and 1 H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  1. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Wojtowicz, Halina [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Wojaczynski, Jacek [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Mariusz [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Kroliczewski, Jaroslaw [Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, 50-148 Wroclaw (Poland); Latos-Grazynski, Lechoslaw [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Teresa [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland)

    2009-05-29

    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and {sup 1}H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  2. Mechanisms of heme utilization by Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Helena Lindgren

    Full Text Available Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.

  3. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: Implications for the enzyme mechanism‡

    Science.gov (United States)

    Ogura, Hiroshi; Evans, John P.; Peng, Dungeng; Satterlee, James D.; de Montellano, Paul R. Ortiz; Mar, Gerd N. La

    2009-01-01

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase-1, (hHO) has been investigated by 1H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. 2D 1H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts, that places the lone iron π-spin in the dxz orbital, rather than the dyz orbital found in the cyanide complex. Comparison of iron spin relaxivity, magnetic anisotropy and magnetic susceptibilities argues for a low-spin, (dxy)2(dyz,dxz)3, ground state in both azide and cyanide complexes. The switch from singly-occupied dyz for the cyanide to dxz for the azide complex of hHO is shown to be consistent with the orbital hole determined by the azide π-plane in the latter complex, which is ∼90° in-plane rotated from that of the imidazole π-plane. The induction of the altered orbital ground state in the azide relative to the cyanide hHO complex, as well as the mean low-field bias of methyl hyperfine shifts and their paramagnetic relaxivity relative to those in globins, indicate that azide exerts a stronger ligand field in hHO than in the globins, or that the distal H-bonding to azide is weaker in hHO than in globins. The Asp140 → Ala hHO mutant that abolishes activity retains the unusual WT azide complex spin/orbital ground state. The relevance of our findings for other HO complexes and the HO mechanism is discussed. PMID:19243105

  4. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: implications for the enzyme mechanism.

    Science.gov (United States)

    Ogura, Hiroshi; Evans, John P; Peng, Dungeng; Satterlee, James D; Ortiz de Montellano, Paul R; La Mar, Gerd N

    2009-04-14

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase 1 (hHO) has been investigated by (1)H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. Two-dimensional (1)H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts that places the lone iron pi-spin in the d(xz) orbital, rather than the d(yz) orbital found in the cyanide complex. Comparison of iron spin relaxivity, magnetic anisotropy, and magnetic susceptibilities argues for a low-spin, (d(xy))(2)(d(yz),d(xz))(3), ground state in both azide and cyanide complexes. The switch from singly occupied d(yz) for the cyanide to d(xz) for the azide complex of hHO is shown to be consistent with the orbital hole determined by the azide pi-plane in the latter complex, which is approximately 90 degrees in-plane rotated from that of the imidazole pi-plane. The induction of the altered orbital ground state in the azide relative to the cyanide hHO complex, as well as the mean low-field bias of methyl hyperfine shifts and their paramagnetic relaxivity relative to those in globins, indicates that azide exerts a stronger ligand field in hHO than in the globins, or that the distal H-bonding to azide is weaker in hHO than in globins. The Asp140 --> Ala hHO mutant that abolishes activity retains the unusual WT azide complex spin/orbital ground state. The relevance of our findings for other HO complexes and the HO mechanism is discussed.

  5. Structure and Heme-Independent Peroxidase Activity of a Fully-Coordinated Mononuclear Mn(II) Complex with a Schiff-Base Tripodal Ligand Containing Three Imidazole Groups

    Energy Technology Data Exchange (ETDEWEB)

    Sarkar, Shuranjan; Lee, Hong In [Kyungpook National University, Daegu (Korea, Republic of); Moon, Do Hyun [Pohang Accelerator Laboratory, Pohang (Korea, Republic of); Lah, Myoung Soo [Ulsan National Institute of Science and Technology, Ulsan (Korea, Republic of)

    2010-11-15

    New complex [Mn(II)H{sub 1.5}L]{sub 2}[Mn(II)H{sub 3}L]{sub 2}(ClO{sub 4}){sub 5}·3H{sub 2}O, where H{sub 3}L is tris{2-(4-imidazolyl)methyliminoethyl} amine (imtren), has been prepared by reacting manganese(II) perchlorate hexahydrate with the imtren ligand in methanol. X-ray crystallographic study revealed that the imtren ligand hexadentately binds to Mn(II) ion through the three Schiff-base imine N atoms and three imidazole N atoms with a distorted octahedral geometry, and the apical tertiary amine N atom of the ligand pseudo-coordinates to Mn(II), forming overall a pseudo-seven coordination environment. The hydrogen-bonds between imidazole and imidazolate of [Mn(II)H{sub 1.5}L]{sup 0.5+} complex ions are extended to build a 2D puckered network with trigonal voids. [Mn(II)H{sub 3}L]{sup 2+} complex ions constitutes another extended 2D puckered layer without hydrogen bonds. Two layers are wedged each other to constitute overall stack of the crystal. Peroxidase activity of complex 1 was examined by observing the oxidation of 2,2'-azinobis(3-ethylbenzothiazoline)- 6-sulfonic acid (ABTS) with hydrogen peroxide in the presence of complex 1. Generation of ABTS{sup +·} was observed by UV-vis and EPR spectroscopies, indicating that the complex 1, a fully-coordinated mononuclear Mn(II) complex with nitrogen-only ligand, has a heme-independent peroxidase activity.

  6. An ethane-bridged porphyrin dimer as a model of di-heme proteins: inorganic and bioinorganic perspectives and consequences of heme-heme interactions.

    Science.gov (United States)

    Sil, Debangsu; Rath, Sankar Prasad

    2015-10-07

    Interaction between heme centers has been cleverly implemented by Nature in order to regulate different properties of multiheme cytochromes, thereby allowing them to perform a wide variety of functions. Our broad interest lies in unmasking the roles played by heme-heme interactions in modulating different properties viz., metal spin state, redox potential etc., of the individual heme centers using an ethane-bridged porphyrin dimer as a synthetic model of dihemes. The large differences in the structure and properties of the diheme complexes, as compared to the monoheme analogs, provide unequivocal evidence of the role played by heme-heme interactions in the dihemes. This Perspective provides a brief account of our recent efforts to explore these interesting aspects and the subsequent outcomes.

  7. Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase

    International Nuclear Information System (INIS)

    Verkman, A.S.; Skorecki, K.L.; Ausiello, D.A.

    1986-01-01

    Radiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone

  8. Binding of molecular oxygen by an artificial heme analogue: investigation on the formation of an Fe–tetracarbene superoxo complex

    KAUST Repository

    Anneser, Markus R.; Haslinger, Stefan; Pö thig, Alexander; Cokoja, Mirza; D'Elia, Valerio; Hö gerl, Manuel P.; Basset, Jean-Marie; Kü hn, Fritz E.

    2016-01-01

    The dioxygen reactivity of a cyclic iron(II) tetra–NHC-complex (NHC: N-heterocyclic carbene) is investigated. Divergent oxidation behavior is observed depending on the choice of the solvent (acetonitrile or acetone). In the first case, exposure

  9. Binding of molecular oxygen by an artificial heme analogue: investigation on the formation of an Fe–tetracarbene superoxo complex

    KAUST Repository

    Anneser, Markus R.

    2016-02-26

    The dioxygen reactivity of a cyclic iron(II) tetra–NHC-complex (NHC: N-heterocyclic carbene) is investigated. Divergent oxidation behavior is observed depending on the choice of the solvent (acetonitrile or acetone). In the first case, exposure to molecular oxygen leads to an oxygen free Fe(III) whereas in the latter case an oxide bridged Fe(III) dimer is formed. In acetone, an Fe(III)-superoxide can be trapped, isolated and characterized as intermediate at low temperatures. An Fe(III)–O–Fe(III) dimer is formed from the Fe(III) superoxide in acetone upon warming and the molecular structure has been revealed by single crystal X-ray diffraction. It is shown that the oxidation of the Fe(II) complex in both solvents is a reversible process. For the regeneration of the initial Fe(II) complex both organic and inorganic reducing agents can be used.

  10. Synthesis and characterization of an iron complex bearing a cyclic tetra-N-heterocyclic carbene ligand: An artifical heme analogue?

    KAUST Repository

    Anneser, Markus R.

    2015-04-20

    An iron(II) complex with a cyclic tetradentate ligand containing four N-heterocyclic carbenes was synthesized and characterized by means of NMR and IR spectroscopies, as well as by single-crystal X-ray structure analysis. The iron center exhibits an octahedral coordination geometry with two acetonitrile ligands in axial positions, showing structural analogies with porphyrine-ligated iron complexes. The acetonitrile ligands can readily be substituted by other ligands, for instance, dimethyl sulfoxide, carbon monoxide, and nitric oxide. Cyclic voltammetry was used to examine the electronic properties of the synthesized compounds. © 2015 American Chemical Society.

  11. The mechanism of stereospecific C-H oxidation by Fe(Pytacn) complexes: bioinspired non-heme iron catalysts containing cis-labile exchangeable sites.

    Science.gov (United States)

    Prat, Irene; Company, Anna; Postils, Verònica; Ribas, Xavi; Que, Lawrence; Luis, Josep M; Costas, Miquel

    2013-05-17

    A detailed mechanistic study of the hydroxylation of alkane C-H bonds using H2O2 by a family of mononuclear non heme iron catalysts with the formula [Fe(II)(CF3SO3)2(L)] is described, in which L is a tetradentate ligand containing a triazacyclononane tripod and a pyridine ring bearing different substituents at the α and γ positions, which tune the electronic or steric properties of the corresponding iron complexes. Two inequivalent cis-labile exchangeable sites, occupied by triflate ions, complete the octahedral iron coordination sphere. The C-H hydroxylation mediated by this family of complexes takes place with retention of configuration. Oxygen atoms from water are incorporated into hydroxylated products and the extent of this incorporation depends in a systematic manner on the nature of the catalyst, and the substrate. Mechanistic probes and isotopic analyses, in combination with detailed density functional theory (DFT) calculations, provide strong evidence that C-H hydroxylation is performed by highly electrophilic [Fe(V)(O)(OH)L] species through a concerted asynchronous mechanism, involving homolytic breakage of the C-H bond, followed by rebound of the hydroxyl ligand. The [Fe(V)(O)(OH)L] species can exist in two tautomeric forms, differing in the position of oxo and hydroxide ligands. Isotopic-labeling analysis shows that the relative reactivities of the two tautomeric forms are sensitively affected by the α substituent of the pyridine, and this reactivity behavior is rationalized by computational methods. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Experimental and Computational Evidence for the Mechanism of Intradiol Catechol Dioxygenation by Non- Heme Iron(III) Complexes

    NARCIS (Netherlands)

    Jastrzebski, Robin; Quesne, Matthew G.; Weckhuysen, Bert M.; de Visser, Sam P.; Bruijnincx, Pieter C. A.

    2014-01-01

    Catechol intradiol dioxygenation is a unique reaction catalyzed by iron-dependent enzymes and nonheme iron(III) complexes. The mechanism by which these systems activate dioxygen in this important metabolic process remains controversial. Using a combination of kinetic measurements and computational

  13. Influence of Ligand Architecture in Tuning Reaction Bifurcation Pathways for Chlorite Oxidation by Non-Heme Iron Complexes

    NARCIS (Netherlands)

    Barman, Prasenjit; Faponle, Abayomi S; Vardhaman, Anil Kumar; Angelone, Davide; Löhr, Anna-Maria; Browne, Wesley R; Comba, Peter; Sastri, Chivukula V; de Visser, Sam P

    2016-01-01

    Reaction bifurcation processes are often encountered in the oxidation of substrates by enzymes and generally lead to a mixture of products. One particular bifurcation process that is common in biology relates to electron transfer versus oxygen atom transfer by high-valent iron(IV)-oxo complexes,

  14. Effects of heme-PrP complex on cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays.

    Science.gov (United States)

    Soutyrine, Andrei; Yogasingam, Nishandan; Huang, Hongsheng; Mitchell, Gordon

    2015-08-01

    Prion protein (PrP) binding to natural and synthetic porphyrins has been previously demonstrated but the effects of endogenous heme interactions with PrP remain uncertain. This study investigated implications of this interaction in blood-based peroxidase-linked prion immunodetection and seeded conversion of cellular prion (PrP(C)) into disease associated form (PrP(Sc)). Heme binding to recombinant PrP(C) enhanced intrinsic peroxidase activity (POD) by 2.5-fold and POD inherent to denatured blood accounted for over 84% of luminol-based substrate oxidation in a prion immunodetection assay. An immuno-capture assay showed that 75-98% of blood POD was attributable to binding of PrP(C) with endogenous heme. Additionally, 10 μM heme inhibited (PPrP(C) to PrP(Sc) through the protein misfolding cycling amplification assay. We conclude that the observed effects can interfere with cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays. These results indicate that heme-PrP interactions could modulate intrinsic POD and protect PrP(C) from conversion into PrP(Sc). Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  15. The Semireduced Mechanism for Nitric Oxide Reduction by Non-Heme Diiron Complexes: Modeling Flavodiiron Nitric Oxide Reductases.

    Science.gov (United States)

    White, Corey J; Speelman, Amy L; Kupper, Claudia; Demeshko, Serhiy; Meyer, Franc; Shanahan, James P; Alp, E Ercan; Hu, Michael; Zhao, Jiyong; Lehnert, Nicolai

    2018-02-21

    Flavodiiron nitric oxide reductases (FNORs) are a subclass of flavodiiron proteins (FDPs) capable of preferential binding and subsequent reduction of NO to N 2 O. FNORs are found in certain pathogenic bacteria, equipping them with resistance to nitrosative stress, generated as a part of the immune defense in humans, and allowing them to proliferate. Here, we report the spectroscopic characterization and detailed reactivity studies of the diiron dinitrosyl model complex [Fe 2 (BPMP)(OPr)(NO) 2 ](OTf) 2 for the FNOR active site that is capable of reducing NO to N 2 O [Zheng et al., J. Am. Chem. Soc. 2013, 135, 4902-4905]. Using UV-vis spectroscopy, cyclic voltammetry, and spectro-electrochemistry, we show that one reductive equivalent is in fact sufficient for the quantitative generation of N 2 O, following a semireduced reaction mechanism. This reaction is very efficient and produces N 2 O with a first-order rate constant k > 10 2 s -1 . Further isotope labeling studies confirm an intramolecular N-N coupling mechanism, consistent with the rapid time scale of the reduction and a very low barrier for N-N bond formation. Accordingly, the reaction proceeds at -80 °C, allowing for the direct observation of the mixed-valent product of the reaction. At higher temperatures, the initial reaction product is unstable and decays, ultimately generating the diferrous complex [Fe 2 (BPMP)(OPr) 2 ](OTf) and an unidentified ferric product. These results combined offer deep insight into the mechanism of NO reduction by the relevant model complex [Fe 2 (BPMP)(OPr)(NO) 2 ] 2+ and provide direct evidence that the semireduced mechanism would constitute a highly efficient pathway to accomplish NO reduction to N 2 O in FNORs and in synthetic catalysts.

  16. Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors.

    Science.gov (United States)

    Hanna, David A; Martinez-Guzman, Osiris; Reddi, Amit R

    2017-04-04

    Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.

  17. Heme metabolism as an integral part of iron homeostasis

    Directory of Open Access Journals (Sweden)

    Paweł Lipiński

    2014-01-01

    Full Text Available Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways – heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S] – is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages.

  18. [Heme metabolism as an integral part of iron homeostasis].

    Science.gov (United States)

    Lipiński, Paweł; Starzyński, Rafał R; Styś, Agnieszka; Gajowiak, Anna; Staroń, Robert

    2014-01-02

    Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways--heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S])--is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis) as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages.

  19. Mononuclear non-heme iron(III)

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 123; Issue 2. Mononuclear non-heme iron(III) complexes of linear and tripodal tridentate ligands as functional models for catechol dioxygenases: Effect of -alkyl substitution on regioselectivity and reaction rate. Mallayan Palaniandavar Kusalendiran Visvaganesan.

  20. Electron Transport in a Dioxygenase-Ferredoxin Complex: Long Range Charge Coupling between the Rieske and Non-Heme Iron Center.

    Directory of Open Access Journals (Sweden)

    Wayne K Dawson

    Full Text Available Dioxygenase (dOx utilizes stereospecific oxidation on aromatic molecules; consequently, dOx has potential applications in bioremediation and stereospecific oxidation synthesis. The reactive components of dOx comprise a Rieske structure Cys2[2Fe-2S]His2 and a non-heme reactive oxygen center (ROC. Between the Rieske structure and the ROC, a universally conserved Asp residue appears to bridge the two structures forming a Rieske-Asp-ROC triad, where the Asp is known to be essential for electron transfer processes. The Rieske and ROC share hydrogen bonds with Asp through their His ligands; suggesting an ideal network for electron transfer via the carboxyl side chain of Asp. Associated with the dOx is an itinerant charge carrying protein Ferredoxin (Fdx. Depending on the specific cognate, Fdx may also possess either the Rieske structure or a related structure known as 4-Cys-[2Fe-2S] (4-Cys. In this study, we extensively explore, at different levels of theory, the behavior of the individual components (Rieske and ROC and their interaction together via the Asp using a variety of density function methods, basis sets, and a method known as Generalized Ionic Fragment Approach (GIFA that permits setting up spin configurations manually. We also report results on the 4-Cys structure for comparison. The individual optimized structures are compared with observed spectroscopic data from the Rieske, 4-Cys and ROC structures (where information is available. The separate pieces are then combined together into a large Rieske-Asp-ROC (donor/bridge/acceptor complex to estimate the overall coupling between individual components, based on changes to the partial charges. The results suggest that the partial charges are significantly altered when Asp bridges the Rieske and the ROC; hence, long range coupling through hydrogen bonding effects via the intercalated Asp bridge can drastically affect the partial charge distributions compared to the individual isolated

  1. Heme Recognition By a Staphylococcus Aureus IsdE

    Energy Technology Data Exchange (ETDEWEB)

    Grigg, J.C.; Vermeiren, C.L.; Heinrichs, D.E.; Murphy, M.E.P.

    2009-06-03

    Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single {alpha}-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met{sup 78} and His{sup 229}. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His{sup 299} is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

  2. The Complexity of Mitochondrial Complex IV: An Update of Cytochrome c Oxidase Biogenesis in Plants

    Science.gov (United States)

    Mansilla, Natanael; Racca, Sofia; Gras, Diana E.; Gonzalez, Daniel H.

    2018-01-01

    Mitochondrial respiration is an energy producing process that involves the coordinated action of several protein complexes embedded in the inner membrane to finally produce ATP. Complex IV or Cytochrome c Oxidase (COX) is the last electron acceptor of the respiratory chain, involved in the reduction of O2 to H2O. COX is a multimeric complex formed by multiple structural subunits encoded in two different genomes, prosthetic groups (heme a and heme a3), and metallic centers (CuA and CuB). Tens of accessory proteins are required for mitochondrial RNA processing, synthesis and delivery of prosthetic groups and metallic centers, and for the final assembly of subunits to build a functional complex. In this review, we perform a comparative analysis of COX composition and biogenesis factors in yeast, mammals and plants. We also describe possible external and internal factors controlling the expression of structural proteins and assembly factors at the transcriptional and post-translational levels, and the effect of deficiencies in different steps of COX biogenesis to infer the role of COX in different aspects of plant development. We conclude that COX assembly in plants has conserved and specific features, probably due to the incorporation of a different set of subunits during evolution. PMID:29495437

  3. Multimeric, Multifunctional Derivatives of Poly(ethylene glycol

    Directory of Open Access Journals (Sweden)

    Gian Maria Bonora

    2011-07-01

    Full Text Available This article reviews the use of multifunctional polymers founded on high-molecular weight poly(ethylene glycol (PEG. The design of new PEG derivatives assembled in a dendrimer-like multimeric fashion or bearing different functionalities on the same molecule is described. Their use as new drug delivery systems based on the conjugation of multiple copies or diversely active drugs on the same biocompatible support is illustrated.

  4. Mini Heme-Proteins: Designability of Structure and Diversity of Functions.

    Science.gov (United States)

    Rai, Jagdish

    2017-08-30

    Natural heme proteins may have heme bound to poly-peptide chain as a cofactor via noncovalent forces or heme as a prosthetic group may be covalently bound to the proteins. Nature has used porphyrins in diverse functions like electron transfer, oxidation, reduction, ligand binding, photosynthesis, signaling, etc. by modulating its properties through diverse protein matrices. Synthetic chemists have tried to utilize these molecules in equally diverse industrial and medical applications due to their versatile electro-chemical and optical properties. The heme iron has catalytic activity which can be modulated and enhanced for specific applications by protein matrix around it. Heme proteins can be designed into novel enzymes for sterio specific catalysis ranging from oxidation to reduction. These designed heme-proteins can have applications in industrial catalysis and biosensing. A peptide folds around heme easily due to hydrophobic effect of the large aromatic ring of heme. The directional property of co-ordinate bonding between peptide and metal ion in heme further specifies the structure. Therefore heme proteins can be easily designed for targeted structure and catalytic activity. The central aromatic chemical entity in heme viz. porphyrin is a very ancient molecule. Its presence in the prebiotic soup and in all forms of life suggests that it has played a vital role in the origin and progressive evolution of living organisms. Porphyrin macrocycles are highly conjugated systems composed of four modified pyrrole subunits interconnected at their α -carbon atoms via methine (=CH-) bridges. Initial minimalist models of hemoproteins focused on effect of heme-ligand co-ordinate bonding on chemical reactivity, spectroscopy, electrochemistry and magnetic properties of heme. The great sensitivity of these spectroscopic features of heme to its surrounding makes them extremely useful in structural elucidation of designed heme-peptide complexes. Therefore heme proteins are

  5. Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

    Science.gov (United States)

    Jiang, Yongying; Trnka, Michael J; Medzihradszky, Katalin F; Ouellet, Hugues; Wang, Yongqiang; Ortiz de Montellano, Paul R

    2009-03-01

    To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.

  6. Multimeric and trimeric subunit SP-D are interconvertible structures with distinct ligand interaction

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Hoegh, Silje V; Leth-Larsen, Rikke

    2009-01-01

    -D compared to Met11 SP-D. Multimerization has proven important for enhancement of microbial phagocytosis. In the present study defined multimeric forms of Met11Thr SP-D were isolated from human amniotic fluid. Implementation of ManNAc-affinity chromatography allowed high recovery of natural trimeric SP......-D multimers. Trimeric SP-D subunits also showed greater binding to endogenous lipoproteins: LDL, oxLDL, and HDL, than multimeric SP-D. In conclusion, purified trimeric and multimeric SP-D represent separate and only partly interconvertible molecular populations with distinct biochemical properties....

  7. One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.

    Science.gov (United States)

    Hamza, Iqbal; Dailey, Harry A

    2012-09-01

    The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Mimicking heme enzymes in the solid state: metal-organic materials with selectively encapsulated heme.

    Science.gov (United States)

    Larsen, Randy W; Wojtas, Lukasz; Perman, Jason; Musselman, Ronald L; Zaworotko, Michael J; Vetromile, Carissa M

    2011-07-13

    To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal-organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a "ship-in-a-bottle" fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

  9. Heme Synthesis and Acquisition in Bacterial Pathogens

    OpenAIRE

    Choby, Jacob E.; Skaar, Eric P.

    2016-01-01

    Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host source...

  10. Heme isomers substantially affect heme's electronic structure and function

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta

    2017-01-01

    Inspection of heme protein structures in the protein data bank reveals four isomers of heme characterized by different relative orientations of the vinyl side chains; remarkably, all these have been reported in multiple protein structures. Density functional theory computations explain this as du...

  11. Wiring of heme enzymes by methylene-blue labeled dendrimers

    DEFF Research Database (Denmark)

    Álvarez-Martos, Isabel; Shahdost-fard, Faezeh; Ferapontova, Elena

    2017-01-01

    Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme- and moli......Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme......- and molibdopterin-containing sulfite oxidase (SOx), wired to gold by the methylene blue (MB)-labeled polyamidoamine (PAMAM) dendrimers. The enzymes’ electrochemical transformation and bioelectrocatalytic function could be followed at both unlabeled and MB-labeled dendrimer-modified electrodes with the formal redox......, optimization of bioelectrocatalysis of complex intermembrane and, possibly, membrane enzymes....

  12. Multimerization of the cytoplasmic domain of syndecan-4 is required for its ability to activate protein kinase C

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Couchman, J R

    1997-01-01

    of syndecan-4 (4L) containing a membrane-proximal basic sequence did not form higher order oligomers and could not regulate the activity of PKCalphabetagamma unless induced to aggregate by phosphatidylinositol 4,5-bisphosphate. Oligomerization and PKC regulatory activity of the 4V peptide were both increased...... by addition of N-terminal cysteine and reduced by phosphorylation of the cysteine thiol group. Concentration of syndecan-4 at sites of focal adhesion formation may enhance multimerization and both localize PKC and potentiate its activity to induce stable complex formation....

  13. A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Samantha C Lewis

    2015-02-01

    Full Text Available Mitochondrial DNA (mtDNA encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.

  14. Introduction of water into the heme distal side by Leu65 mutations of an oxygen sensor, YddV, generates verdoheme and carbon monoxide, exerting the heme oxygenase reaction.

    Science.gov (United States)

    Stranava, Martin; Martínková, Markéta; Stiborová, Marie; Man, Petr; Kitanishi, Kenichi; Muchová, Lucie; Vítek, Libor; Martínek, Václav; Shimizu, Toru

    2014-11-01

    The globin-coupled oxygen sensor, YddV, is a heme-based oxygen sensor diguanylate cyclase. Oxygen binding to the heme Fe(II) complex in the N-terminal sensor domain of this enzyme substantially enhances its diguanylate cyclase activity which is conducted in the C-terminal functional domain. Leu65 is located on the heme distal side and is important for keeping the stability of the heme Fe(II)-O2 complex by preventing the entry of the water molecule to the heme complex. In the present study, it was found that (i) Escherichia coli-overexpressed and purified L65N mutant of the isolated heme-bound domain of YddV (YddV-heme) contained the verdoheme iron complex and other modified heme complexes as determined by optical absorption spectroscopy and mass spectrometry; (ii) CO was generated in the reconstituted system composed of heme-bound L65N and NADPH:cytochrome P450 reductase as confirmed by gas chromatography; (iii) CO generation of heme-bound L65N in the reconstituted system was inhibited by superoxide dismutase and catalase. In a concordance with the result, the reactive oxygen species increased the CO generation; (iv) the E. coli cells overexpressing the L65N protein of YddV-heme also formed significant amounts of CO compared to the cells overexpressing the wild type protein; (v) generation of verdoheme and CO was also observed for other mutants at Leu65 as well, but to a lesser extent. Since Leu65 mutations are assumed to introduce the water molecule into the heme distal side of YddV-heme, it is suggested that the water molecule would significantly contribute to facilitating heme oxygenase reactions for the Leu65 mutants. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

    Science.gov (United States)

    Matuschewski, Kai; Haussig, Joana M.

    2016-01-01

    Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

  16. The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake.

    Directory of Open Access Journals (Sweden)

    Marcelo L Merli

    2016-01-01

    Full Text Available Trypanosoma cruzi, the etiological agent of Chagas' disease, presents nutritional requirements for several metabolites. It requires heme for the biosynthesis of several heme-proteins involved in essential metabolic pathways like mitochondrial cytochromes and respiratory complexes, as well as enzymes involved in the biosynthesis of sterols and unsaturated fatty acids. However, this parasite lacks a complete route for its synthesis. In view of these facts, T. cruzi has to incorporate heme from the environment during its life cycle. In other words, their hosts must supply the heme for heme-protein synthesis. Although the acquisition of heme is a fundamental issue for the parasite's replication and survival, how this cofactor is imported and distributed is poorly understood. In this work, we used different fluorescent heme analogs to explore heme uptake along the different life-cycle stages of T. cruzi, showing that this parasite imports it during its replicative stages: the epimastigote in the insect vector and the intracellular amastigote in the mammalian host. Also, we identified and characterized a T. cruzi protein (TcHTE with 55% of sequence similarity to LHR1 (protein involved in L. amazonensis heme transport, which is located in the flagellar pocket, where the transport of nutrients proceeds in trypanosomatids. We postulate TcHTE as a protein involved in improving the efficiency of the heme uptake or trafficking in T. cruzi.

  17. The ability of multimerized cyclophilin A to restrict retrovirus infection

    International Nuclear Information System (INIS)

    Javanbakht, Hassan; Diaz-Griffero, Felipe; Yuan Wen; Yeung, Darwin F.; Li Xing; Song Byeongwoon; Sodroski, Joseph

    2007-01-01

    In owl monkeys, the typical retroviral restriction factor of primates, TRIM5α, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection

  18. The heme-heme oxygenase system: a molecular switch in wound healing.

    NARCIS (Netherlands)

    Wagener, F.A.D.T.G.; Beurden, H.E. van; Hoff, J.W. Von den; Adema, G.J.; Figdor, C.G.

    2003-01-01

    When cells are injured they release their contents, resulting in a local accumulation of free heme proteins and heme. Here, we investigated the involvement of heme and its degrading enzyme heme oxygenase (HO) in the inflammatory process during wound healing. We observed that heme directly

  19. Isocyanides inhibit human heme oxygenases at the verdoheme stage.

    Science.gov (United States)

    Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

    2009-09-22

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

  20. Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

    Science.gov (United States)

    Evans, John P.; Kandel, Sylvie; Ortiz de Montellano, Paul R.

    2010-01-01

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides; isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides, and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 μM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

  1. Cyanide binding to human plasma heme-hemopexin: A comparative study

    Energy Technology Data Exchange (ETDEWEB)

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Laboratorio Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Roma (Italy); Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Leboffe, Loris [Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Polticelli, Fabio [Dipartimento di Biologia, Universita Roma Tre, Roma (Italy)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  2. Heme Synthesis and Acquisition in Bacterial Pathogens.

    Science.gov (United States)

    Choby, Jacob E; Skaar, Eric P

    2016-08-28

    Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host sources, particularly hemoglobin, and both heme acquisition and synthesis are important for pathogenesis. Paradoxically, excess heme is toxic to bacteria and pathogens must rely on heme detoxification strategies. Heme is a key nutrient in the struggle for survival between host and pathogen, and its study has offered significant insight into the molecular mechanisms of bacterial pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. HemeBIND: a novel method for heme binding residue prediction by combining structural and sequence information

    Directory of Open Access Journals (Sweden)

    Hu Jianjun

    2011-05-01

    Full Text Available Abstract Background Accurate prediction of binding residues involved in the interactions between proteins and small ligands is one of the major challenges in structural bioinformatics. Heme is an essential and commonly used ligand that plays critical roles in electron transfer, catalysis, signal transduction and gene expression. Although much effort has been devoted to the development of various generic algorithms for ligand binding site prediction over the last decade, no algorithm has been specifically designed to complement experimental techniques for identification of heme binding residues. Consequently, an urgent need is to develop a computational method for recognizing these important residues. Results Here we introduced an efficient algorithm HemeBIND for predicting heme binding residues by integrating structural and sequence information. We systematically investigated the characteristics of binding interfaces based on a non-redundant dataset of heme-protein complexes. It was found that several sequence and structural attributes such as evolutionary conservation, solvent accessibility, depth and protrusion clearly illustrate the differences between heme binding and non-binding residues. These features can then be separately used or combined to build the structure-based classifiers using support vector machine (SVM. The results showed that the information contained in these features is largely complementary and their combination achieved the best performance. To further improve the performance, an attempt has been made to develop a post-processing procedure to reduce the number of false positives. In addition, we built a sequence-based classifier based on SVM and sequence profile as an alternative when only sequence information can be used. Finally, we employed a voting method to combine the outputs of structure-based and sequence-based classifiers, which demonstrated remarkably better performance than the individual classifier alone

  4. The haptoglobin-CD163-heme oxygenase-1 pathway for hemoglobin scavenging

    DEFF Research Database (Denmark)

    Thomsen, Jens Haugbølle; Etzerodt, Anders; Svendsen, Pia

    2013-01-01

    The haptoglobin- (Hp-) CD163-heme oxygenase-1 (HO-1) pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex...

  5. Synthesis and Evaluation of Amyloid β Derived and Amyloid β Independent Enhancers of the Peroxidase-like Activity of Heme.

    Science.gov (United States)

    Wißbrock, Amelie; Kühl, Toni; Silbermann, Katja; Becker, Albert J; Ohlenschläger, Oliver; Imhof, Diana

    2017-01-12

    Labile heme has been suggested to have an impact in several severe diseases. In the context of Alzheimer's disease (AD), however, decreased levels of free heme have been reported. Therefore, we were looking for an assay system that can be used for heme concentration determination. From a biochemical point of view the peroxidase activity of the Aβ-heme complex seemed quite attractive to pursue this goal. As a consequence, a peptide that is able to increase the readout even in the case of a low heme concentration is favorable. The examination of Aβ- and non-Aβ-derived peptides in complex with heme revealed that the peroxidase-like activity significantly depends on the peptide sequence and length. A 23mer His-based peptide derived from human fatty acyl-CoA reductase 1 in complex with heme exhibited a significantly higher peroxidase activity than Aβ(40)-heme. Structural modeling of both complexes demonstrated that heme binding via a histidine can be supported by hydrogen bond interactions of a basic residue near the propionate carboxyl function of protoporphyrin IX. Furthermore, the interplay of Aβ-heme and the lipoprotein LDL as a potential physiological effector of Aβ was examined.

  6. Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.

    Science.gov (United States)

    Babbitt, Shalon E; San Francisco, Brian; Bretsnyder, Eric C; Kranz, Robert G

    2014-08-19

    C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical "correction" supports the proposed role of heme binding for the corresponding residues.

  7. Heme Mobilization in Animals: A Metallolipid's Journey.

    Science.gov (United States)

    Reddi, Amit R; Hamza, Iqbal

    2016-06-21

    Heme is universally recognized as an essential and ubiquitous prosthetic group that enables proteins to carry out a diverse array of functions. All heme-dependent processes, from protein hemylation to heme signaling, require the dynamic and rapid mobilization of heme to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitates that heme mobilization is carefully controlled at the cellular and systemic level. However, the molecules and mechanisms that mediate heme homeostasis are poorly understood. In this Account, we provide a heuristic paradigm with which to conceptualize heme trafficking and highlight the most recent developments in the mechanisms underlying heme trafficking. As an iron-containing tetrapyrrole, heme exhibits properties of both transition metals and lipids. Accordingly, we propose its transport and trafficking will reflect principles gleaned from the trafficking of both metals and lipids. Using this conceptual framework, we follow the flow of heme from the final step of heme synthesis in the mitochondria to hemoproteins present in various subcellular organelles. Further, given that many cells and animals that cannot make heme can assimilate it intact from nutritional sources, we propose that intercellular heme trafficking pathways must exist. This necessitates that heme be able to be imported and exported from cells, escorted between cells and organs, and regulated at the organismal level via a coordinated systemic process. In this Account, we highlight recently discovered heme transport and trafficking factors and provide the biochemical foundation for the cell and systems biology of heme. Altogether, we seek to reconceptualize heme from an exchange inert cofactor buried in hemoprotein active sites to an exchange labile and mobile metallonutrient.

  8. Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

    Directory of Open Access Journals (Sweden)

    Norifumi Muraki

    2016-05-01

    Full Text Available Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

  9. Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

    Science.gov (United States)

    Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

    2013-08-01

    To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Human heme oxygenase oxidation of 5- and 15-phenylhemes.

    Science.gov (United States)

    Wang, Jinling; Niemevz, Fernando; Lad, Latesh; Huang, Liusheng; Alvarez, Diego E; Buldain, Graciela; Poulos, Thomas L; de Montellano, Paul R Ortiz

    2004-10-08

    Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.

  11. TLR Stimulation Dynamically Regulates Heme and Iron Export Gene Expression in Macrophages

    Directory of Open Access Journals (Sweden)

    Mary Philip

    2016-01-01

    Full Text Available Pathogenic bacteria have evolved multiple mechanisms to capture iron or iron-containing heme from host tissues or blood. In response, organisms have developed defense mechanisms to keep iron from pathogens. Very little of the body’s iron store is available as free heme; rather nearly all body iron is complexed with heme or other proteins. The feline leukemia virus, subgroup C (FeLV-C receptor, FLVCR, exports heme from cells. It was unknown whether FLVCR regulates heme-iron availability after infection, but given that other heme regulatory proteins are upregulated in macrophages in response to bacterial infection, we hypothesized that macrophages dynamically regulate FLVCR. We stimulated murine primary macrophages or macrophage cell lines with LPS and found that Flvcr is rapidly downregulated in a TLR4/MD2-dependent manner; TLR1/2 and TLR3 stimulation also decreased Flvcr expression. We identified several candidate TLR-activated transcription factors that can bind to the Flvcr promoter. Macrophages must balance the need to sequester iron from systemic circulating or intracellular pathogens with the macrophage requirement for heme and iron to produce reactive oxygen species. Our findings underscore the complexity of this regulation and point to a new role for FLVCR and heme export in macrophages responses to infection and inflammation.

  12. Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.

    Science.gov (United States)

    Ogura, Hiroshi; Evans, John P; de Montellano, Paul R Ortiz; La Mar, Gerd N

    2008-01-08

    The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.

  13. Potent heme-degrading action of antimony and antimony-containing parasiticidal agents.

    Science.gov (United States)

    Drummond, G S; Kappas, A

    1981-02-01

    The ability of antimony and antimony-containing parasiticidal agents to enhance the rate of heme degradation in liver and kidney was investigated. Trivalent antimony was shown to be an extremely potent inducer of heme oxygenase, the initial and rate-limiting enzyme in heme degradation, in both organs, whereas the pentavalent form was a weak inducer of this enzyme. The ability of antimony to induce heme oxygenase was dose-dependent, independent of the salt used, and not a result of a direct activation of the enzyme in vitro. Concomitant with heme oxygenase induction by antimony, microsomal heme and cytochrome P-450 contents decreased, the cyto-chrome P-450-dependent mixed function oxidase system was impaired, and delta-ami-nolevulinate synthase (ALAS), the rate-limiting enzyme of heme synthesis, underwent the sequential changes-initial inhibition followed by rebound induction-usually associated with the administration of transition elements such as cobalt. Antimony induction of heme oxygenase however, unlike the enzyme induction elicited by cobalt, was not prevented either by cysteine administered orally or as a cysteine metal complex, or by simultaneous zinc administration. Desferoxamine also did not block heme oxygenase induction by antimony, but this chelator did prevent the rebound increase in ALAS activity associated with antimony or cobalt treatment. Antimony-containing parasiticidal drugs were also potent inducers of heme oxygenase in liver and kidney. The heme degradative action of these drugs may be related in part to the jaundice commonly associated with the prolonged therapeutic use of these agents. The heme-oxygenase-inducing action of antimony-containing parasiticidal drugs is a newly defined biological property of these compounds. The relation between the parasiticidal and the heme-oxygenase-inducing actions of such drugs is unknown. However, certain parasites contain hemoproteins or require heme compounds during their life cycle. It may therefore be

  14. Hemoglobin and heme scavenger receptors

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Møller, Holger Jon; Moestrup, Søren Kragh

    2010-01-01

    Heme, the functional group of hemoglobin, myoglobin, and other hemoproteins, is a highly toxic substance when it appears in the extracellular milieu. To circumvent potential harmful effects of heme from hemoproteins released during physiological or pathological cell damage (such as hemolysis...... and rhabdomyolysis), specific high capacity scavenging systems have evolved in the mammalian organism. Two major systems, which essentially function in a similar way by means of a circulating latent plasma carrier protein that upon ligand binding is recognized by a receptor, are represented by a) the hemoglobin...

  15. Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles

    Science.gov (United States)

    Lee, Jiho; Chang, Jeong Ho

    2014-12-01

    This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively.

  16. Transmutation of a heme protein.

    Science.gov (United States)

    Barker, P D; Ferrer, J C; Mylrajan, M; Loehr, T M; Feng, R; Konishi, Y; Funk, W D; MacGillivray, R T; Mauk, A G

    1993-01-01

    Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (E(m) = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the alpha carbon of the heme 4-vinyl group. Form BI has a covalently bound heme group coupled through a thioether linkage involving the beta carbon of the heme 4-vinyl group. Form BII is similar to BI except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (E(m) = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed. PMID:8341666

  17. Modeling the Syn-Disposition of Nitrogen Donors in Non-Heme Diiron Enzymes. Synthesis, Characterization, and Hydrogen Peroxide Reactivity of Diiron(III) Complexes with the Syn N-Donor Ligand H2BPG2DEV

    Science.gov (United States)

    Friedle, Simone; Kodanko, Jeremy J.; Morys, Anna J.; Hayashi, Takahiro; Moënne-Loccoz, Pierre; Lippard, Stephen J.

    2009-01-01

    In order to model the syn disposition of histidine residues in carboxylate-bridged non-heme diiron enzymes, we prepared a new dinucleating ligand, H2BPG2DEV, that provides this geometric feature. The ligand incorporates biologically relevant carboxylate functionalities, which have not been explored as extensively as nitrogen-only analogs. Three novel oxo-bridged diiron(III) complexes [Fe2(μ-O)(H2O)2-(BPG2DEV)](ClO4)2 (6), [Fe2(μ-O)(μ-O CAriPrO)(BPG2DEV)](ClO4) (7), and [Fe2(μ-O)(μ-CO3)(BPG2DEV)] (8) were prepared. Single crystal X-ray structural characterization confirms that two pyridines are bound syn with respect to the Fe–Fe vector in these compounds. The carbonato-bridged complex 8 forms quantitatively from 6 in a rapid reaction with gaseous CO2 in organic solvents. A common maroon-colored intermediate (λmax = 490 nm; ε = 1500 M−1 cm−1) forms in reactions of 6, 7, or 8 with H2O2 and NEt3 in CH3CN/H2O solutions. Mass spectrometric analyses of this species, formed using 18O-labeled H2O2, indicate the presence of a peroxide ligand bound to the oxo-bridged diiron(III) center. The Mössbauer spectrum at 90 K of the EPR-silent intermediate exhibits a quadrupole doublet with δ. = 0.58 mm/s and ΔEQ = 0.58 mm/s. The isomer shift is typical for a peroxodiiron(III) species, but the quadrupole splitting parameter is unusually small compared to related complexes. These Mössbauer parameters are comparable to those observed for a peroxo intermediate formed in the reaction of reduced toluene/o-xylene monooxygenase hydroxylase (ToMOH) with dioxygen. Resonance Raman studies reveal an unusually low-energy O–O stretching mode in the peroxo intermediate that is consistent with a short diiron distance. Although peroxodiiron(III) intermediates generated from 6, 7, and 8 are poor O-atom transfer catalysts, they display highly efficient catalase activity, with turnover numbers up to 10,000. In contrast to hydrogen peroxide reactions of diiron(III) complexes that lack

  18. Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

    Science.gov (United States)

    Joubert, Laetitia; Dagieu, Jean-Baptiste; Fernandez, Annabelle; Derré-Bobillot, Aurélie; Borezée-Durant, Elise; Fleurot, Isabelle; Gruss, Alexandra; Lechardeur, Delphine

    2017-01-16

    Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

  19. Interaction of nitric oxide with human heme oxygenase-1.

    Science.gov (United States)

    Wang, Jinling; Lu, Shen; Moënne-Loccoz, Pierre; Ortiz de Montellano, Paul R

    2003-01-24

    NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

  20. Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

    Science.gov (United States)

    Inforzato, Antonio; Rivieccio, Vincenzo; Morreale, Antonio P; Bastone, Antonio; Salustri, Antonietta; Scarchilli, Laura; Verdoliva, Antonio; Vincenti, Silvia; Gallo, Grazia; Chiapparino, Caterina; Pacello, Lucrezia; Nucera, Eleonora; Serlupi-Crescenzi, Ottaviano; Day, Anthony J; Bottazzi, Barbara; Mantovani, Alberto; De Santis, Rita; Salvatori, Giovanni

    2008-04-11

    PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

  1. Surfactant protein D multimerization and gene polymorphism in COPD and asthma

    DEFF Research Database (Denmark)

    Fakih, Dalia; Akiki, Zeina; Junker, Kirsten

    2018-01-01

    BACKGROUND AND OBJECTIVE: A structural single nucleotide polymorphism rs721917 in the surfactant protein D (SP-D) gene, known as Met11Thr, was reported to influence the circulating levels and degree of multimerization of SP-D and was associated with both COPD and atopy in asthma. Moreover, diseas...

  2. Isoporphyrin Intermediate in Heme Oxygenase Catalysis

    Science.gov (United States)

    Evans, John P.; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

    2008-01-01

    Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. PMID:18487208

  3. Heme Oxygenase-1 and breast cancer resistance protein protect against heme-induced toxicity

    NARCIS (Netherlands)

    Wagener, Frank A D T G; Dankers, Anita C A; van Summeren, Frank; Scharstuhl, Alwin; van den Heuvel, Jeroen J M W; Koenderink, Jan B; Pennings, Sebastiaan W C; Russel, Frans G M; Masereeuw, R.

    2013-01-01

    Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating

  4. Allocation of Heme is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells

    Directory of Open Access Journals (Sweden)

    Nino Asuela Espinas

    2016-08-01

    Full Text Available Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1 and null (fc1-2 mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1 and null (fc2-2 mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.

  5. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    Science.gov (United States)

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  6. Nitric oxide heme interactions in nitrophorin from Cimex lectularius

    Energy Technology Data Exchange (ETDEWEB)

    Christmann, R.; Auerbach, H., E-mail: auerbach@physik.uni-kl.de [University of Kaiserslautern, Department of Physics (Germany); Berry, R. E.; Walker, F. A. [The University of Arizona, Department of Chemistry and Biochemistry (United States); Schünemann, V. [University of Kaiserslautern, Department of Physics (Germany)

    2016-12-15

    The nitrophorin from the bedbug Cimex lectularius (cNP) is a nitric oxide (NO) carrying protein. Like the nitrophorins (rNPs) from the kissing bug Rhodnius prolixus, cNP forms a stable heme Fe(III)-NO complex, where the NO can be stored reversibly for a long period of time. In both cases, the NPs are found in the salivary glands of blood-sucking bugs. The insects use the nitrophorins to transport the NO to the victim’s tissues, resulting in vasodilation and reduced blood coagulation. However, the structure of cNP is significantly different to those of the rNPs from Rhodnius prolixus. Furthermore, the cNP can bind a second NO molecule to the proximal heme cysteine when present at higher concentrations. High field Mössbauer spectroscopy on {sup 57}Fe enriched cNP complexed with NO shows reduction of the heme iron and formation of a ferrous nitric oxide (Fe(II)-NO) complex. Density functional theory calculations reproduce the experimental Mössbauer parameters and confirm this observation.

  7. The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Marcia Cristina Paes

    2011-01-01

    Full Text Available Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

  8. Heme A synthesis and CcO activity are essential for Trypanosoma cruzi infectivity and replication.

    Science.gov (United States)

    Merli, Marcelo L; Cirulli, Brenda A; Menéndez-Bravo, Simón M; Cricco, Julia A

    2017-06-27

    Trypanosoma cruzi , the causative agent of Chagas disease, presents a complex life cycle and adapts its metabolism to nutrients' availability. Although T. cruzi is an aerobic organism, it does not produce heme. This cofactor is acquired from the host and is distributed and inserted into different heme-proteins such as respiratory complexes in the parasite's mitochondrion. It has been proposed that T. cruzi's energy metabolism relies on a branched respiratory chain with a cytochrome c oxidase-type aa 3 (C c O) as the main terminal oxidase. Heme A, the cofactor for all eukaryotic C c O, is synthesized via two sequential enzymatic reactions catalyzed by heme O synthase (HOS) and heme A synthase (HAS). Previously, TcCox10 and TcCox15 ( Trypanosoma cruzi Cox10 and Cox15 proteins) were identified in T. cruzi They presented HOS and HAS activity, respectively, when they were expressed in yeast. Here, we present the first characterization of TcCox15 in T. cruzi , confirming its role as HAS. It was differentially detected in the different T. cruzi stages, being more abundant in the replicative forms. This regulation could reflect the necessity of more heme A synthesis, and therefore more C c O activity at the replicative stages. Overexpression of a non-functional mutant caused a reduction in heme A content. Moreover, our results clearly showed that this hindrance in the heme A synthesis provoked a reduction on C c O activity and, in consequence, an impairment on T. cruzi survival, proliferation and infectivity. This evidence supports that T. cruzi depends on the respiratory chain activity along its life cycle, being C c O an essential terminal oxidase. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  9. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    Science.gov (United States)

    de Visser, Sam P.; Stillman, Martin J.

    2016-01-01

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties. PMID:27070578

  10. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    Directory of Open Access Journals (Sweden)

    Sam P. de Visser

    2016-04-01

    Full Text Available In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1. This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties.

  11. Prebiotics increase heme iron bioavailability and do not affect non-heme iron bioavailability in humans.

    Science.gov (United States)

    Weinborn, Valerie; Valenzuela, Carolina; Olivares, Manuel; Arredondo, Miguel; Weill, Ricardo; Pizarro, Fernando

    2017-05-24

    The aim of this study was to establish the effect of a prebiotic mix on heme and non-heme iron (Fe) bioavailability in humans. To this purpose, twenty-four healthy women were randomized into one of two study groups. One group ate one yogurt per day for 12 days with a prebiotic mix (prebiotic group) and the other group received the same yogurt but without the prebiotic mix (control group). Before and after the intake period, the subjects participated in Fe absorption studies. These studies used 55 Fe and 59 Fe radioactive isotopes as markers of heme Fe and non-heme Fe, respectively, and Fe absorption was measured by the incorporation of radioactive Fe into erythrocytes. The results showed that there were no significant differences in heme and non-heme Fe bioavailability in the control group. Heme Fe bioavailability of the prebiotic group increased significantly by 56% post-prebiotic intake. There were no significant differences in non-heme Fe bioavailability in this group. We concluded that daily consumption of a prebiotic mix increases heme Fe bioavailability and does not affect non-heme iron bioavailability.

  12. Hemopexin and haptoglobin: allies against heme toxicity from hemoglobin not contenders.

    Directory of Open Access Journals (Sweden)

    Ann eSmith

    2015-06-01

    Full Text Available The goal here is to describe our current understanding of heme metabolism and the deleterious effects of free heme on immunological processes, endothelial function, systemic inflammation, and various end-organ tissues (e.g. kidney, lung, liver, etc., with particular attention paid to the role of hemopexin (HPX. Because heme toxicity is the impetus for much of the pathology in sepsis, sickle cell disease, and other hemolytic conditions, the biological importance and clinical relevance of HPX, the predominant heme binding protein, is reinforced. A perspective on the function of HPX and haptoglobin (Hp is presented, updating how these two proteins and their respective receptors act simultaneously to protect the body in clinical conditions that entail hemolysis and/or systemic intravascular inflammation. Evidence from longitudinal studies in patients supports that HPX plays a Hp-independent role in genetic and non-genetic hemolytic diseases without the need for global Hp depletion. Evidence also supports that HPX has an important role in the prognosis of complex illnesses characterized predominantly by the presence of hemolysis, such as sickle cell disease, sepsis, hemolytic-uremic syndrome, and conditions involving intravascular and extravascular hemolysis, such as that generated by extracorporeal circulation during cardiopulmonary bypass and from blood transfusions. We propose that quantitating the amounts of plasma heme, HPX, Hb-Hp, heme-HPX and heme-albumin levels in various disease states may aid in the diagnosis and treatment of the above-mentioned conditions, which is crucial to developing targeted plasma protein supplementation (i.e. replenishment therapies for patients with heme toxicity due to HPX depletion.

  13. Heme oxygenase-1: a metabolic nike.

    Science.gov (United States)

    Wegiel, Barbara; Nemeth, Zsuzsanna; Correa-Costa, Matheus; Bulmer, Andrew C; Otterbein, Leo E

    2014-04-10

    Heme degradation, which was described more than 30 years ago, is still very actively explored with many novel discoveries on its role in various disease models every year. The heme oxygenases (HO) are metabolic enzymes that utilize NADPH and oxygen to break apart the heme moiety liberating biliverdin (BV), carbon monoxide (CO), and iron. Heme that is derived from hemoproteins can be toxic to the cells and if not removed immediately, it causes cell apoptosis and local inflammation. Elimination of heme from the milieu enables generation of three products that influences numerous metabolic changes in the cell. CO has profound effects on mitochondria and cellular respiration and other hemoproteins to which it can bind and affect their function, while BV and bilirubin (BR), the substrate and product of BV, reductase, respectively, are potent antioxidants. Sequestration of iron into ferritin and its recycling in the tissues is a part of the homeodynamic processes that control oxidation-reduction in cellular metabolism. Further, heme is an important component of a number of metabolic enzymes, and, therefore, HO-1 plays an important role in the modulation of cellular bioenergetics. In this review, we describe the cross-talk between heme oxygenase-1 (HO-1) and its products with other metabolic pathways. HO-1, which we have labeled Nike, the goddess who personified victory, dictates triumph over pathophysiologic conditions, including diabetes, ischemia, and cancer.

  14. A rapid, simple method for obtaining radiochemically pure hepatic heme

    International Nuclear Information System (INIS)

    Bonkowski, H.L.; Bement, W.J.; Erny, R.

    1978-01-01

    Radioactively-labelled heme has usually been isolated from liver to which unlabelled carrier has been added by long, laborious techniques involving organic solvent extraction followed by crystallization. A simpler, rapid method is devised for obtaining radiochemically-pure heme synthesized in vivo in rat liver from delta-amino[4- 14 C]levulinate. This method, in which the heme is extracted into ethyl acetate/glacial acetic acid and in which porphyrins are removed from the heme-containing organic phase with HCl washes, does not require addition of carrier heme. The new method gives better heme recoveries than and heme specific activities identical to, those obtained using the crystallization method. In this new method heme must be synthesized from delta-amino[4- 14 C]levulinate; it is not satisfactory to use [2- 14 C]glycine substrate because non-heme counts are isolated in the heme fraction. (Auth.)

  15. Insights on Heme Synthesis in the Malaria Parasite.

    Science.gov (United States)

    Nagaraj, Viswanathan A; Padmanaban, Govindarajan

    2017-08-01

    The malaria parasite has a functional heme-biosynthetic pathway, although it can access host hemoglobin-heme. The heme pathway is dispensable for blood stages, but essential in the mosquito stages which do not acquire hemoglobin-heme. We propose that the blood stage parasites maintain a dynamic heme pool through multiple back-up mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Studying disorders of vertebrate iron and heme metabolism using zebrafish.

    Science.gov (United States)

    van der Vorm, Lisa N; Paw, Barry H

    2017-01-01

    Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be - and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Mechanism governing heme synthesis reveals a GATA factor/heme circuit that controls differentiation.

    Science.gov (United States)

    Tanimura, Nobuyuki; Miller, Eli; Igarashi, Kazuhiko; Yang, David; Burstyn, Judith N; Dewey, Colin N; Bresnick, Emery H

    2016-02-01

    Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governing heme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis elements strongly reduces GATA-1-induced Alas2 transcription, heme biosynthesis, and surprisingly, GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing ALAS-2 function in Alas2 cis element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescues heme biosynthesis and the GATA-1-dependent genetic network. Heme amplifies GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Through this dual mechanism, heme and a master regulator collaborate to orchestrate a cell type-specific transcriptional program that promotes cellular differentiation. © 2015 The Authors.

  18. Measurement of Heme Synthesis Levels in Mammalian Cells.

    Science.gov (United States)

    Hooda, Jagmohan; Alam, Maksudul; Zhang, Li

    2015-07-09

    Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-(14)C] 5-aminolevulinic acid ([(14)C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [(14)C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

  19. Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.

    NARCIS (Netherlands)

    S. de Weert; P.J. Punt; Christien Lokman; C.A. van den Hondel; A.C. Franken; A.F. Ram

    2011-01-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

  20. Heme biosynthesis and its regulation: Towards understanding and improvement of heme biosynthesis in filamentous fungi

    NARCIS (Netherlands)

    Franken, A.C.W.; Lokman, B.C.; Ram, A.F.J.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Weert, S. de

    2011-01-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

  1. The Bryopsis hypnoides plastid genome: multimeric forms and complete nucleotide sequence.

    Directory of Open Access Journals (Sweden)

    Fang Lü

    Full Text Available BACKGROUND: Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga. PRINCIPAL FINDINGS: A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp, arrangement, and inverted-repeat (IR-lacking structure of the B. hypnoides chloroplast DNA (cpDNA closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support. CONCLUSION: All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.

  2. Enhancement of Muramyldipeptide (MDP) Immunostimulatory Activity by Controlled Multimerization on Dendrimers

    DEFF Research Database (Denmark)

    Sørensen, Nanna Skall; Boas, Ulrik; Heegaard, Peter M. H.

    2011-01-01

    Peptidoglycan is a widespread bacterial PAMP molecule and a powerful initiator of innate immune responses. It consists of repeating units of MDP, which as a monomer is only weakly immunostimulatory. Here, MDP-coupled dendrimers were prepared and investigated for stimulation of pig blood mononuclear...... cells. Compared to monomeric MDP, MDP-dendrimers induced a markedly enhanced production of IL-12 p40, IL-1β and IL-6 and completely down-regulated surface expression of B7 and MHC class II. These results suggest a possible novel strategy based on controlled multimerization of minimal PAMP motifs...... on dendrimers for preparing molecularly defined immunostimulators with predictable bioactivities....

  3. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    Science.gov (United States)

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  4. Heme synthesis in normal mouse liver and mouse liver tumors

    International Nuclear Information System (INIS)

    Stout, D.L.; Becker, F.F.

    1990-01-01

    Hepatic cancers from mice and rats demonstrate decreased levels of delta-aminolevulinic acid synthase, the rate-limiting enzyme in the heme synthetic pathway, and increased heme oxygenase, the heme-catabolizing enzyme. These findings suggest that diminution of P-450, b5, and catalase in these lesions may result from a heme supply that is limited by decreased heme synthesis and increased heme catabolism. Heme synthesis was measured in mouse liver tumors (MLT) and adjacent tumor-free lobes (BKG) by administering the radiolabeled heme precursors 55 FeCl3 and [2- 14 C]glycine and subsequently extracting the heme for determination of specific activity. Despite reduced delta-aminolevulinic acid synthase activity in MLT, both tissues incorporated [2-14C]glycine into heme at similar rates. At early time points, heme extracted from MLT contained less 55Fe than that from BKG. This was attributed to the findings that MLT took up 55Fe at a slower rate than BKG and had larger iron stores than BKG. The amount of heme per milligram of protein was also similar in both tissues. These findings militate against the hypothesis that diminished hemoprotein levels in MLT result from limited availability of heme. It is probable, therefore, that decreased hemoprotein levels in hepatic tumors are linked to a general program of dedifferentiation associated with the cancer phenotype. Diminution of hemoprotein in MLT may result in a relatively increased intracellular heme pool. delta-Aminolevulinic acid synthase and heme oxygenase are, respectively, negatively and positively regulated by heme. Thus, their alteration in MLT may be due to the regulatory influences of the heme pool

  5. Heme and non-heme iron transporters in non-polarized and polarized cells

    Directory of Open Access Journals (Sweden)

    Yasui Yumiko

    2010-06-01

    Full Text Available Abstract Background Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs, and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs. Results In this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1, and 2 candidate heme transporters--heme carrier protein 1 (HCP1 and heme responsive gene-1 (HRG-1--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase. Conclusions HCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is

  6. Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs.

    Science.gov (United States)

    Hultsch, Christina; Pawelke, Beate; Bergmann, Ralf; Wuest, Frank

    2006-09-01

    Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.

  7. A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

    Science.gov (United States)

    Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

    2016-03-01

    Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

    Czech Academy of Sciences Publication Activity Database

    Du, Y.; Liu, G.; Yan, Y.; Huang, D.; Luo, W.; Martínková, M.; Man, Petr; Shimizu, T.

    2013-01-01

    Roč. 26, č. 5 (2013), s. 839-852 ISSN 0966-0844 Institutional support: RVO:61388971 Keywords : Heme oxygenase * Heme protein * Hydrogen sulfide Subject RIV: CE - Biochemistry Impact factor: 2.689, year: 2013

  9. Heme biomolecule as redox mediator and oxygen shuttle for efficient charging of lithium-oxygen batteries

    Science.gov (United States)

    Ryu, Won-Hee; Gittleson, Forrest S.; Thomsen, Julianne M.; Li, Jinyang; Schwab, Mark J.; Brudvig, Gary W.; Taylor, André D.

    2016-01-01

    One of the greatest challenges with lithium-oxygen batteries involves identifying catalysts that facilitate the growth and evolution of cathode species on an oxygen electrode. Heterogeneous solid catalysts cannot adequately address the problematic overpotentials when the surfaces become passivated. However, there exists a class of biomolecules which have been designed by nature to guide complex solution-based oxygen chemistries. Here, we show that the heme molecule, a common porphyrin cofactor in blood, can function as a soluble redox catalyst and oxygen shuttle for efficient oxygen evolution in non-aqueous Li-O2 batteries. The heme's oxygen binding capability facilitates battery recharge by accepting and releasing dissociated oxygen species while benefiting charge transfer with the cathode. We reveal the chemical change of heme redox molecules where synergy exists with the electrolyte species. This study brings focus to the rational design of solution-based catalysts and suggests a sustainable cross-link between biomolecules and advanced energy storage. PMID:27759005

  10. Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

    Science.gov (United States)

    Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

    2016-02-15

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. A Non-Heme Iron Photocatalyst for Light-Driven Aerobic Oxidation of Methanol

    NARCIS (Netherlands)

    Chen, Juan; Stepanovic, Stepan; Draksharapu, Apparao; Gruden, Maja; Browne, Wesley R

    2018-01-01

    Non-heme (L)FeIIIand (L)FeIII-O-FeIII(L) complexes (L=1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)ethan-1-amine) underwent reduction under irradiation to the FeIIstate with concomitant oxidation of methanol to methanal, without the need for a secondary photosensitizer. Spectroscopic and DFT

  12. Molecular hijacking of siroheme for the synthesis of heme and d1 heme.

    Science.gov (United States)

    Bali, Shilpa; Lawrence, Andrew D; Lobo, Susana A; Saraiva, Lígia M; Golding, Bernard T; Palmer, David J; Howard, Mark J; Ferguson, Stuart J; Warren, Martin J

    2011-11-08

    Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

  13. Kidney injury and heme oxygenase-1

    Directory of Open Access Journals (Sweden)

    Hai-xing MAI

    2012-02-01

    Full Text Available     Heme oxygenase-1 (HO-1 is one of the main pathways to degrade heme in mammals, and the main degradation products are free iron (Fe2+, carbon monoxide (CO, and bilirubin. Heme plays an important role in promoting cell survival, circulation of intracellular substrates, and immune regulation. Previous studies suggest that HO-1 pathway is an important internal factor in determining the susceptibility and severity of acute kidney injury (AKI. The induction of HO-1 expression can attenuate the severity of renal ischemia-reperfusion injury (IRI, and the inhibition of HO-1 expression will aggravate IRI. The present article summarizes the latest advances in research abroad and at home on protective mechanism by which HO-1 prevents AKI to further deepen our understanding of the role of HO-1 in the treatment of AKI.   

  14. Heme and erythropoieis: more than a structural role

    OpenAIRE

    Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own ...

  15. Interactions between 4-aminoquinoline and heme: Promising mechanism against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Guilherme Curty Lechuga

    2016-12-01

    Full Text Available Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 μM, with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 μM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasite's lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies.

  16. Interactions between 4-aminoquinoline and heme: Promising mechanism against Trypanosoma cruzi.

    Science.gov (United States)

    Lechuga, Guilherme Curty; Borges, Júlio Cesar; Calvet, Claudia Magalhães; de Araújo, Humberto Pinheiro; Zuma, Aline Araujo; do Nascimento, Samara Braga; Motta, Maria Cristina Machado; Bernardino, Alice Maria Rolim; Pereira, Mirian Claudia de Souza; Bourguignon, Saulo Cabral

    2016-12-01

    Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 μM), with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 μM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasite's lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies. Copyright © 2016 The Authors. Published by Elsevier

  17. Red meat and colon cancer : how dietary heme initiates hyperproliferation

    NARCIS (Netherlands)

    IJssennagger, N.

    2012-01-01

    Colorectal cancer is a leading cause of cancer deaths in Western countries. The risk to develop colorectal cancer is associated with the intake of red meat. Red meat contains the porphyrin pigment heme. Heme is an irritant for the colonic wall and it is previously shown that the addition of heme

  18. Heme oxygenase activity increases after exercise in healthy volunteers

    Science.gov (United States)

    AbstractHeme oxygenase (HO) is an essential, rate-limiting protein which participates in the catabolism of heme to iron, carbon monoxide (CO), and biliverdin. The alpha methene bridge carbon of the heme is eliminated as CO which can be measured as blood carboxyhemoglobin (COHb)....

  19. Dibromine radical anion reactions with heme enzymes

    International Nuclear Information System (INIS)

    Gebicka, L.; Gebicki, J.L.

    1996-01-01

    Reactions of Br 2 radical anion with heme enzymes, catalase horseradish peroxidase, have been studied by pulse radiolysis. It has been found that Br 2 - does not react with the heme centre of investigated enzymes. Dibromine radical anion reacts with tryptophan residues of catalase without any influence on the activity of catalase. It is suggested that in pulse radiolysis studies, where horseradish peroxidase is at about tenfold excess toward Br 2 - , the enzyme is modified rather by Br 2 , than by Br 2 - . (author). 26 refs., 3 figs

  20. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    OpenAIRE

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We inv...

  1. Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

    Science.gov (United States)

    Zhang, Jingbo; Baetz, Ulrike; Krügel, Undine; Martinoia, Enrico; De Angeli, Alexis

    2013-10-01

    Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

  2. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

    NARCIS (Netherlands)

    Sha, H.; Yang, L.; Liu, M.; Xia, S.; Liu, Y.; Liu, F.; Kersten, A.H.; Qi, L.

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in

  3. Distinct subcellular trafficking resulting from monomeric vs multimeric targeting to endothelial ICAM-1: implications for drug delivery.

    Science.gov (United States)

    Ghaffarian, Rasa; Muro, Silvia

    2014-12-01

    Ligand-targeted, receptor-mediated endocytosis is commonly exploited for intracellular drug delivery. However, cells-surface receptors may follow distinct endocytic fates when bound by monomeric vs multimeric ligands. Our purpose was to study this paradigm using ICAM-1, an endothelial receptor involved in inflammation, to better understand its regulation and potential for drug delivery. Our procedure involved fluorescence microscopy of human endothelial cells to determine the endocytic behavior of unbound ICAM-1 vs ICAM-1 bound by model ligands: monomeric (anti-ICAM) vs multimeric (anti-ICAM biotin-streptavidin conjugates or anti-ICAM coated onto 100 nm nanocarriers). Our findings suggest that both monomeric and multimeric ligands undergo a similar endocytic pathway sensitive to amiloride (∼50% inhibition), but not inhibitors of clathrin-pits or caveoli. After 30 min, ∼60-70% of both ligands colocalized with Rab11a-compartments. By 3-5 h, ∼65-80% of multimeric anti-ICAM colocalized with perinuclear lysosomes with ∼60-80% degradation, while 70% of monomeric anti-ICAM remained associated with Rab11a at the cell periphery and recycled to and from the cell-surface with minimal (drug delivery.

  4. Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

    Czech Academy of Sciences Publication Activity Database

    Knejzlík, Z.; Ulbrich, P.; Strohalm, Martin; Laštůvková, H.; Kodíček, M.; Sakalian, M.; Ruml, T.

    2009-01-01

    Roč. 393, č. 1 (2009), s. 168-176 ISSN 0042-6822 Institutional research plan: CEZ:AV0Z50200510 Keywords : alpha-Helix * Multimerization * CD spectroscopy Subject RIV: EE - Microbiology, Virology Impact factor: 3.042, year: 2009

  5. Coordination Chemistry of Linear Oligopyrrolic Fragments Inspired by Heme Metabolites

    Science.gov (United States)

    Gautam, Ritika

    Linear oligopyrroles are degradation products of heme, which is converted in the presence of heme oxygenase to bile pigments, such as biliverdin and bilirubin. These tetrapyrrolic oligopyrroles are ubiquitously present in biological systems and find applications in the fields of catalysis and sensing. These linear tetrapyrrolic scaffolds are further degraded into linear tripyrrolic and dipyrrolic fragments. Although these lower oligopyrroles are abundantly present, their coordination chemistry requires further characterization. This dissertation focuses mainly on two classes of bioinspired linear oligopyrroles, propentdyopent and tripyrrindione, and their transition metal complexes, which present a rich ligand-based redox chemistry. Chapter 1 offers an overview of heme degradation to different classes of linear oligopyrroles and properties of their transition metal complexes. Chapter 2 is focused on the tripyrrin-1,14-dione scaffold of the urinary pigment uroerythrin, which coordinates divalent transition metals palladium and copper with square planar geometry. Specifically, the tripyrrin-1, 14-dione ligand binds Cu(II) and Pd(II) as a dianionic organic radical under ambient conditions. The electrochemical study confirms the presence of ligand based redox chemistry, and one electron oxidation or reduction reactions do not alter the planar geometry around the metal center. The X-Ray analysis and the electron paramagnetic resonance (EPR) studies of the complexes in the solid and solution phase reveals intermolecular interactions between the ligand based unpaired electrons and therefore formation of neutral pi-pi dimers. In Chapter 3, the antioxidant activity and the fluorescence sensor properties of the tripyrrin-1,14-dione ligand in the presence of superoxide are described. We found that the tripyrrindione ligand undergoes one-electron reduction in the presence of the superoxide radical anion (O2•- ) to form highly fluorescent H3TD1•- radical anion, which emits

  6. Heme pathway evolution in kinetoplastid protists

    Czech Academy of Sciences Publication Activity Database

    Cenci, U.; Moog, D.; Curtis, B.A.; Tanifuji, G.; Eme, L.; Lukeš, Julius; Archibald, J.M.

    2016-01-01

    Roč. 16, MAY 18 (2016), č. článku 109. ISSN 1471-2148 Institutional support: RVO:60077344 Keywords : heme * kinetoplastea * Paramoeba pemaquidensis * Perkinsela * evolution * endosymbiosis * Prokinetoplastina * lateral gene transfer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.221, year: 2016

  7. Heme and erythropoieis: more than a structural role.

    Science.gov (United States)

    Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

    2014-06-01

    Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own synthesis and regulates the expression of several erythroid-specific genes. Heme is synthesized in developing erythroid progenitors by the stage of proerythroblast, through a series of eight enzymatic reactions divided between mitochondria and cytosol. Defects of heme synthesis in the erythroid lineage result in sideroblastic anemias, characterized by microcytic anemia associated to mitochondrial iron overload, or in erythropoietic porphyrias, characterized by porphyrin deposition in erythroid cells. Here, we focus on the heme biosynthetic pathway and on human erythroid disorders due to defective heme synthesis. The regulatory role of heme during erythroid differentiation is discussed as well as the heme-mediated regulatory mechanisms that allow the orchestration of the adaptive cell response to heme deficiency. Copyright© Ferrata Storti Foundation.

  8. Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

    Science.gov (United States)

    Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

    2017-06-01

    Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

  9. The Chemistry and Biochemistry of Heme c: Functional Bases for Covalent Attachment

    OpenAIRE

    Bowman, Sarah E. J.; Bren, Kara L.

    2008-01-01

    A discussion of the literature concerning the synthesis, function, and activity of heme c-containing proteins is presented. Comparison of the properties of heme c, which is covalently bound to protein, is made to heme b, which is bound noncovalently. A question of interest is why nature uses biochemically expensive heme c in many proteins when its properties are expected to be similar to heme b. Considering the effects of covalent heme attachment on heme conformation and on the proximal histi...

  10. Heme-containing enzymes and inhibitors for tryptophan metabolism.

    Science.gov (United States)

    Yan, Daojing; Lin, Ying-Wu; Tan, Xiangshi

    2017-09-20

    Iron-containing enzymes such as heme enzymes play crucial roles in biological systems. Three distinct heme-containing dioxygenase enzymes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the initial and rate-limiting step of l-tryptophan catabolism through the kynurenine pathway in mammals. Overexpression of these enzymes causes depletion of tryptophan and the accumulation of metabolic products, which contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. In the past few decades, IDO1 has garnered the most attention as a therapeutic target with great potential in cancer immunotherapy. Many potential inhibitors of IDO1 have been designed, synthesized and evaluated, among which indoximod (d-1-MT), INCB024360, GDC-0919 (formerly NLG-919), and an IDO1 peptide-based vaccine have advanced to the clinical trial stage. However, recently, the roles of TDO and IDO2 have been elucidated in immune suppression. In this review, the current drug discovery landscape for targeting TDO, IDO1 and IDO2 is highlighted, with particular attention to the recent use of drugs in clinical trials. Moreover, the crystal structures of these enzymes, in complex with inhibitors, and the mechanisms of Trp catabolism in the first step, are summarized to provide information for facilitating the discovery of new enzyme inhibitors.

  11. Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

    Science.gov (United States)

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-05-01

    The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  12. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    Science.gov (United States)

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

  13. Single or functionalized fullerenes interacting with heme group

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Wallison Chaves; Diniz, Eduardo Moraes, E-mail: eduardo.diniz@ufma.br [Departamento de Física, Universidade Federal do Maranhão, Avenida dos Portugueses, 1966, CEP 65080-805, São Luís - MA (Brazil)

    2014-09-15

    The heme group is responsible for iron transportation through the bloodstream, where iron participates in redox reactions, electron transfer, gases detection etc. The efficiency of such processes can be reduced if the whole heme molecule or even the iron is somehow altered from its original oxidation state, which can be caused by interactions with nanoparticles as fullerenes. To verify how such particles alter the geometry and electronic structure of heme molecule, here we report first principles calculations based on density functional theory of heme group interacting with single C{sub 60} fullerene or with C{sub 60} functionalized with small functional groups (−CH{sub 3}, −COOH, −NH{sub 2}, −OH). The calculations shown that the system heme + nanoparticle has a different spin state in comparison with heme group if the fullerene is functionalized. Also a functional group can provide a stronger binding between nanoparticle and heme molecule or inhibit the chemical bonding in comparison with single fullerene results. In addition heme molecule loses electrons to the nanoparticles and some systems exhibited a geometry distortion in heme group, depending on the binding energy. Furthermore, one find that such nanoparticles induce a formation of spin up states in heme group. Moreover, there exist modifications in density of states near the Fermi energy. Although of such changes in heme electronic structure and geometry, the iron atom remains in the heme group with the same oxidation state, so that processes that involve the iron might not be affected, only those that depend on the whole heme molecule.

  14. Study on models of O2 binding to heme using density functional theory

    Directory of Open Access Journals (Sweden)

    Hovorun D. M.

    2009-08-01

    Full Text Available Aim. To study a mechanism of molecular oxygen binding to heme three models of geometry structure of the complex are considered: the axis of O2 molecule is situated perpendicularly to the porphin macrocycle, parallel, and angularly. Methods. The Fe(II porphin complexes with dioxygen are calculated by the quantum-chemical method of density functional theory with the UB3LYP/6-311G approximation. Results. The optimized geometry and electron structures as well as the absorption IR spectra of the complexes in the high-spin (septet state are described. Conclusions. It is shown that the main mechanism of spin-orbit coupling during the O2 binding to heme is connected with peculiarity of the O2 molecule electronic structure.

  15. The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for Hemoglobin Scavenging

    Directory of Open Access Journals (Sweden)

    Jens Haugbølle Thomsen

    2013-01-01

    Full Text Available The haptoglobin- (Hp- CD163-heme oxygenase-1 (HO-1 pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO, biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6, but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.

  16. Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

    International Nuclear Information System (INIS)

    Gardner, L.C.; Cox, T.M.

    1988-01-01

    Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular free heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation

  17. Effect of lead on heme synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Neuberger, A.

    1975-01-01

    Recently, a fair amount of work has been done on the effect of lead on porphobilinogen dehydratase, which has been used as a sensitive indicator of lead poisoning. How far this is in itself harmful depends on the Michaelis constants of both the aminolaevulinic synthetase and of the dehydratase, and in addition on the relative activities of the two enzymes in a cell and also on the tissue concentration of glycine. Information on some of these points is still fragmentary, and a reliable judgement is at the present not very easy. Another step in the heme synthesis, which is sensitive to low concentrations of lead, is the incorporation of iron into protoporphyrin. Inhibition of this step may be important in accounting to a large extent for the anaemia found in individuals with lead poisoning. Reduction in the tissue concentration of heme or of heme-like compounds may also explain, through the mechanism of de-repression, the excretion of increased amounts of aminolaevulinic acid in the urine observed in cases of lead poisoning. A third step in heme synthesis, which might be sensitive to lead, is the oxidative decarboxylation of coproporphyrin to protoporphyrin, and this may explain why the former derivative is excreted in the urine. Recent work of the Harvard Medical School has indicated that greatly reduced levels of ALA dehydratase may be found in most cases of severe liver damage due to alcoholism. In most of these cases the level of lead in the blood is within normal limits, and there is no history of exposure to toxic amounts of lead. We therefore have to assume that a reduction in the blood level of this enzyme is not necessarily an indication of lead poisoning.

  18. Heme-based sensors in biological systems.

    Science.gov (United States)

    Rodgers, K R

    1999-04-01

    The past several years have been witness to a staggering rate of advancement in the understanding of how organisms respond to changes in the availability of diatomic molecules that are toxic and/or crucial to survival. Heme-based sensors presently constitute the majority of the proteins known to sense NO, O2 and CO and to initiate the chemistry required to adapt to changes in their availabilities. Knowledge of the three characterized members of this class, soluble guanylate cyclase, FixL and CooA, has grown substantially during the past year. The major advances have resulted from a broad range of approaches to elucidation of both function and mechanism. They include growth in the understanding of the interplay between the heme and protein in soluble guanylate cyclase, as well as alternate means for its stimulation. Insight into the O2-induced structural changes in FixL has been supplied by the single crystal structure of the heme domain of Bradyrhizobium japonicum. Finally, the ligation environment and ligand interchange that facilitates CO sensing by CooA has been established by spectroscopic and mutagenesis techniques.

  19. Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

    Science.gov (United States)

    Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

    2009-05-27

    The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

  20. Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

    Directory of Open Access Journals (Sweden)

    Karina E. J. Tripodi

    2011-01-01

    Full Text Available Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi, leishmaniasis (Leishmania spp., and African trypanosomiasis (Trypanosoma brucei. Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents.

  1. Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp.

    Science.gov (United States)

    Taengchaiyaphum, Suparat; Nakayama, Hideki; Srisala, Jiraporn; Khiev, Ratny; Aldama-Cano, Diva January; Thitamadee, Siripong; Sritunyalucksana, Kallaya

    2017-11-01

    To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. TIA-1 Self-Multimerization, Phase Separation, and Recruitment into Stress Granules Are Dynamically Regulated by Zn2+

    Directory of Open Access Journals (Sweden)

    Joseph B. Rayman

    2018-01-01

    Full Text Available Summary: Stress granules are non-membranous structures that transiently form in the cytoplasm during cellular stress, where they promote translational repression of non-essential RNAs and modulate cell signaling by sequestering key signal transduction proteins. These and other functions of stress granules facilitate an adaptive cellular response to environmental adversity. A key component of stress granules is the prion-related RNA-binding protein, T cell intracellular antigen-1 (TIA-1. Here, we report that recombinant TIA-1 undergoes rapid multimerization and phase separation in the presence of divalent zinc, which can be reversed by the zinc chelator, TPEN. Similarly, the formation and maintenance of TIA-1-positive stress granules in arsenite-treated cells are inhibited by TPEN. In addition, Zn2+ is released in cells treated with arsenite, before stress granule formation. These findings suggest that Zn2+ is a physiological ligand of TIA-1, acting as a stress-inducible second messenger to promote multimerization of TIA-1 and subsequent localization into stress granules. : Rayman et al. show that Zn2+ is a stress-inducible second messenger that triggers self-multimerization and phase separation of TIA-1 and regulates dynamic recruitment of TIA-1 into stress granules. This mechanism is part of an adaptive cellular response to environmental adversity. Keywords: TIA-1, TIA1, stress granules, cellular stress, functional prion, phase separation, zinc regulation

  3. Silencing of Iron and Heme-Related Genes Revealed a Paramount Role of Iron in the Physiology of the Hematophagous Vector Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Ana B. Walter-Nuno

    2018-02-01

    Full Text Available Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of Rhodnius prolixus, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP, Heme Oxygenase (HO and the heme exporter Feline Leukemia Virus C Receptor (FLVCR, components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM, where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for R. prolixus physiology and imbalances in the levels of

  4. Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris

    International Nuclear Information System (INIS)

    Acosta-Rivero, Nelson; Rodriguez, Armando; Musacchio, Alexis; Falcon, Viviana; Suarez, Viana M.; Chavez, Liudmila; Morales-Grillo, Juan; Duenas-Carrera, Santiago

    2004-01-01

    Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure

  5. Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression.

    Science.gov (United States)

    Zhang, Tiantian; Bu, Pengli; Zeng, Joey; Vancura, Ales

    2017-10-13

    Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4 , the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1 , which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

    Science.gov (United States)

    Stranava, Martin; Man, Petr; Skálová, Tereza; Kolenko, Petr; Blaha, Jan; Fojtikova, Veronika; Martínek, Václav; Dohnálek, Jan; Lengalova, Alzbeta; Rosůlek, Michal; Shimizu, Toru; Martínková, Markéta

    2017-12-22

    The heme-based oxygen sensor histidine kinase Af GcHK is part of a two-component signal transduction system in bacteria. O 2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His 183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH - and -CN - complexes of Af GcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN - and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length Af GcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of Af GcHK. We conclude that Af GcHK functions as an ensemble of molecules sampling at least two conformational states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Synthetic heme/copper assemblies: toward an understanding of cytochrome c oxidase interactions with dioxygen and nitrogen oxides.

    Science.gov (United States)

    Hematian, Shabnam; Garcia-Bosch, Isaac; Karlin, Kenneth D

    2015-08-18

    Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper or iron ions or both, those reacting with dioxygen (O2) or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2(-))) or both. As inspiration for this work, we turn to mitochondrial cytochrome c oxidase, which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis, and characterization of new O2 adducts whose further study will add insights into O2 reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO's function, which is intimately tied to cellular O2 balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-Fe(III)-O-Cu(II)(L) products; their properties are discussed. The O-atom is derived from dioxygen, and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a "naked" synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active sites. The other sector of research is focused on heme/Cu assemblies mediating the redox

  8. Relationship between natural and heme-mediated antibody polyreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Hadzhieva, Maya; Vassilev, Tchavdar [Stephan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113 (Bulgaria); Bayry, Jagadeesh; Kaveri, Srinivas; Lacroix-Desmazes, Sébastien [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France); Dimitrov, Jordan D., E-mail: jordan.dimitrov@crc.jussieu.fr [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France)

    2016-03-25

    Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.

  9. Formation of nitrosyl non-heme iron-sulphur complexes of a mitrochondria electron-transport chain in a liver and kidneys under prolonged permanent action of radiation contamination in the Chernobyl region

    International Nuclear Information System (INIS)

    Sidorik, E.P.; Burlaka, A.P.; Druzhina, N.A.

    1995-01-01

    No-complexes with iron-sulfur protein of the N-type (EPR signal g=2.03 at 77 K) have been revealed in a mitochondria electron transport chain in a liver and kidneys of animals which were hold for 1.5 years in the Chernobyl area under action of low intensity ionizing radiation as a result of incorporated radionuclides. These alterations in protein give evidence of changes in oxidation and phosphorylation in tissues

  10. Synthesis, antimalarial activity, heme binding and docking studies of N-substituted 4-aminoquinoline-pyrimidine molecular hybrids.

    Science.gov (United States)

    Maurya, Shiv Shyam; Khan, Shabana I; Bahuguna, Aparna; Kumar, Deepak; Rawat, Diwan S

    2017-03-31

    A series of novel N-substituted 4-aminoquinoline-pyrimidine hybrids have been synthesized via simple and economic route and evaluated for their antimalarial activity. Most compounds showed potent antimalarial activity against both CQ-sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-toxic to the mammalian cell lines. The most active compound 7b was analysed for heme binding activity using UV-spectrophotometer. Compound was found to interact with heme and a complex formation between compound and heme in a 1:1 stoichiometry ratio was determined using job plots. The interaction of these hybrids was also investigated by the molecular docking studies in the binding site of wild type Pf-DHFR-TS and quadruple mutant Pf-DHFR-TS. The pharmacokinetic property analysis of best active compounds was also studied by ADMET prediction. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. Synthesis of a Non-Heme Template for Attaching Four Peptides : An Approach to Artificial Iron(II)-Containing Peroxidases

    NARCIS (Netherlands)

    Heuvel, Marco van den; Berg, Tieme A. van den; Kellogg, Richard M.; Choma, Christin T.; Feringa, Bernard

    2004-01-01

    We are developing all-synthetic model cofactor-protein complexes in order to define the parameters controlling non-natural cofactor activity. The long-term objective is to establish the theoretical and practical basis for designing novel enzymes. A non-heme pentadentate ligand (N4Py) is being

  12. Heme oxygenase-1, oxidation, inflammation and atherosclerosis

    Directory of Open Access Journals (Sweden)

    Jesus A Araujo

    2012-07-01

    Full Text Available Atherosclerosis is an inflammatory process of the vascular wall characterized by the infiltration of lipids and inflammatory cells. Oxidative modifications of infiltrating low density lipoproteins and induction of oxidative stress play a major role in lipid retention in the vascular wall, uptake by macrophages and generation of foam cells, a hallmark of this disorder. The vasculature has a plethora of protective resources against oxidation and inflammation, many of them regulated by the Nrf2 transcription factor. Heme oxygenase-1 (HO-1 is a Nrf2-regulated gene that plays a critical role in the prevention of vascular inflammation. It is the inducible isoform of heme oxygenase, responsible for the oxidative cleavage of heme groups leading to the generation of biliverdin, carbon monoxide and release of ferrous iron. HO-1 has important antioxidant, antiinflammatory, antiapoptotic, antiproliferative and immunomodulatory effects in vascular cells, most of which play a significant role in the protection against atherogenesis. HO-1 may also be an important feature in macrophage differentiation and polarization to certain subtypes. The biological effects of HO-1 are largely attributable to its enzymatic activity, which can be conceived as a system with three arms of action, corresponding to its three enzymatic byproducts. HO-1 mediated vascular protection may be due to a combination of systemic and vascular local effects. It is usually expressed at low levels but can be highly upregulated in the presence of several proatherogenic stimuli. The HO-1 system is amenable for use in the development of new therapies, some of them currently under experimental and clinical trials. Interestingly, in contrast to the HO-1 antiatherogenic actions, the expression of its transcriptional regulator Nrf2 leads to proatherogenic effects instead. This article reviews the evidence that supports the antiatherogenic role of HO-1, potential pathways and mechanisms mediating

  13. Dietary heme-mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon.

    Directory of Open Access Journals (Sweden)

    Noortje Ijssennagger

    Full Text Available Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARα target genes. The aim of this study was to investigate the role of PPARα in heme-induced hyperproliferation and hyperplasia. Male PPARα KO and WT mice received a purified diet with or without heme. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARα leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARα in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARα KO mice. We conclude that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.

  14. Heme acquisition mechanisms of Porphyromonas gingivalis - strategies used in a polymicrobial community in a heme-limited host environment.

    Science.gov (United States)

    Smalley, J W; Olczak, T

    2017-02-01

    Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell-surface heme-containing pigment in the form of μ-oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB-dependent outer-membrane receptors (HmuR, HusB, IhtA), and hemophore-like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)-containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine- and lysine-specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Role of heme in bromine-induced lung injury

    Science.gov (United States)

    Lam, Adam; Vetal, Nilam; Matalon, Sadis; Aggarwal, Saurabh

    2016-01-01

    Bromine (Br2) gas inhalation poses an environmental and occupational hazard resulting in high morbidity and mortality. In this review, we underline the acute lung pathology (within 24 hours of exposure) and potential therapeutic interventions that may be utilized to mitigate Br2-induced human toxicity. We will discuss our latest published data, which suggests that an increase in heme-dependent tissue injury underlies the pathogenesis of Br2 toxicity. Our study was based on previous findings that demonstrated that Br2 upregulates the heme-degrading enzyme heme oxygenase-1 (HO-1), which converts toxic heme into billiverdin. Interestingly, following Br2 inhalation, heme levels were indeed elevated in bronchoalveolar lavage fluid, plasma, and whole lung tissue in C57BL/6 mice. High heme levels correlated with increased lung oxidative stress, lung inflammation, respiratory acidosis, lung edema, higher airway resistance, and mortality. However, therapeutic reduction of heme levels, by either scavenging with hemopexin or degradation by HO-1, improved lung function and survival. Therefore, heme attenuation may prove a useful adjuvant therapy to treat patients after Br2 exposure. PMID:27244263

  16. Heme: From quantum spin crossover to oxygen manager of life

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta

    2016-01-01

    The review discusses how the electronic structure of heme explains its central importance to oxygen-based life on Earth. Emphasis is on the chemical bonding of heme, its spin crossover, reversible O2 binding, and O-O bond activation, put in relation to its physiological functions. The review disc...

  17. Mechanism of H2O2 dismutation catalyzed by a new catalase mimic (a non-heme dibenzotetraaza[14]annulene-Fe(III) complex): a density functional theory investigation.

    Science.gov (United States)

    Wang, Xin; Li, Shuhua; Jiang, Yuansheng

    2004-10-04

    The mechanism of H(2)O(2) dismutation catalyzed by the dibenzotetraaza[14]annulene-Fe(III) complex ([Fe(C(24)H(22)N(4)O(4))](+)) which was recently reported (Paschke, J.; Kirsch, M.; Korth, H. G.; de Groot, H.; Sustmann, R. J. Am. Chem. Soc. 2001, 123, 11099) has been investigated by density functional theory using the B3LYP hybrid functional. The quartet potential energy profile of the catalytic reaction has been explored. In the whole catalytic cycle, the rate-determining step is found to be the O-O bond homolytic cleavage, without the assistance of solvent molecules in the second coordination shell. The calculated free energy barrier for this step is 10.8 kcal/mol, which is in reasonable agreement with the experimental facts. The calculations also show that the hydroxyl and hydroperoxyl radicals may be generated in the reaction processes, but they can be efficiently quenched in strongly exothermic steps. This study provides a satisfactory explanation to the observed efficiency of the H(2)O(2) dismutation catalyzed by this complex.

  18. Role of distal arginine in early sensing intermediates in the heme domain of the oxygen sensor FixL.

    Science.gov (United States)

    Jasaitis, Audrius; Hola, Klara; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe; Balland, Veronique; Vos, Marten H; Liebl, Ursula

    2006-05-16

    FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O(2), NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O(2), a 5-8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase ( approximately 10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O(2) and arginine 220 away from the heme

  19. Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy.

    Science.gov (United States)

    Kruglik, Sergei G; Jasaitis, Audrius; Hola, Klara; Yamashita, Taku; Liebl, Ursula; Martin, Jean-Louis; Vos, Marten H

    2007-05-01

    Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.

  20. 4-Aminoquinoline-pyrimidine hybrids: synthesis, antimalarial activity, heme binding and docking studies.

    Science.gov (United States)

    Kumar, Deepak; Khan, Shabana I; Tekwani, Babu L; Ponnan, Prija; Rawat, Diwan S

    2015-01-07

    A series of novel 4-aminoquinoline-pyrimidine hybrids has been synthesized and evaluated for their antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-toxic to the mammalian cell lines. Selected compound 7g exhibited significant suppression of parasitemia in the in vivo assay. The heme binding studies were conducted to determine the mode of action of these hybrid molecules. These compounds form a stable 1:1 complex with hematin suggesting that heme may be one of the possible targets of these hybrids. The interaction of these conjugate hybrids was also investigated by the molecular docking studies in the binding site of PfDHFR. The pharmacokinetic property analysis of best active compounds was also studied using ADMET prediction. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. X-ray absorption spectroscopic studies of mononuclear non-heme iron enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Westre, Tami E. [Stanford Univ., CA (United States)

    1996-01-01

    Fe-K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the electronic and geometric structure of the iron active site in non-heme iron enzymes. A new theoretical extended X-ray absorption fine structure (EXAFS) analysis approach, called GNXAS, has been tested on data for iron model complexes to evaluate the utility and reliability of this new technique, especially with respect to the effects of multiple-scattering. In addition, a detailed analysis of the 1s→3d pre-edge feature has been developed as a tool for investigating the oxidation state, spin state, and geometry of iron sites. Edge and EXAFS analyses have then been applied to the study of non-heme iron enzyme active sites.

  2. Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

    Science.gov (United States)

    Novak, David; Mojovic, Milos; Pavicevic, Aleksandra; Zatloukalova, Martina; Hernychova, Lenka; Bartosik, Martin; Vacek, Jan

    2018-02-01

    Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Increase on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro.

    Directory of Open Access Journals (Sweden)

    Renata Stiebler

    Full Text Available BACKGROUND: Hemozoin (Hz is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH (the synthetic counterpart of Hz formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO and a series of polyethyleneglycols (PEGs. We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000 increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300 caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels.

  4. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    Science.gov (United States)

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-04

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

  5. [Update on the biology of heme synthesis in erythroid cells].

    Science.gov (United States)

    Fujiwara, Tohru; Harigae, Hideo

    2015-02-01

    Heme is a prosthetic group of hemoproteins playing important roles in oxygen transport, detoxification, circadian rhythm, microRNA processing, regulation of transcription, and translation. The majority of heme (-85%) is synthesized in red blood cells mainly for hemoglobin production, whereas hepatocytes account for most of the rest, functioning primarily in the synthesis of cytochrome P450 enzymes and mitochondrial respiratory enzymes. Thus, failure of heme biosynthesis causes severe inherited or acquired disorders in humans, including porphyria and sideroblastic anemia. The heme biosynthetic pathway is composed of eight enzymes that work in either mitochondria or the cytoplasm, which have been extensively researched and frequently reviewed. On the other hand, the mechanisms governing transport and intracellular trafficking of heme intermediates, as well as their potential links to human diseases, are poorly understood. Herein, we focus on recent understanding of the heme biosynthetic pathway and on human disorders due to defective heme synthesis in erythroid cells, such as X-linked sideroblastic anemia and erythropoietic protoporphyria.

  6. Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

    International Nuclear Information System (INIS)

    Lara, F.A.; Sant'Anna, C.; Lemos, D.; Laranja, G.A.T.; Coelho, M.G.P.; Reis Salles, I.; Michel, A.; Oliveira, P.L.; Cunha-e-Silva, N.; Salmon, D.; Paes, M.C.

    2007-01-01

    Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane

  7. The effect of proteins from animal source foods on heme iron bioavailability in humans.

    Science.gov (United States)

    Pizarro, Fernando; Olivares, Manuel; Valenzuela, Carolina; Brito, Alex; Weinborn, Valerie; Flores, Sebastián; Arredondo, Miguel

    2016-04-01

    Forty-five women (35-45 year) were randomly assigned to three iron (Fe) absorption sub-studies, which measured the effects of dietary animal proteins on the absorption of heme Fe. Study 1 was focused on heme, red blood cell concentrate (RBCC), hemoglobin (Hb), RBCC+beef meat; study 2 on heme, heme+fish, chicken, and beef; and study 3 on heme and heme+purified animal protein (casein, collagen, albumin). Study 1: the bioavailability of heme Fe from Hb was similar to heme only (∼13.0%). RBCC (25.0%) and RBCC+beef (21.3%) were found to be increased 2- and 1.6-fold, respectively, when compared with heme alone (pProteins from animal source foods and their digestion products did not enhance heme Fe absorption. Copyright © 2015. Published by Elsevier Ltd.

  8. Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

    Science.gov (United States)

    Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

    2014-01-01

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

  9. Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

    Science.gov (United States)

    Babbitt, Shalon E; San Francisco, Brian; Mendez, Deanna L; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; Bretsnyder, Eric C; Kranz, Robert G

    2014-10-17

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b.

    Science.gov (United States)

    Streit, Bennett R; Celis, Arianna I; Shisler, Krista; Rodgers, Kenton R; Lukat-Rodgers, Gudrun S; DuBois, Jennifer L

    2017-01-10

    A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O 2 and H 2 O 2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O 2 to the ferric state. The subsequent second-order reaction between the ferric complex and H 2 O 2 is slow, pH-dependent, and further decelerated by D 2 O 2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H 2 O 2 . Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H 2 O 2 cleavage is therefore unclear. From a cellular perspective, the use of H 2 O 2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

  11. Wearing red for signaling: the heme-bach axis in heme metabolism, oxidative stress response and iron immunology.

    Science.gov (United States)

    Igarashi, Kazuhiko; Watanabe-Matsui, Miki

    2014-04-01

    The connection between gene regulation and metabolism is an old issue that warrants revisiting in order to understand both normal as well as pathogenic processes in higher eukaryotes. Metabolites affect the gene expression by either binding to transcription factors or serving as donors for post-translational modification, such as that involving acetylation and methylation. The focus of this review is heme, a prosthetic group of proteins that includes hemoglobin and cytochromes. Heme has been shown to bind to several transcription factors, including Bach1 and Bach2, in higher eukaryotes. Heme inhibits the transcriptional repressor activity of Bach1, resulting in the derepression of its target genes, such as globin in erythroid cells and heme oxygenase-1 in diverse cell types. Since Bach2 is important for class switch recombination and somatic hypermutation of immunoglobulin genes as well as regulatory and effector T cell differentiation and the macrophage function, the heme-Bach2 axis may regulate the immune response as a signaling cascade. We discuss future issues regarding the topic of the iron/heme-gene regulation network based on current understanding of the heme-Bach axis, including the concept of "iron immunology" as the synthesis of the iron metabolism and the immune response.

  12. Introduction of a covalent histidine-heme linkage in a hemoglobin: a promising tool for heme protein engineering.

    Science.gov (United States)

    Rice, Selena L; Preimesberger, Matthew R; Johnson, Eric A; Lecomte, Juliette T J

    2014-12-01

    The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine-heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (>90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His-heme modification will be applicable to the design of proteins with a non-dissociable heme group. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

    International Nuclear Information System (INIS)

    Nagao, Satoshi; Hirai, Yueki; Kawano, Shin; Imai, Kiyohiro; Suzuki, Akihiro; Yamamoto, Yasuhiko

    2007-01-01

    A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12, 18-trimethyl-porphyrin-atoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using 19 F NMR and the O 2 binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in α- and β- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O 2 affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O 2 affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O 2 affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A

  14. Characterisation of Anopheles gambiae heme oxygenase and metalloporphyrin feeding suggests a potential role in reproduction.

    Science.gov (United States)

    Spencer, Christopher S; Yunta, Cristina; de Lima, Glauber Pacelli Gomes; Hemmings, Kay; Lian, Lu-Yun; Lycett, Gareth; Paine, Mark J I

    2018-05-03

    The mosquito Anopheles gambiae is the principal vector for malaria in sub-Saharan Africa. The ability of A. gambiae to transmit malaria is strictly related to blood feeding and digestion, which releases nutrients for oogenesis, as well as substantial amounts of highly toxic free heme. Heme degradation by heme oxygenase (HO) is a common protective mechanism, and a gene for HO exists in the An. gambiae genome HO (AgHO), although it has yet to be functionally examined. Here, we have cloned and expressed An. gambiae HO (AgHO) in E. coli. Purified recombinant AgHO bound hemin stoichiometrically to form a hemin-enzyme complex similar to other HOs, with a K D of 3.9 ± 0.6 μM; comparable to mammalian and bacterial HOs, but 7-fold lower than that of Drosophila melanogaster HO. AgHO also degraded hemin to biliverdin and released CO and iron in the presence of NADPH cytochrome P450 oxidoreductase (CPR). Optimal AgHO activity was observed at 27.5 °C and pH 7.5. To investigate effects of AgHO inhibition, adult female A. gambiae were fed heme analogues Sn- and Zn-protoporphyrins (SnPP and ZnPP), known to inhibit HO. These led to a dose dependent decrease in oviposition. Cu-protoporphyrin (CuPP), which does not inhibit HO had no effect. These results demonstrate that AgHO is a catalytically active HO and that it may play a key role in egg production in mosquitoes. It also presents a potential target for the development of compounds aimed at sterilising mosquitoes for vector control. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Explaining the atypical reaction profiles of heme enzymes with a novel mechanistic hypothesis and kinetic treatment.

    Directory of Open Access Journals (Sweden)

    Kelath Murali Manoj

    Full Text Available Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP, uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the "steady state kinetics" of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of "the product of interest" in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of "substrate concentration at maximum observed rate". The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.

  16. TIA-1 Self-Multimerization, Phase Separation, and Recruitment into Stress Granules Are Dynamically Regulated by Zn2.

    Science.gov (United States)

    Rayman, Joseph B; Karl, Kevin A; Kandel, Eric R

    2018-01-02

    Stress granules are non-membranous structures that transiently form in the cytoplasm during cellular stress, where they promote translational repression of non-essential RNAs and modulate cell signaling by sequestering key signal transduction proteins. These and other functions of stress granules facilitate an adaptive cellular response to environmental adversity. A key component of stress granules is the prion-related RNA-binding protein, T cell intracellular antigen-1 (TIA-1). Here, we report that recombinant TIA-1 undergoes rapid multimerization and phase separation in the presence of divalent zinc, which can be reversed by the zinc chelator, TPEN. Similarly, the formation and maintenance of TIA-1-positive stress granules in arsenite-treated cells are inhibited by TPEN. In addition, Zn 2+ is released in cells treated with arsenite, before stress granule formation. These findings suggest that Zn 2+ is a physiological ligand of TIA-1, acting as a stress-inducible second messenger to promote multimerization of TIA-1 and subsequent localization into stress granules. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage.

    Science.gov (United States)

    Kim, Jung Min; Jang, Su A; Yu, Byung Jo; Sung, Bong Hyun; Cho, Ju Hyun; Kim, Sun Chang

    2008-02-01

    Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.

  18. Heme synthesis in the lead-intoxicated mouse embryo

    Energy Technology Data Exchange (ETDEWEB)

    Gerber, G B; Maes, J

    1978-02-01

    Incorporation of /sup 55/Fe and of (/sup 14/C) glycine was studied in control embryos and mothers and in those which had received lead in the diet from day 7 of pregnancy. Incorporation of Fe into heme of embryonic liver which increases markedly for controls on day 17 of pregnancy was depressed greatly and showed no such increase in lead-intoxicated embryos. These embryos were retarded in growth but had normal heme concentrations in body and liver. Incorporation of glycine into embryonic heme and proteins was not affected. Data on incorporation in the mothers are also presented. It is thought that the impaired synthesis of heme in lead-intoxicated embryos limits their body growth during the late phase of pregnancy.

  19. Effect of a heme oxygenase-1 inducer on NADPH oxidase ...

    African Journals Online (AJOL)

    Effect of a heme oxygenase-1 inducer on NADPH oxidase expression in ... and immunohistochemistry of hepatic NOX1 and NOX4 were investigated in week 4. ... (HO-1 inhibitor) administration caused upregulation of NOX gene expression ...

  20. Heme and blood-feeding parasites: friends or foes?

    Directory of Open Access Journals (Sweden)

    Glanfield Amber

    2010-11-01

    Full Text Available Abstract Hemoparasites, like malaria and schistosomes, are constantly faced with the challenges of storing and detoxifying large quantities of heme, released from their catabolism of host erythrocytes. Heme is an essential prosthetic group that forms the reactive core of numerous hemoproteins with diverse biological functions. However, due to its reactive nature, it is also a potentially toxic molecule. Thus, the acquisition and detoxification of heme is likely to be paramount for the survival and establishment of parasitism. Understanding the underlying mechanism involved in this interaction could possibly provide potential novel targets for drug and vaccine development, and disease treatment. However, there remains a wide gap in our understanding of these mechanisms. This review summarizes the biological importance of heme for hemoparasite, and the adaptations utilized in its sequestration and detoxification.

  1. Heme and blood-feeding parasites: friends or foes?

    Science.gov (United States)

    2010-01-01

    Hemoparasites, like malaria and schistosomes, are constantly faced with the challenges of storing and detoxifying large quantities of heme, released from their catabolism of host erythrocytes. Heme is an essential prosthetic group that forms the reactive core of numerous hemoproteins with diverse biological functions. However, due to its reactive nature, it is also a potentially toxic molecule. Thus, the acquisition and detoxification of heme is likely to be paramount for the survival and establishment of parasitism. Understanding the underlying mechanism involved in this interaction could possibly provide potential novel targets for drug and vaccine development, and disease treatment. However, there remains a wide gap in our understanding of these mechanisms. This review summarizes the biological importance of heme for hemoparasite, and the adaptations utilized in its sequestration and detoxification. PMID:21087517

  2. Immunolocalization of heme oxygenase-1 in periodontal diseases

    Directory of Open Access Journals (Sweden)

    G Gayathri

    2014-01-01

    Conclusion: The results of our study is an increasing evidence of involvement of antioxidant enzymes like heme oxygenase-1 in periodontal inflammation and their implication for treatment of chronic periodontitis.

  3. Hal Is a Bacillus anthracis Heme Acquisition Protein

    Science.gov (United States)

    Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

    2012-01-01

    The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

  4. Heme and menaquinone induced electron transport in lactic acid bacteria

    OpenAIRE

    Brooijmans, Rob; Smit, Bart; Santos, Filipe; van Riel, Jan; de Vos, Willem M; Hugenholtz, Jeroen

    2009-01-01

    Abstract Background For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Results Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacill...

  5. Acquisition of iron from transferrin regulates reticulocyte heme synthesis

    International Nuclear Information System (INIS)

    Ponka, P.; Schulman, H.M.

    1985-01-01

    Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59 Fe from [ 59 Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [ 59 Fe]transferrin. Also, Fe-SIH stimulates [2- 14 C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59 Fe incorporation into heme from either [ 59 Fe]transferrin or [ 59 Fe]SIH but does reverse the inhibition of 59 Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes

  6. TMEM14C is required for erythroid mitochondrial heme metabolism.

    Science.gov (United States)

    Yien, Yvette Y; Robledo, Raymond F; Schultz, Iman J; Takahashi-Makise, Naoko; Gwynn, Babette; Bauer, Daniel E; Dass, Abhishek; Yi, Gloria; Li, Liangtao; Hildick-Smith, Gordon J; Cooney, Jeffrey D; Pierce, Eric L; Mohler, Kyla; Dailey, Tamara A; Miyata, Non; Kingsley, Paul D; Garone, Caterina; Hattangadi, Shilpa M; Huang, Hui; Chen, Wen; Keenan, Ellen M; Shah, Dhvanit I; Schlaeger, Thorsten M; DiMauro, Salvatore; Orkin, Stuart H; Cantor, Alan B; Palis, James; Koehler, Carla M; Lodish, Harvey F; Kaplan, Jerry; Ward, Diane M; Dailey, Harry A; Phillips, John D; Peters, Luanne L; Paw, Barry H

    2014-10-01

    The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

  7. Engineering Non-Heme Mono- and Dioxygenases for Biocatalysis

    Directory of Open Access Journals (Sweden)

    Adi Dror

    2012-09-01

    Full Text Available Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.

  8. Effective electrochemical method for investigation of hemoglobin unfolding based on the redox property of heme groups at glassy carbon electrodes.

    Science.gov (United States)

    Li, Xianchan; Zheng, Wei; Zhang, Limin; Yu, Ping; Lin, Yuqing; Su, Lei; Mao, Lanqun

    2009-10-15

    This study demonstrates a facile and effective electrochemical method for investigation of hemoglobin (Hb) unfolding based on the electrochemical redox property of heme groups in Hb at bare glassy carbon (GC) electrodes. In the native state, the heme groups are deeply buried in the hydrophobic pockets of Hb with a five-coordinate high-spin complex and thus show a poor electrochemical property at bare GC electrodes. Upon the unfolding of Hb induced by the denaturant of guanidine hydrochloride (GdnHCl), the fifth coordinative bond between the heme groups and the residue of the polypeptides (His-F8) is broken, and as a result, the heme groups initially buried deeply in the hydrophobic pockets dissociate from the polypeptide chains and are reduced electrochemically at GC electrodes, which can be used to probe the unfolding of Hb. The results on the GdnHCl-induced Hb unfolding obtained with the electrochemical method described here well coincide with those studied with other methods, such as UV-vis spectroscopy, fluorescence, and circular dichroism. The application of the as-established electrochemical method is illustrated to study the kinetics of GdnHCl-induced Hb unfolding, the GdnHCl-induced unfolding of another kind of hemoprotein, catalase, and the pH-induced Hb unfolding/refolding.

  9. Using porphyrin-amino acid pairs to model the electrochemistry of heme proteins: experimental and theoretical investigations.

    Science.gov (United States)

    Samajdar, Rudra N; Manogaran, Dhivya; Yashonath, S; Bhattacharyya, Aninda J

    2018-04-18

    Quasi reversibility in electrochemical cycling between different oxidation states of iron is an often seen characteristic of iron containing heme proteins that bind dioxygen. Surprisingly, the system becomes fully reversible in the bare iron-porphyrin complex: hemin. This leads to the speculation that the polypeptide bulk (globin) around the iron-porphyrin active site in these heme proteins is probably responsible for the electrochemical quasi reversibility. To understand the effect of such polypeptide bulk on iron-porphyrin, we study the interaction of specific amino acids with the hemin center in solution. We choose three representative amino acids-histidine (a well-known iron coordinator in bio-inorganic systems), tryptophan (a well-known fluoroprobe for proteins), and cysteine (a redox-active organic molecule). The interactions of these amino acids with hemin are studied using electrochemistry, spectroscopy, and density functional theory. The results indicate that among these three, the interaction of histidine with the iron center is strongest. Further, histidine maintains the electrochemical reversibility of iron. On the other hand, tryptophan and cysteine interact weakly with the iron center but disturb the electrochemical reversibility by contributing their own redox active processes to the system. Put together, this study attempts to understand the molecular interactions that can control electrochemical reversibility in heme proteins. The results obtained here from the three representative amino acids can be scaled up to build a heme-amino acid interaction database that may predict the electrochemical properties of any protein with a defined polypeptide sequence.

  10. Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

    Science.gov (United States)

    Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

    2008-02-26

    Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

  11. Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

    International Nuclear Information System (INIS)

    Majuri, R.

    1989-01-01

    Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of 35 S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with 35 S. The same two bands were observed if the cell surface proteins were labeled with 125 I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author)

  12. Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

    International Nuclear Information System (INIS)

    Lukat, G.S.; Rodgers, K.R.; Jabro, M.N.; Goff, H.M.

    1989-01-01

    Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, the authors characterize the H 2 O 2 -oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Caprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum

  13. Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

    International Nuclear Information System (INIS)

    Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin; Lastuvkova, Hana; Kodicek, Milan; Sakalian, Michael; Ruml, Tomas

    2009-01-01

    The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibers is linked to a conformational transition from unfolded or another non-periodical state to α-helix during multimerization.

  14. The Role of Heme Chirality in the Circular Dichroism of Heme Proteins

    Science.gov (United States)

    Woody, Robert W.; Pescitelli, Gennaro

    2014-07-01

    The rotational strength (R) of the Soret transition in sperm-whale myoglobin (SW Mb), the hemoglobin from Chironomus thummi thummi (CTT Hb), and human hemoglobin (hHb) has been calculated using 20 high-resolution ( Raro > Rpep. For CTT Hb and hHB, the orders were, respectively, Rint > Rpep > Raro and Rint > Raro ≈ Rpep. Human Hb ɑ chains showed the same trend as CTT Hb. Only in the hHb β chains did Raro predominate, with the order Raro > Rint > Rpep. The total predicted Rtot for SW Mb, CTT Hb, and hHb averaged +0.77±0.10 (0.56 - 0.80), -0.37±0.12 (-0.5), and +0.31±0.17 DBM (0.23 - 0.50), respectively. (Values in parentheses are experimental values.) Thus, contrary to the currently accepted view, coupling with aromatic side-chain or peptide transitions is not the dominant factor in the Soret circular dichroism (CD) of these proteins. The Soret CD is dominated by intrinsic CD of the heme chromophore, of which vinyl torsion is the major determinant. This result suggests an explanation for the large effect of heme isomerism on the Soret CD of Mb and Hb. Rotation about the ɑ-γ axis may be associated with large changes in vinyl torsion and thus substantially alter the intrinsic CD, even reversing its sign.

  15. Structure of the haptoglobin-haemoglobin complex

    DEFF Research Database (Denmark)

    Andersen, Christian Brix Folsted; Torvund-Jensen, Morten; Nielsen, Marianne Jensby

    2012-01-01

    to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger...... that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4)....

  16. Dietary iron controls circadian hepatic glucose metabolism through heme synthesis.

    Science.gov (United States)

    Simcox, Judith A; Mitchell, Thomas Creighton; Gao, Yan; Just, Steven F; Cooksey, Robert; Cox, James; Ajioka, Richard; Jones, Deborah; Lee, Soh-Hyun; King, Daniel; Huang, Jingyu; McClain, Donald A

    2015-04-01

    The circadian rhythm of the liver maintains glucose homeostasis, and disruption of this rhythm is associated with type 2 diabetes. Feeding is one factor that sets the circadian clock in peripheral tissues, but relatively little is known about the role of specific dietary components in that regard. We assessed the effects of dietary iron on circadian gluconeogenesis. Dietary iron affects circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its cosuppressor nuclear receptor corepressor 1 (NCOR). Loss of regulated heme synthesis was achieved by aminolevulinic acid (ALA) treatment of mice or cultured cells to bypass the rate-limiting enzyme in hepatic heme synthesis, ALA synthase 1 (ALAS1). ALA treatment abolishes differences in hepatic glucose production and in the expression of gluconeogenic enzymes seen with variation of dietary iron. The differences among diets are also lost with inhibition of heme synthesis with isonicotinylhydrazine. Dietary iron modulates levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a transcriptional activator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1α variation observed among the iron diets, suggesting that iron is acting through reactive oxygen species signaling. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  17. Heme oxygenase-1 deletion affects stress erythropoiesis.

    Directory of Open Access Journals (Sweden)

    Yu-An Cao

    Full Text Available Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1 deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/- or hmox(+/+ bone marrow cells, we evaluated (i the erythrocyte parameters in the peripheral blood; (ii the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii the patterns of histological iron staining; and (iv the number of Mac-1(+-cells expressing TNF-α. In the spleens of mice that received hmox(+/- cells, we show (i decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii increases in the insoluble iron levels and decreases in the soluble iron levels; (iii increased numbers of Mac-1(+-cells expressing TNF-α; and (iv decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.

  18. Heme oxygenase activity correlates with serum indices of iron homeostasis in healthy nonsmokers

    Science.gov (United States)

    Heme oxygenase (HO) catalyzes the breakdown of heme to carbon monoxide, iron, and biliverdin. While the use of genetically altered animal models in investigation has established distinct associations between HO activity and systemic iron availability, studies have not yet confirm...

  19. Novel Insights in Mammalian Catalase Heme Maturation: Effect of NO and Thioredoxin-1

    OpenAIRE

    Chakravarti, Ritu; Gupta, Karishma; Majors, Alana; Ruple, Lisa; Aronica, Mark; Stuehr, Dennis J.

    2015-01-01

    Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorpo...

  20. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

    OpenAIRE

    Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multi...

  1. Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis

    OpenAIRE

    Jacob E. Choby; Caroline M. Grunenwald; Arianna I. Celis; Svetlana Y. Gerdes; Jennifer L. DuBois; Eric P. Skaar; Kimberly A. Kline

    2018-01-01

    Staphylococcus aureus is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized de novo from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme hom...

  2. Considerations on the mechanism of action of artemisinin antimalarials: part 1--the 'carbon radical' and 'heme' hypotheses.

    Science.gov (United States)

    Haynes, Richard K; Cheu, Kwan-Wing; N'Da, David; Coghi, Paolo; Monti, Diego

    2013-08-01

    - reactions employing catalytic reagents under aqueous or semi-aqueous conditions - to those conducted under highly reducing and eminently artificial conditions, usually in the solvent dimethyl sulfoxide (DMSO) that both forms well characterized complexes with heme-Fe(2+) and actually assists in driving single electron transfer processes. It is noted that alkylated products tend to form in high yields under the last conditions, and this aspect is readily explained. Irrespective of product yields obtained under various conditions, an overarching correlation between facility of the reaction of the peroxide with heme and their antimalarial activities does not exist. The is underscored by the reproducible outcomes of reactions conducted under biomimetic conditions indicating adducts cannot form in physiologically meaningful concentrations and that heme is a recalcitrant reaction partner to artemisinins in general. Again, as in the case of the C-radical hypothesis, structure-activity data from a wide variety of artemisinins and synthetic peroxides is difficult to reconcile with the heme hypothesis. This applies in particular to dimeric and trimeric artemisinin derivatives where the ascribing of biological activity to reactions of the derived radicals or to the vastly encumbered artemisinin-heme adducts is physically unrealistic. Finally, the facile metabolism and induction of metabolism of the current clinically used artemisinins by members of the CYP superfamily - heme proteins that require an intimate interaction of the heme with the artemisinin for metabolism to occur - is incompatible with the oft-cited proclivity of the peroxide to associate via complex formation with heme as a prelude to its 'activation' as an antimalarial agent within the malaria parasite. (ABSTRACT TRUNCATED)

  3. Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.

    Science.gov (United States)

    Kumar, Devesh; de Visser, Samuël P; Shaik, Sason

    2005-06-08

    The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem. The degradation is initiated by complexation of the heme, then formation of the iron-hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O-O bond homolysis of iron-hydroperoxide. The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H(3)O(+)(H(2)O)(n)(), n = 0-2. The effect of the acid strength is tested using the H(4)N(+)(H(2)O)(2) cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O-O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H(3)O(+)(H(2)O)(2) cluster. The calculated alpha-deuterium secondary kinetic isotope effect is inverse (0.95-0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism.

  4. Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

    Directory of Open Access Journals (Sweden)

    Viswanathan Arun Nagaraj

    Full Text Available Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS, and the last enzyme, ferrochelatase (FC, in the heme-biosynthetic pathway of Plasmodium berghei (Pb. The wild-type and knockout (KO parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14C] aminolevulinic acid (ALA. We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

  5. Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but not Disruption of the Distal Hydrogen Bonding Network†

    Science.gov (United States)

    Wang, Jinling; Evans, John P.; Ogura, Hiroshi; La Mar, Gerd N.; Ortiz de Montellano, Paul R.

    2008-01-01

    Heme oxygenase regiospecifically oxidizes heme at the α-meso position to give biliverdin IXα, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry, but partially shifts the oxidation to the β/δ-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by ~90°, causes a slight loss of regiospecificity, but combined with the R183E and K18E mutations results primarily in β/δ-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network, impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane. PMID:16388581

  6. Heme and menaquinone induced electron transport in lactic acid bacteria

    NARCIS (Netherlands)

    Brooijmans, R.J.W.; Smit, B.; Santos, dos F.; Riel, van J.; Vos, de W.M.; Hugenholtz, J.

    2009-01-01

    ABSTRACT: BACKGROUND: For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait.

  7. Cysteine-independent activation/inhibition of heme oxygenase-2

    Directory of Open Access Journals (Sweden)

    Dragic Vukomanovic

    2016-01-01

    Full Text Available Reactive thiols of cysteine (cys residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2 isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2 and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

  8. Heme and HO-1 inhibition of HCV, HBV, and HIV

    Directory of Open Access Journals (Sweden)

    Warren N Schmidt

    2012-10-01

    Full Text Available Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system are virucidal for all three viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections.

  9. Cysteine-independent activation/inhibition of heme oxygenase-2.

    Science.gov (United States)

    Vukomanovic, Dragic; Rahman, Mona N; Maines, Mahin D; Ozolinš, Terence Rs; Szarek, Walter A; Jia, Zongchao; Nakatsu, Kanji

    2016-03-01

    Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

  10. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    Science.gov (United States)

    An ELISA assay for heme oxygenase (HO-l ) Abstract A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  11. Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

    Science.gov (United States)

    Manoj, Kelath Murali; Parashar, Abhinav; Venkatachalam, Avanthika; Goyal, Sahil; Satyalipsu; Singh, Preeti Gunjan; Gade, Sudeep K; Periyasami, Kalaiselvi; Jacob, Reeba Susan; Sardar, Debosmita; Singh, Shanikant; Kumar, Rajan; Gideon, Daniel A

    2016-06-01

    Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  12. Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

    Science.gov (United States)

    Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

    2018-05-15

    Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

  13. Coordinate expression of heme and globin is essential for effective erythropoiesis.

    Science.gov (United States)

    Doty, Raymond T; Phelps, Susan R; Shadle, Christina; Sanchez-Bonilla, Marilyn; Keel, Siobán B; Abkowitz, Janis L

    2015-12-01

    Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.

  14. ATP-binding cassette B10 regulates early steps of heme synthesis.

    Science.gov (United States)

    Bayeva, Marina; Khechaduri, Arineh; Wu, Rongxue; Burke, Michael A; Wasserstrom, J Andrew; Singh, Neha; Liesa, Marc; Shirihai, Orian S; Langer, Nathaniel B; Paw, Barry H; Ardehali, Hossein

    2013-07-19

    Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established. The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

  15. Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

    Science.gov (United States)

    Posayapisit, Navaporn; Songsungthong, Warangkhana; Koonyosying, Pongpisid; Falade, Mofolusho O; Uthaipibull, Chairat; Yuthavong, Yongyuth; Shaw, Philip J; Kamchonwongpaisan, Sumalee

    Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc 1 hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc 1 hl are both essential and thus may be antimalarial targets. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems.

    Science.gov (United States)

    Cavallaro, Gabriele; Decaria, Leonardo; Rosato, Antonio

    2008-11-01

    Heme is the prosthetic group of many proteins that carry out a variety of key biological functions. In addition, for many pathogenic organisms, heme (acquired from the host) may constitute a very important source of iron. Organisms can meet their heme demands by taking it up from external sources, by producing the cofactor through a dedicated biosynthetic pathway, or both. Here we analyzed the distribution of proteins specifically involved in the processes of heme biosynthesis and heme uptake in 474 prokaryotic organisms. These data allowed us to identify which organisms are capable of performing none, one, or both processes, based on the similarity to known systems. Some specific instances where one or more proteins along the pathways had unusual modifications were singled out. For two key protein domains involved in heme uptake, we could build a series of structural models, which suggested possible alternative modes of heme binding. Future directions for experimental work are given.

  17. CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123

    OpenAIRE

    Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

    2010-01-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biot...

  18. Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.

    Science.gov (United States)

    Surdel, Matthew C; Dutter, Brendan F; Sulikowski, Gary A; Skaar, Eric P

    2016-08-12

    Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.

  19. Binding analysis of ferritin with heme using α-casein and biotinylated-hemin: detection of heme-binding capacity of Dpr derived from heme synthesis-deficient Streptococcus mutans.

    Science.gov (United States)

    Mieno, Ayako; Yamamoto, Yuji; Yoshikawa, Yasunaga; Watanabe, Kiyotaka; Mukai, Takao; Orino, Koichi

    2013-01-01

    Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to an α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Escherichia coli, did not bind to an α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After preincubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin and that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.

  20. Dietary heme mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon

    NARCIS (Netherlands)

    IJssennagger, Noortje; Wit, de Nicole; Muller, Michael; Meer, van der Roelof

    2012-01-01

    Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome

  1. Dietary heme-mediated PPARa activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon

    NARCIS (Netherlands)

    IJssenagger, N.; Wit, de N.J.W.; Muller, M.R.; Meer, van der R.

    2012-01-01

    Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome

  2. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

    Directory of Open Access Journals (Sweden)

    Anusha Panjwani

    2014-08-01

    Full Text Available Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

  3. Significant role of PB1 and UBA domains in multimerization of Joka2, a selective autophagy cargo receptor from tobacco

    Directory of Open Access Journals (Sweden)

    Katarzyna eZientara-Rytter

    2014-01-01

    Full Text Available Tobacco Joka2 protein is a hybrid homolog of two mammalian selective autophagy cargo receptors, p62 and NBR1. These proteins can directly interact with the members of ATG8 family and the polyubiquitinated cargoes designed for degradation. Function of the selective autophagy cargo receptors relies on their ability to form protein aggregates. It has been shown that the N-terminal PB1 domain of p62 is involved in formation of aggregates, while the UBA domains of p62 and NBR1 have been associated mainly with cargo binding. Here we focus on roles of PB1 and UBA domains in localization and aggregation of Joka2 in plant cells. We show that Joka2 can homodimerize not only through its N-terminal PB1-PB1 interactions but also via interaction between N-terminal PB1 and C-terminal UBA domains. We also demonstrate that Joka2 co-localizes with recombinant ubiquitin and sequestrates it into aggregates and that C-terminal part (containing UBA domains is sufficient for this effect. Our results indicate that Joka2 accumulates in cytoplasmic aggregates and suggest that in addition to these multimeric forms it also exists in the nucleus and cytoplasm in a monomeric form.

  4. AMOEBA Polarizable Force Field Parameters of the Heme Cofactor in Its Ferrous and Ferric Forms.

    Science.gov (United States)

    Wu, Xiaojing; Clavaguera, Carine; Lagardère, Louis; Piquemal, Jean-Philip; de la Lande, Aurélien

    2018-04-16

    We report the first parameters of the heme redox cofactors for the polarizable AMOEBA force field in both the ferric and ferrous forms. We consider two types of complexes, one with two histidine side chains as axial ligands and one with a histidine and a methionine side chain as ligands. We have derived permanent multipoles from second-order Møller-Plesset perturbation theory (MP2). The sets of parameters have been validated in a first step by comparison of AMOEBA interaction energies of heme and a collection of biologically relevant molecules with MP2 and Density Functional Theory (DFT) calculations. In a second validation step, we consider interaction energies with large aggregates comprising around 80 H 2 O molecules. These calculations are repeated for 30 structures extracted from semiempirical PM7 DM simulations. Very encouraging agreement is found between DFT and the AMOEBA force field, which results from an accurate treatment of electrostatic interactions. We finally report long (10 ns) MD simulations of cytochromes in two redox states with AMOEBA testing both the 2003 and 2014 AMOEBA water models. These simulations have been carried out with the TINKER-HP (High Performance) program. In conclusion, owing to their ubiquity in biology, we think the present work opens a wide array of applications of the polarizable AMOEBA force field on hemeproteins.

  5. A Novel, “Double-Clamp” Binding Mode for Human Heme Oxygenase-1 Inhibition

    Science.gov (United States)

    Rahman, Mona N.; Vlahakis, Jason Z.; Vukomanovic, Dragic; Lee, Wallace; Szarek, Walter A.; Nakatsu, Kanji; Jia, Zongchao

    2012-01-01

    The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC50 = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC50 = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors. PMID:22276118

  6. A novel, "double-clamp" binding mode for human heme oxygenase-1 inhibition.

    Directory of Open Access Journals (Sweden)

    Mona N Rahman

    Full Text Available The development of heme oxygenase (HO inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl-4,4-diphenyl-2-butanone (QC-308. Using a carbon monoxide (CO formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC(50 = 0.27±0.07 µM than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC(50 = 4.0±1.8 µM. The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This "double-clamp" binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.

  7. A novel, "double-clamp" binding mode for human heme oxygenase-1 inhibition.

    Science.gov (United States)

    Rahman, Mona N; Vlahakis, Jason Z; Vukomanovic, Dragic; Lee, Wallace; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

    2012-01-01

    The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC(50) = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC(50) = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This "double-clamp" binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.

  8. Syntheses of carbon-13 labeled protoporphyrin-IX for spectroscopic studies of heme proteins

    International Nuclear Information System (INIS)

    Fujinari, E.M.

    1985-01-01

    The development of various methodologies for synthesis of selectively tailored protoporphyrin-IX dimethyl ester are presented. The iron(II) complex of protoporphyrin-IX is the heme, the prosthetic group for Hb, Mb, cytochromes and peroxidases. The significance of this research is to provide direct means to establish definitive carbon-13 NMR assignments of heme proteins in order to study not only the structure-function relationships, but also protein dynamics of these vital systems. Carbon-13 labeling at the beta-vinyl position was first achieved by ozonolysis of protoporphyrin-IX dimethyl ester. Column LC method were used to first isolate 2,4-diformyldeuteroporphyrin-IX dimethyl ester. Concomitantly, monofomyl-monovinyl porphyrins were obtained as a mixture of two isomers. This mixture was separated by MPLC or prep HPLC to afford the isomerically pure products, Spirographis porphyrin dimethyl ester and Iso-Spirographis porphyrin dimethyl ester. A Wittig reaction to each of these porphyrins with 13 C-methyltriphenylphosphonium iodide gave 2,4-bis[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, 2-[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, and the 4-[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, respectively

  9. Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin.

    Science.gov (United States)

    Vu, B Christie; Nothnagel, Henry J; Vuletich, David A; Falzone, Christopher J; Lecomte, Juliette T J

    2004-10-05

    The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed

  10. Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Christine A.; Zhou, Mowei; Song, Yang; Wysocki, Vicki H.; Dohnalkova, Alice C.; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M.

    2017-09-29

    Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase, Mnx, in Bacillus sp. PL-12 is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. However, MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. In this study, native mass spectrometry defines the subunit topology and copper binding of the Mnx complex, while high resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for conceptualizing how Mnx produces nanoparticulate Mn oxides.

  11. Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx.

    Science.gov (United States)

    Romano, Christine A; Zhou, Mowei; Song, Yang; Wysocki, Vicki H; Dohnalkova, Alice C; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M

    2017-09-29

    Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products.

  12. The interaction of equine lysozyme:oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism

    DEFF Research Database (Denmark)

    Nielsen, Søren Bang; Wilhelm, Kristina; Vad, Brian

    2010-01-01

    The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes...

  13. Heme degrading protein HemS is involved in oxidative stress response of Bartonella henselae.

    Directory of Open Access Journals (Sweden)

    MaFeng Liu

    Full Text Available Bartonellae are hemotropic bacteria, agents of emerging zoonoses. These bacteria are heme auxotroph Alphaproteobacteria which must import heme for supporting their growth, as they cannot synthesize it. Therefore, Bartonella genome encodes for a complete heme uptake system allowing the transportation of this compound across the outer membrane, the periplasm and the inner membranes. Heme has been proposed to be used as an iron source for Bartonella since these bacteria do not synthesize a complete system required for iron Fe³⁺ uptake. Similarly to other bacteria which use heme as an iron source, Bartonellae must transport this compound into the cytoplasm and degrade it to allow the release of iron from the tetrapyrrole ring. For Bartonella, the gene cluster devoted to the synthesis of the complete heme uptake system also contains a gene encoding for a polypeptide that shares homologies with heme trafficking or degrading enzymes. Using complementation of an E. coli mutant strain impaired in heme degradation, we demonstrated that HemS from Bartonella henselae expressed in E. coli allows the release of iron from heme. Purified HemS from B. henselae binds heme and can degrade it in the presence of a suitable electron donor, ascorbate or NADPH-cytochrome P450 reductase. Knocking down the expression of HemS in B. henselae reduces its ability to face H₂O₂ induced oxidative stress.

  14. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    Science.gov (United States)

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury.

    Science.gov (United States)

    Sawicki, Konrad Teodor; Shang, Meng; Wu, Rongxue; Chang, Hsiang-Chun; Khechaduri, Arineh; Sato, Tatsuya; Kamide, Christine; Liu, Ting; Naga Prasad, Sathyamangla V; Ardehali, Hossein

    2015-07-31

    Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the

  16. Alteration by irradiation and storage at amount of heme iron in poultry meat; Alteracoes provocadas pela irradiacao e armazenamento nos teores de ferro heme em carne de frango

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Adriana Regia Marques de; Arthur, Valter Arthur [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Lab. de Irradiacao de Alimentos e Radioentomologia; Canniatti-Brazaca, Solange Guidolin [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil). Dept. de Agroindustria, Alimentos e Nutricao]. E-mail: sgcbraza@esalq.usp.br

    2007-04-15

    Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 deg C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author)

  17. Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

    Energy Technology Data Exchange (ETDEWEB)

    Hammerstrom, Troy G.; Lori, Horton B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activity in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

  18. Dietary Heme Induces Gut Dysbiosis, Aggravates Colitis, and Potentiates the Development of Adenomas in Mice

    Directory of Open Access Journals (Sweden)

    Marco Constante

    2017-09-01

    Full Text Available Dietary heme can be used by colonic bacteria equipped with heme-uptake systems as a growth factor and thereby impact on the microbial community structure. The impact of heme on the gut microbiota composition may be particularly pertinent in chronic inflammation such as in inflammatory bowel disease (IBD, where a strong association with gut dysbiosis has been consistently reported. In this study we investigated the influence of dietary heme on the gut microbiota and inferred metagenomic composition, and on chemically induced colitis and colitis-associated adenoma development in mice. Using 16S rRNA gene sequencing, we found that mice fed a diet supplemented with heme significantly altered their microbiota composition, characterized by a decrease in α-diversity, a reduction of Firmicutes and an increase of Proteobacteria, particularly Enterobacteriaceae. These changes were similar to shifts seen in dextran sodium sulfate (DSS-treated mice to induce colitis. In addition, dietary heme, but not systemically delivered heme, contributed to the exacerbation of DSS-induced colitis and facilitated adenoma formation in the azoxymethane/DSS colorectal cancer (CRC mouse model. Using inferred metagenomics, we found that the microbiota alterations elicited by dietary heme resulted in non-beneficial functional shifts, which were also characteristic of DSS-induced colitis. Furthermore, a reduction in fecal butyrate levels was found in mice fed the heme supplemented diet compared to mice fed the control diet. Iron metabolism genes known to contribute to heme release from red blood cells, heme uptake, and heme exporter proteins, were significantly enriched, indicating a shift toward favoring the growth of bacteria able to uptake heme and protect against its toxicity. In conclusion, our data suggest that luminal heme, originating from dietary components or gastrointestinal bleeding in IBD and, to lesser extent in CRC, directly contributes to microbiota dysbiosis

  19. Irradiation of bovine meat: effect of heme-iron concentration

    International Nuclear Information System (INIS)

    Mistura, Liliana Perazzini Furtado

    2002-01-01

    The irradiation is often used, nowadays, for meat conservation and it is important to know how much this process interferes with the nutritional quality of the meat. In this study round cut meat, ground and steaks (from a local supermarket) was irradiated with doses of O; 1; 2; 3; 4; 5; 7,5 and 10 kGy (JS-7500 Nordium Inc -Canada) and the interference of irradiation and the process of food preparation on heme-iron (H Fe) content was determined. Half of the sample was kept raw and the other half was grilled in a pre-warmed oven at 250 deg C for 9 min and a controlled humidity of 70%. The chemical composition, the total iron (T Fe) (EM) and the heme iron concentration were determined (Hornsey,1956) and the sensorial quality evaluated. The average T Fe concentration of raw and ground , ground and grilled, raw steaks and grilled steak meat, on dry and degreased basis was 113 mug/g, 121 mug/g , 91 mug/g and 77 mug/g; and the H Fe concentration 105 mug/g (93% of T Fe) , 88 mug/g (73% of T Fe), 90 mug/g (99% of T Fe) and 52 mug/g (68% of T Fe) respectively. Data were evaluated by ANOVA with fixed effects and multiple comparisons. The irradiation neither altered the chemical composition nor the proportion of heme iron of meat. The preparation conditions (temperature, cooking time, environment humidity, meat presentation) of the sample interfered more with the heme iron content than the irradiation. With the sensorial analysis we verified that meats irradiated with doses of 3 kGy were better evaluated in softness and succulency attributes than the others. Meat submitted to irradiation doses up to 3 kGy were accepted by the specialists' panel. (author)

  20. Functional imaging: monitoring heme oxygenase-1 gene expression in vivo

    Science.gov (United States)

    Zhang, Weisheng; Reilly-Contag, Pamela; Stevenson, David K.; Contag, Christopher H.

    1999-07-01

    The regulation of genetic elements can be monitored in living animals using photoproteins as reporters. Heme oxygenase (HO) is the key catabolic enzyme in the heme degradation pathway. Here, HO expression serves as a model for in vivo functional imaging of transcriptional regulation of a clinically relevant gene. HO enzymatic activity is inhibited by heme analogs, metalloporphyrins, but many members of this family of compounds also activate transcription of the HO-1 promoter. The degree of transcriptional activation by twelve metalloporphyrins, differing at the central metal and porphyrin ring substituents, was evaluated in both NIH 3T3 stable lines and transgenic animals containing HO-1 promoter-luciferase gene fusions. In the correlative cell culture assays, the metalloporphyrins increased transcription form the full length HO promoter fusion to varying degrees, but none increased transcription from a truncated HO-1 promoter. These results suggested that one or both of the two distal enhancer elements located at -4 and -10 Kb upstream from transcriptional start are required for HO-1 induction by heme and its analogs. The full-length HO-1-luc fusion was then evaluated as a transgene in mice. It was possible to monitor the effects of the metalloporphyrins, SnMP and ZnPP, in living animals over time. This spatiotemporal analyses of gene expression in vivo implied that alterations in porphyrin ring substituents and the central metal may affect the extent of gene activation. These data further indicate that using photoprotein reporters, subtle differences in gene expression can be monitored in living animals.

  1. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

    Science.gov (United States)

    Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

    2015-02-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. © 2014 John Wiley & Sons Ltd.

  2. Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

    Science.gov (United States)

    Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S

    2015-01-05

    Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Schizosaccharomyces pombe Rad22A and Rad22B have similar biochemical properties and form multimeric structures

    Energy Technology Data Exchange (ETDEWEB)

    Vries, Femke A.T. de [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Zonneveld, Jose B.M. [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Groot, Anton J. de [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Koning, Roman I. [Department of Molecular Cell Biology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert A. van [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Pastink, Albert [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands)]. E-mail: A.Pastink@lumc.nl

    2007-02-03

    The Saccharomyces cerevisiae Rad52 protein has a crucial role in the repair of DNA double-strand breaks by homologous recombination. In vitro, Rad52 displays DNA binding and strand annealing activities and promotes Rad51-mediated strand exchange. Schizosaccharomyces pombe has two Rad52 homologues, Rad22A and Rad22B. Whereas rad22A deficient strains exhibit severe defects in repair and recombination, rad22B mutants have a much less severe phenotype. To better understand the role of Rad22A and Rad22B in double-strand break repair, both proteins were purified to near homogeneity. Using gel retardation and filter binding assays, binding of Rad22A and Rad22B to short single-stranded DNAs was demonstrated. Binding of Rad22A to double-stranded oligonucleotides or linearized plasmid molecules containing blunt ends or short single-stranded overhangs could not be detected. Rad22B also does not bind efficiently to short duplex oligonucleotides but binds readily to DNA fragments containing 3'-overhangs. Rad22A as well as Rad22B efficiently promote annealing of complementary single-stranded DNAs. In the presence of Rad22A annealing of complementary DNAs is almost 90%. Whereas in reactions containing Rad22B the maximum level of annealing is 60%, most likely due to inhibition of the reaction by duplex DNA. Gel-filtration experiments and electron microscopic analyses indicate self-association of Rad22A and Rad22B and the formation of multimeric structures as has been observed for Rad52 in yeast and man.

  4. Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

    Science.gov (United States)

    Dailey, Harry A; Dailey, Tamara A; Gerdes, Svetlana; Jahn, Dieter; Jahn, Martina; O'Brian, Mark R; Warren, Martin J

    2017-03-01

    The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. Copyright © 2017 American Society for Microbiology.

  5. Stability enhancement of cytochrome c through heme deprotonation and mutations.

    Science.gov (United States)

    Sonoyama, Takafumi; Hasegawa, Jun; Uchiyama, Susumu; Nakamura, Shota; Kobayashi, Yuji; Sambongi, Yoshihiro

    2009-01-01

    The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.

  6. In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

    International Nuclear Information System (INIS)

    Mack, John; Vermeiren, Christie; Heinrichs, David E.; Stillman, Martin J.

    2004-01-01

    We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins

  7. Isoporphyrin intermediate in heme oxygenase catalysis. Oxidation of alpha-meso-phenylheme.

    Science.gov (United States)

    Evans, John P; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

    2008-07-11

    Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the alpha-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin pi-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of alpha-meso-phenylheme-IX, alpha-meso-(p-methylphenyl)-mesoheme-III, and alpha-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593-42604), only the alpha-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced alpha-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation.

  8. Enhancement of nitrite on heme-induced oxidative reactions: A potential toxicological implication.

    Science.gov (United States)

    Lu, Naihao; Chen, Wei; Zhu, Jingjie; Peng, Yi-Yuan

    2012-02-01

    Evidence to support the role of heme as major inducers of oxidative damage is increasingly present. Nitrite (NO(2)(-)) is one of the major end products of NO metabolism. Although the biological significance of heme/NO(2)(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO(2)(-) on heme-dependent redox reaction have been greatly underestimated. In this study, we investigated the influence of NO(2)(-) on heme -dependent oxidative reactions. It was found that NO(2)(-) had the capacity to act as a reducing agent to remove high oxidation states of heme iron. In the reduction of ferryl heme to ferric heme, NO(2)(-) was oxidized to a nitrating agent NO(2), and subsequently, tyrosine residues in bovine serum albumin (BSA) were nitrated. However, the presence of NO(2)(-) surprisingly exerted pro-oxidant effect on heme-H(2)O(2)-induced formation of BSA carbonyls at lower concentrations and enhanced the loss of HepG2 cell viability dose-dependently, which was probably due to the ability of this inorganic compound to efficiently enhance the peroxidase activity and oxidative degradation of heme. These data provide novel evidence that the dietary intake and experimental use of NO(2)(-) in vivo and in vitro would possess the pro-oxidant activity through interfering in heme-dependent oxidative reactions. Besides the classic role in protein tyrosine nitration, the deleterious effects on heme redox reactions may provide new insights into the toxicological implications of NO(2)(-) with cellular heme proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Effects of Metalloporphyrins on Heme Oxygenase-1 Transcription: Correlative Cell Culture Assays Guide in Vivo Imaging

    OpenAIRE

    Monica Hajdena-Dawson; Weisheng Zhang; Pamela R. Contag; Ronald J. Wong; Hendrik J. Vreman; David K. Stevenson; Christopher H. Contag

    2003-01-01

    Heme oxygenase (HO) is the rate-limiting step in the heme degradation pathway and is a potential target for the control, or prevention, of pathologic jaundice in neonates. Metalloporphyrins (Mps), a diverse set of synthetic derivatives of heme, can competitively inhibit the HO enzymes. However, certain Mps are phototoxic and some increase transcription of HO-1, the inducible HO isozyme. Therefore, effective development of this class of compounds as therapeutics for treating pathologic jaundic...

  10. Irradiation of bovine meat: effect of heme-iron concentration.; Irradiacao de carne bovina: efeito na concentracao de ferro heme

    Energy Technology Data Exchange (ETDEWEB)

    Mistura, Liliana Perazzini Furtado

    2002-07-01

    The irradiation is often used, nowadays, for meat conservation and it is important to know how much this process interferes with the nutritional quality of the meat. In this study round cut meat, ground and steaks (from a local supermarket) was irradiated with doses of O; 1; 2; 3; 4; 5; 7,5 and 10 kGy (JS-7500 Nordium Inc -Canada) and the interference of irradiation and the process of food preparation on heme-iron (H Fe) content was determined. Half of the sample was kept raw and the other half was grilled in a pre-warmed oven at 250 deg C for 9 min and a controlled humidity of 70%. The chemical composition, the total iron (T Fe) (EM) and the heme iron concentration were determined (Hornsey,1956) and the sensorial quality evaluated. The average T Fe concentration of raw and ground , ground and grilled, raw steaks and grilled steak meat, on dry and degreased basis was 113 mug/g, 121 mug/g , 91 mug/g and 77 mug/g; and the H Fe concentration 105 mug/g (93% of T Fe) , 88 mug/g (73% of T Fe), 90 mug/g (99% of T Fe) and 52 mug/g (68% of T Fe) respectively. Data were evaluated by ANOVA with fixed effects and multiple comparisons. The irradiation neither altered the chemical composition nor the proportion of heme iron of meat. The preparation conditions (temperature, cooking time, environment humidity, meat presentation) of the sample interfered more with the heme iron content than the irradiation. With the sensorial analysis we verified that meats irradiated with doses of 3 kGy were better evaluated in softness and succulency attributes than the others. Meat submitted to irradiation doses up to 3 kGy were accepted by the specialists' panel. (author)

  11. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    Science.gov (United States)

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  12. Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase.

    Directory of Open Access Journals (Sweden)

    Quang M Tran

    Full Text Available The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.

  13. A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

    Science.gov (United States)

    Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

    2014-10-01

    Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

  14. In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

    Science.gov (United States)

    Coelho, Pedro S; Brustad, Eric M; Arnold, Frances H; Wang, Zhan; Lewis, Jared C

    2015-03-31

    The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

  15. O2-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-01-01

    Research highlights: → Human serum heme-albumin displays globin-like properties. → O 2 -mediated oxidation of ferrous nitrosylated human serum heme-albumin. → Allosteric modulation of human serum heme-albumin reactivity. → Rifampicin is an allosteric effector of human serum heme-albumin. → Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O 2 -mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O 2 -mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10 -5 and 8.3 x 10 -4 s -1 , and h = 1.3 x 10 -4 and 8.5 x 10 -4 s -1 , in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 o C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O 2 -mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O 2 does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O 2 -mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  16. Dietary hemoglobin rescues young piglets from severe iron deficiency anemia: Duodenal expression profile of genes involved in heme iron absorption.

    Directory of Open Access Journals (Sweden)

    Robert Staroń

    Full Text Available Heme is an efficient source of iron in the diet, and heme preparations are used to prevent and cure iron deficiency anemia in humans and animals. However, the molecular mechanisms responsible for heme absorption remain only partially characterized. Here, we employed young iron-deficient piglets as a convenient animal model to determine the efficacy of oral heme iron supplementation and investigate the pathways of heme iron absorption. The use of bovine hemoglobin as a dietary source of heme iron was found to efficiently counteract the development of iron deficiency anemia in piglets, although it did not fully rebalance their iron status. Our results revealed a concerted increase in the expression of genes responsible for apical and basolateral heme transport in the duodenum of piglets fed a heme-enriched diet. In these animals the catalytic activity of heme oxygenase 1 contributed to the release of elemental iron from the protoporphyrin ring of heme within enterocytes, which may then be transported by the strongly expressed ferroportin across the basolateral membrane to the circulation. We hypothesize that the well-recognized high bioavailability of heme iron may depend on a split pathway mediating the transport of heme-derived elemental iron and intact heme from the interior of duodenal enterocytes to the bloodstream.

  17. The "innocent" role of Sc3+ on a non-heme Fe catalyst in an O2 environment

    KAUST Repository

    Poater, Albert; Chaitanya Vummaleti, Sai Vikrama; Cavallo, Luigi

    2014-01-01

    Density functional theory calculations have been used to investigate the reaction mechanism proposed for the formation of an oxoiron(iv) complex [Fe IV(TMC)O]2+ (P) (TMC = 1,4,8,11-tetramethylcyclam) starting from a non-heme reactant complex [FeII(TMC)]2+ (R) and O2 in the presence of acid H+ and reductant BPh4 -. We also addressed the possible role of redox-inactive Sc3+ as a replacement for H+ acid in this reaction to trigger the formation of P. Our computational results substantially confirm the proposed mechanism and, more importantly, support that Sc 3+ could trigger the O2 activation, mainly dictated by the availability of two electrons from BPh4 -, by forming a thermodynamically stable Sc3+-peroxo-Fe3+ core that facilitates O-O bond cleavage to generate P by reducing the energy barrier. These insights may pave the way to improve the catalytic reactivity of metal-oxo complexes in O2 activation at non-heme centers. This journal is © the Partner Organisations 2014.

  18. Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal.

    Science.gov (United States)

    Correia, Maria Almira; Sinclair, Peter R; De Matteis, Francesco

    2011-02-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers, with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein.

  19. Pilot-scale tests of HEME and HEPA dissolution process

    Energy Technology Data Exchange (ETDEWEB)

    Qureshi, Z.H.; Strege, D.K.

    1994-06-01

    A series of pilot-scale demonstration tests for the dissolution of High Efficiency Mist Eliminators (HEME`s) and High Efficiency Particulate Airfilters (HEPA) were performed on a 1/5th linear scale. These fiberglass filters are to be used in the Defense Waste Processing Facility (DWPF) to decontaminate the effluents from the off-gases generated during the feed preparation process and vitrification. When removed, these filters will be dissolved in the Decontamination Waste Treatment Tank (DWTT) using 5 wt% NaOH solution. The contaminated fiberglass is converted to an aqueous stream which will be transferred to the waste tanks. The filter metal structure will be rinsed with process water before its disposal as low-level solid waste. The pilot-scale study reported here successfully demonstrated a simple one step process using 5 wt% NaOH solution. The proposed process requires the installation of a new water spray ring with 30 nozzles. In addition to the reduced waste generated, the total process time is reduced to 48 hours only (66% saving in time). The pilot-scale tests clearly demonstrated that the dissolution process of HEMEs has two stages - chemical digestion of the filter and mechanical erosion of the digested filter. The digestion is achieved by a boiling 5 wt% caustic solutions, whereas the mechanical break down of the digested filter is successfully achieved by spraying process water on the digested filter. An alternate method of breaking down the digested filter by increased air sparging of the solution was found to be marginally successful are best. The pilot-scale tests also demonstrated that the products of dissolution are easily pumpable by a centrifugal pump.

  20. Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer.

    Science.gov (United States)

    Sunamura, Makoto; Duda, Dan G; Ghattas, Maivel H; Lozonschi, Lucian; Motoi, Fuyuhiko; Yamauchi, Jun-Ichiro; Matsuno, Seiki; Shibahara, Shigeki; Abraham, Nader G

    2003-01-01

    Angiogenesis is necessary for the continued growth of solid tumors, invasion and metastasis. Several studies clearly showed that heme oxygenase-1 (HO-1) plays an important role in angiogenesis. In this study, we used the vital microscope system, transparent skinfold model, lung colonization model and transduced pancreatic cancer cell line (Panc-1)/human heme oxygenase-1 (hHO-1) cells, to precisely analyze, for the first time, the effect of hHO-1 gene on tumor growth, angiogenesis and metastasis. Our results revealed that HO-1 stimulates angiogenesis of pancreatic carcinoma in severe combined immune deficient mice. Overexpression of human hHO-1 after its retroviral transfer into Panc-1 cells did not interfere with tumor growth in vitro. While in vivo the development of tumors was accelerated upon transfection with hHO-1. On the other hand, inhibition of heme oxygenase (HO) activity by stannous mesoporphyrin was able transiently to delay tumor growth in a dose dependent manner. Tumor angiogenesis was markedly increased in Panc-1/hHO-1 compared to mock transfected and wild type. Lectin staining and Ki-67 proliferation index confirmed these results. In addition hHO-1 stimulated in vitro tumor angiogenesis and increased endothelial cell survival. In a lung colonization model, overexpression of hHO-1 increased the occurrence of metastasis, while inhibition of HO activity by stannous mesoporphyrin completely inhibited the occurrence of metastasis. In conclusion, overexpression of HO-1 genes potentiates pancreatic cancer aggressiveness, by increasing tumor growth, angiogenesis and metastasis and that the inhibition of the HO system may be of useful benefit for the future treatment of the disease.

  1. Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

    International Nuclear Information System (INIS)

    Westberg, Johan A.; Jiang, Ji; Andersson, Leif C.

    2011-01-01

    Highlights: → Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. → Central iron atom of heme and cysteine-114 of STC1 are essential for binding. → STC1 binds Fe 2+ and Fe 3+ heme. → STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys 114 as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H 2 O 2 induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

  2. Heme metabolism in stress regulation and protein production: from Cinderella to a key player

    DEFF Research Database (Denmark)

    Martinez Ruiz, José Luis; Petranovic, D.; Nielsen, Jens

    2016-01-01

    Heme biosynthesis is a highly conserved pathway which is present in all kingdoms, from Archaea to higher organisms such as plants and mammals. The heme molecule acts as a prosthetic group for different proteins and enzymes involved in energy metabolism and reactions involved in electron transfer....

  3. The effect of irradiation and thermal process on beef heme iron concentration and color properties

    International Nuclear Information System (INIS)

    Mistura, Liliana Perazzini Furtado; Colli, Celia

    2009-01-01

    The aim of this study was to evaluate the influence of irradiation and thermal process on the heme iron (heme-Fe) concentration and color properties of Brazilian cattle beef. Beef samples (patties and steaks) were irradiated at 0-10 kGy and cooked in a combination oven at 250 deg C for 9 minutes with 70% humidity. Total iron and heme iron (heme-Fe) concentrations were determined. The data were compared by multiple comparisons and fixed- effects ANOVA. Irradiation at doses higher than 5 kGy significantly altered the heme-Fe concentration. However, the sample preparation conditions interfered more in the heme-Fe content than did the irradiation. Depending on the animal species, meat heme iron levels between 35 and 52% of the total iron are used for dietetic calculations. In this study the percentage of heme-iron was, on average, 70% of the total iron showing that humidity is an important factor for its preservation. The samples were analyzed instrumentally for CIE L * , a * , and b * values. (author)

  4. Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

    Energy Technology Data Exchange (ETDEWEB)

    Westberg, Johan A., E-mail: johan.westberg@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Jiang, Ji, E-mail: ji.jiang@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Andersson, Leif C., E-mail: leif.andersson@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland)

    2011-06-03

    Highlights: {yields} Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. {yields} Central iron atom of heme and cysteine-114 of STC1 are essential for binding. {yields} STC1 binds Fe{sup 2+} and Fe{sup 3+} heme. {yields} STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys{sup 114} as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H{sub 2}O{sub 2} induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

  5. Electrochemical and spectroscopic investigations of immobilized de novo designed heme proteins on metal electrodes

    DEFF Research Database (Denmark)

    Albrecht, Tim; Li, WW; Ulstrup, Jens

    2005-01-01

    On the basis of rational design principles, template-assisted four-helix-bundle proteins that include two histidines for coordinative binding of a heme were synthesized. Spectroscopic and thermodynamic characterization of the proteins in solution reveals the expected bis-histidine coordinated heme...

  6. Alteration by irradiation and storage at amount of heme iron in poultry meat

    International Nuclear Information System (INIS)

    Souza, A.R.M. de; Arthur, V.; Canniatti-Brazaca, S.G.

    2007-01-01

    Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 °C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author) [pt

  7. Alteration by irradiation and storage at amount of heme iron in poultry meat

    International Nuclear Information System (INIS)

    Souza, Adriana Regia Marques de; Arthur, Valter Arthur; Canniatti-Brazaca, Solange Guidolin

    2007-01-01

    Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 deg C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author)

  8. Ironing out the Details: Exploring the Role of Iron and Heme in Blood-Sucking Arthropods

    Science.gov (United States)

    Whiten, Shavonn R.; Eggleston, Heather; Adelman, Zach N.

    2018-01-01

    Heme and iron are essential molecules for many physiological processes and yet have the ability to cause oxidative damage such as lipid peroxidation, protein degradation, and ultimately cell death if not controlled. Blood-sucking arthropods have evolved diverse methods to protect themselves against iron/heme-related damage, as the act of bloodfeeding itself is high risk, high reward process. Protective mechanisms in medically important arthropods include the midgut peritrophic matrix in mosquitoes, heme aggregation into the crystalline structure hemozoin in kissing bugs and hemosomes in ticks. Once heme and iron pass these protective mechanisms they are presumed to enter the midgut epithelial cells via membrane-bound transporters, though relatively few iron or heme transporters have been identified in bloodsucking arthropods. Upon iron entry into midgut epithelial cells, ferritin serves as the universal storage protein and transport for dietary iron in many organisms including arthropods. In addition to its role as a nutrient, heme is also an important signaling molecule in the midgut epithelial cells for many physiological processes including vitellogenesis. This review article will summarize recent advancements in heme/iron uptake, detoxification and exportation in bloodfeeding arthropods. While initial strides have been made at ironing out the role of dietary iron and heme in arthropods, much still remains to be discovered as these molecules may serve as novel targets for the control of many arthropod pests. PMID:29387018

  9. Unsaturated Glycerophospholipids Mediate Heme Crystallization: Biological Implications for Hemozoin Formation in the Kissing Bug Rhodnius prolixus

    DEFF Research Database (Denmark)

    Stiebler, R.; Majerowicz, David; Knudsen, Jens

    2014-01-01

    Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membrane...

  10. Spray nozzle pattern test for the DWPF HEME Task QA Plan

    International Nuclear Information System (INIS)

    Lee, L.

    1991-01-01

    The DWPF melter off-gas systems have two High Efficiency Mist Eliminators (HEME) upstream of the High-Efficiency Particulates Air filters (HEPA) to remove fine mists and particulates from the off-gas. To have an acceptable filter life and an efficient operation, an air atomized water is spray on the HEME. The water spray keeps the HEME wet and dissolves the soluble particulates and enhances and HEME efficiency. DWPF Technical asked SRL to determine the conditions which will give satisfactory atomization and distribution of water so that the HEME will operate efficiently. The purpose of this document is to identify, QA controls to be applied in the pursuit of this task (WSRC-RP-91-1151)

  11. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Energy Technology Data Exchange (ETDEWEB)

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  12. Hemoglobin fructation promotes heme degradation through the generation of endogenous reactive oxygen species

    Science.gov (United States)

    Goodarzi, M.; Moosavi-Movahedi, A. A.; Habibi-Rezaei, M.; Shourian, M.; Ghourchian, H.; Ahmad, F.; Farhadi, M.; Saboury, A. A.; Sheibani, N.

    2014-09-01

    Protein glycation is a cascade of nonenzymatic reactions between reducing sugars and amino groups of proteins. It is referred to as fructation when the reducing monosaccharide is fructose. Some potential mechanisms have been suggested for the generation of reactive oxygen species (ROS) by protein glycation reactions in the presence of glucose. In this state, glucose autoxidation, ketoamine, and oxidative advance glycation end products (AGEs) formation are considered as major sources of ROS and perhaps heme degradation during hemoglobin glycation. However, whether fructose mediated glycation produces ROS and heme degradation is unknown. Here we report that ROS (H2O2) production occurred during hemoglobin fructation in vitro using chemiluminescence methods. The enhanced heme exposure and degradation were determined using UV-Vis and fluorescence spectrophotometry. Following accumulation of ROS, heme degradation products were accumulated reaching a plateau along with the detected ROS. Thus, fructose may make a significant contribution to the production of ROS, glycation of proteins, and heme degradation during diabetes.

  13. Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent

    Directory of Open Access Journals (Sweden)

    Christian A. Schaer

    2013-01-01

    Full Text Available The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb, which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM. We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1 response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

  14. Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury

    Science.gov (United States)

    Aggarwal, Saurabh; Lam, Adam; Bolisetty, Subhashini; Carlisle, Matthew A.; Traylor, Amie; Agarwal, Anupam

    2016-01-01

    Abstract Aims: Exposure to irritant gases, such as bromine (Br2), poses an environmental and occupational hazard that results in severe lung and systemic injury. However, the mechanism(s) of Br2 toxicity and the therapeutic responses required to mitigate lung damage are not known. Previously, it was demonstrated that Br2 upregulates the heme degrading enzyme, heme oxygenase-1 (HO-1). Since heme is a major inducer of HO-1, we determined whether an increase in heme and heme-dependent oxidative injury underlies the pathogenesis of Br2 toxicity. Results: C57BL/6 mice were exposed to Br2 gas (600 ppm, 30 min) and returned to room air. Thirty minutes postexposure, mice were injected intraperitoneally with a single dose of the heme scavenging protein, hemopexin (Hx) (3 μg/gm body weight), or saline. Twenty-four hours postexposure, saline-treated mice had elevated total heme in bronchoalveolar lavage fluid (BALF) and plasma and acute lung injury (ALI) culminating in 80% mortality after 10 days. Hx treatment significantly lowered heme, decreased evidence of ALI (lower protein and inflammatory cells in BALF, lower lung wet-to-dry weight ratios, and decreased airway hyperreactivity to methacholine), and reduced mortality. In addition, Br2 caused more severe ALI and mortality in mice with HO-1 gene deletion (HO-1−/−) compared to wild-type controls, while transgenic mice overexpressing the human HO-1 gene (hHO-1) showed significant protection. Innovation: This is the first study delineating the role of heme in ALI caused by Br2. Conclusion: The data suggest that attenuating heme may prove to be a useful adjuvant therapy to treat patients with ALI. Antioxid. Redox Signal. 24, 99–112. PMID:26376667

  15. Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury.

    Science.gov (United States)

    Aggarwal, Saurabh; Lam, Adam; Bolisetty, Subhashini; Carlisle, Matthew A; Traylor, Amie; Agarwal, Anupam; Matalon, Sadis

    2016-01-10

    Exposure to irritant gases, such as bromine (Br2), poses an environmental and occupational hazard that results in severe lung and systemic injury. However, the mechanism(s) of Br2 toxicity and the therapeutic responses required to mitigate lung damage are not known. Previously, it was demonstrated that Br2 upregulates the heme degrading enzyme, heme oxygenase-1 (HO-1). Since heme is a major inducer of HO-1, we determined whether an increase in heme and heme-dependent oxidative injury underlies the pathogenesis of Br2 toxicity. C57BL/6 mice were exposed to Br2 gas (600 ppm, 30 min) and returned to room air. Thirty minutes postexposure, mice were injected intraperitoneally with a single dose of the heme scavenging protein, hemopexin (Hx) (3 μg/gm body weight), or saline. Twenty-four hours postexposure, saline-treated mice had elevated total heme in bronchoalveolar lavage fluid (BALF) and plasma and acute lung injury (ALI) culminating in 80% mortality after 10 days. Hx treatment significantly lowered heme, decreased evidence of ALI (lower protein and inflammatory cells in BALF, lower lung wet-to-dry weight ratios, and decreased airway hyperreactivity to methacholine), and reduced mortality. In addition, Br2 caused more severe ALI and mortality in mice with HO-1 gene deletion (HO-1-/-) compared to wild-type controls, while transgenic mice overexpressing the human HO-1 gene (hHO-1) showed significant protection. This is the first study delineating the role of heme in ALI caused by Br2. The data suggest that attenuating heme may prove to be a useful adjuvant therapy to treat patients with ALI.

  16. Enhanced Heme Function and Mitochondrial Respiration Promote the Progression of Lung Cancer Cells

    Science.gov (United States)

    Alam, Md Maksudul; Shah, Ajit; Cao, Thai M.; Sullivan, Laura A.; Brekken, Rolf; Zhang, Li

    2013-01-01

    Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer

  17. Host heme oxygenase-1: Friend or foe in tackling pathogens?

    Science.gov (United States)

    Singh, Nisha; Ahmad, Zeeshan; Baid, Navin; Kumar, Ashwani

    2018-05-14

    Infectious diseases are a major challenge in management of human health worldwide. Recent literature suggests that host immune system could be modulated to ameliorate the pathogenesis of infectious disease. Heme oxygenase (HMOX1) is a key regulator of cellular signaling and it could be modulated using pharmacological reagents. HMOX1 is a cytoprotective enzyme that degrades heme to generate carbon monoxide (CO), biliverdin, and molecular iron. CO and biliverdin (or bilirubin derived from it) can restrict the growth of a few pathogens. Both of these also induce antioxidant pathways and anti-inflammatory pathways. On the other hand, molecular iron can induce proinflammatory pathway besides making the cellular environment oxidative in nature. Since microbial infections often induce oxidative stress in host cells/tissues, role of HMOX1 has been analyzed in the pathogenesis of number of infections. In this review, we have described the role of HMOX1 in pathogenesis of bacterial infections caused by Mycobacterium species, Salmonella and in microbial sepsis. We have also provided a succinct overview of the role of HMOX1 in parasitic infections such as malaria and leishmaniasis. In the end, we have also elaborated the role of HMOX1 in viral infections such as AIDS, hepatitis, dengue, and influenza. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

  18. Heme and menaquinone induced electron transport in lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Santos Filipe

    2009-05-01

    Full Text Available Abstract Background For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Results Heme- (and menaquinone stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. Conclusion We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species.

  19. The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication.

    Science.gov (United States)

    Hill-Batorski, Lindsay; Halfmann, Peter; Neumann, Gabriele; Kawaoka, Yoshihiro

    2013-12-01

    Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

  20. Heme and menaquinone induced electron transport in lactic acid bacteria.

    Science.gov (United States)

    Brooijmans, Rob; Smit, Bart; Santos, Filipe; van Riel, Jan; de Vos, Willem M; Hugenholtz, Jeroen

    2009-05-29

    For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species.

  1. Energy transfer at the active sites of heme proteins

    International Nuclear Information System (INIS)

    Dlott, D.D.; Hill, J.R.

    1995-01-01

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

  2. Heme oxygenase-1 comes back to endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hong Pyo [School of Biological Sciences, Ulsan University (Korea, Republic of); Pae, Hyun-Ock [Department of Immunology, Wonkwang University School of Medicine (Korea, Republic of); Back, Sung Hun; Chung, Su Wol [School of Biological Sciences, Ulsan University (Korea, Republic of); Woo, Je Moon [Department of Opthalmology, Ulasn University Hospital (Korea, Republic of); Son, Yong [Department of Anesthesiology and Pain Medicine, Wonkwang University School of Medicine (Korea, Republic of); Chung, Hun-Taeg, E-mail: chung@ulsan.ac.kr [School of Biological Sciences, Ulsan University (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  3. Heme oxygenase and carbon monoxide protect from muscle dystrophy.

    Science.gov (United States)

    Chan, Mun Chun; Ziegler, Olivia; Liu, Laura; Rowe, Glenn C; Das, Saumya; Otterbein, Leo E; Arany, Zoltan

    2016-11-28

    Duchenne muscle dystrophy (DMD) is one of the most common lethal genetic diseases of children worldwide and is 100% fatal. Steroids, the only therapy currently available, are marred by poor efficacy and a high side-effect profile. New therapeutic approaches are urgently needed. Here, we leverage PGC-1α, a powerful transcriptional coactivator known to protect against dystrophy in the mdx murine model of DMD, to search for novel mechanisms of protection against dystrophy. We identify heme oxygenase-1 (HO-1) as a potential novel target for the treatment of DMD. Expression of HO-1 is blunted in the muscles from the mdx murine model of DMD, and further reduction of HO-1 by genetic haploinsufficiency worsens muscle damage in mdx mice. Conversely, induction of HO-1 pharmacologically protects against muscle damage. Mechanistically, HO-1 degrades heme into biliverdin, releasing in the process ferrous iron and carbon monoxide (CO). We show that exposure to a safe low dose of CO protects against muscle damage in mdx mice, as does pharmacological treatment with CO-releasing molecules. These data identify HO-1 and CO as novel therapeutic agents for the treatment of DMD. Safety profiles and clinical testing of inhaled CO already exist, underscoring the translational potential of these observations.

  4. Antibiotic suppression of intestinal microbiota reduces heme-induced lipoperoxidation associated with colon carcinogenesis in rats.

    Science.gov (United States)

    Martin, O C B; Lin, C; Naud, N; Tache, S; Raymond-Letron, I; Corpet, D E; Pierre, F H

    2015-01-01

    Epidemiological studies show that heme iron from red meat is associated with increased colorectal cancer risk. In carcinogen-induced-rats, a heme iron-rich diet increases the number of precancerous lesions and raises associated fecal biomarkers. Heme-induced lipoperoxidation measured by fecal thiobarbituric acid reagents (TBARs) could explain the promotion of colon carcinogenesis by heme. Using a factorial design we studied if microbiota could be involved in heme-induced carcinogenesis, by modulating peroxidation. Rats treated or not with an antibiotic cocktail were given a control or a hemoglobin-diet. Fecal bacteria were counted on agar and TBARs concentration assayed in fecal water. The suppression of microbiota by antibiotics was associated with a reduction of crypt height and proliferation and with a cecum enlargement, which are characteristics of germ-free rats. Rats given hemoglobin diets had increased fecal TBARs, which were suppressed by the antibiotic treatment. A duplicate experiment in rats given dietary hemin yielded similar results. These data show that the intestinal microbiota is involved in enhancement of lipoperoxidation by heme iron. We thus suggest that microbiota could play a role in the heme-induced promotion of colorectal carcinogenesis.

  5. Regulation of human heme oxygenase-1 gene expression under thermal stress.

    Science.gov (United States)

    Okinaga, S; Takahashi, K; Takeda, K; Yoshizawa, M; Fujita, H; Sasaki, H; Shibahara, S

    1996-06-15

    Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.

  6. A Heme-Sensing Mechanism in the Translational Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

    Science.gov (United States)

    Soto, Iliana C.; Fontanesi, Flavia; Myers, Richard S.; Hamel, Patrice; Barrientos, Antoni

    2012-01-01

    Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, is a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N-terminus. Using a combination of in vitro and in vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production. PMID:23217259

  7. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    International Nuclear Information System (INIS)

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-01-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and 14 C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of 14 C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose

  8. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    Directory of Open Access Journals (Sweden)

    Christopher M Brennan

    Full Text Available The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA, the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  9. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    International Nuclear Information System (INIS)

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-01-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  10. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    Energy Technology Data Exchange (ETDEWEB)

    Souza, C.F. [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Carneiro, A.B.; Silveira, A.B. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); Laranja, G.A.T. [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); Silva-Neto, M.A.C. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); INCT, Entomologia Molecular (Brazil); Costa, S.C. Goncalves da [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Paes, M.C., E-mail: mcpaes@uerj.br [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); INCT, Entomologia Molecular (Brazil)

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  11. Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

    Science.gov (United States)

    Mouriño, Susana; Rodríguez-Ares, Isabel; Osorio, Carlos R.; Lemos, Manuel L.

    2005-01-01

    The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity. PMID:16332832

  12. Relationship between Antimalarial Activity and Heme Alkylation for Spiro- and Dispiro-1,2,4-Trioxolane Antimalarials▿

    Science.gov (United States)

    Creek, Darren J.; Charman, William N.; Chiu, Francis C. K.; Prankerd, Richard J.; Dong, Yuxiang; Vennerstrom, Jonathan L.; Charman, Susan A.

    2008-01-01

    The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study. PMID:18268087

  13. Heme as a danger molecule in pathogen recognition.

    Science.gov (United States)

    Wegiel, Barbara; Hauser, Carl J; Otterbein, Leo E

    2015-12-01

    Appropriate control of redox mechanisms are critical for and effective innate immune response, which employs multiple cell types, receptors and molecules that recognize danger signals when they reach the host. Recognition of pathogen-associated pattern molecules (PAMPs) is a fundamental host survival mechanism for efficient elimination of invading pathogens and resolution of the infection and inflammation. In addition to PAMPs, eukaryotic cells contain a plethora of intracellular molecules that are normally secured within the confines of the plasma membrane, but if liberated and encountered in the extracellular milieu can provoke rapid cell activation. These are known as Alarmins or Danger-Associated Molecular Patterns (DAMPs) and can be released actively by cells or passively as a result of sterile cellular injury after trauma, ischemia, or toxin-induced cell rupture. Both PAMPs and DAMPs are recognized by a series of cognate receptors that increase the generation of free radicals and activate specific signaling pathways that result in regulation of a variety of stress response, redox sensitive genes. Multiple mediators released, as cells die include, but are not limited to ATP, hydrogen peroxide, heme, formyl peptides, DNA or mitochondria provide the second signal to amplify immune responses. In this review, we will focus on how sterile and infective stimuli activate the stress response gene heme oxygenase-1 (Hmox1, HO-1), a master gene critical to an appropriate host response that is now recognized as one with enormous therapeutic potential. HO-1 gene expression is regulated in large part by redox-sensitive proteins including but not limited to nrf2. Both PAMPs and DAMPs increase the activation of nrf2 and HO-1. Heme is a powerful pro-oxidant and as such should be qualified as a DAMP. With its degradation by HO-1a molecule of carbon monoxide (CO) is generated that in turn serves as a bioactive signaling molecule. PAMPs such as bacterial endotoxin activate HO-1

  14. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

    Science.gov (United States)

    Kathiresan, Meena; Martins, Dorival; English, Ann M.

    2014-01-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

  15. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

    Science.gov (United States)

    Kathiresan, Meena; Martins, Dorival; English, Ann M

    2014-12-09

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

  16. Monomeric, dimeric and multimeric system of RGD peptides radiolabeled with 177Lu for tumors therapy that expressing αβ integrin s

    International Nuclear Information System (INIS)

    Luna G, M. A.

    2014-01-01

    The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target-specific molecular recognition. Peptides based on the cyclic Arg-Gly-Asp (RGD) sequence have been reported as high affinity agents for the α(v)β(3) and α(v)β(5) integrin. The aim of this research was to prepare a multimeric system of 177 Lu-labeled gold nanoparticles conjugated to c[RGDfK(C)] [cyclo(Arg-Gly-Asp-Phe-Lys(Cys)] peptides and to compare the radiation absorbed dose with that of 177 Lu-labeled monomeric and dimeric RGD peptides to α(v)β(3) integrin-positive U87MG tumors in mice, as well as, evaluate the in vitro potential 177 Lu-AuNP-c[RGDfK(C)] as a plasmonic photothermal therapy and targeted radiotherapy system in MCF7 breast cancer cells. DOTA-GGC (1,4,7,10-tetraaza cyclododecane-N,N,N-tetraacetic-Gly-Gly-Cys) and c[RGDfK(C)] peptides were synthesized and conjugated to AuNPs by the spontaneous reaction of the thiol groups. Tem, UV-Vis, XP S, Raman and Far-IR spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. To obtain 177 Lu-AuNP-c[RGDfK(C)], the 177 Lu-DOTA-GGC radio peptide was first prepared and added to a solution of AuNPs followed by c[RGDfK(C)] (25 μL, 5 μM) at 18 grades C for 15 min. 177 Lu-DOTA-GGC, 177 Lu- DOTA-cRGDfK and 177 Lu-DOTA-E-c(RGDfK) 2 were prepared by adding 177 LuCl 3 (370 MBq) to 5 μL (1 mg/ml) of the DOTA derivative diluted with 50 μL of 1 M acetate buffer at ph 5. The mixture was incubated at 90 grades C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. After laser irradiation, the presence of c[RGDfK(C)]-AuNP in cells caused a significant increase in the temperature of the medium (50.5 grades C, compared to 40.3 grades C without AuNPs) resulting in a significant decrease in MCF7 cell viability down to 9 %. After treatment with 177 Lu-AuNP-c[RGDfK(C)], the MCF7 cell proliferation was significantly inhibited

  17. Effects of 1,2-dibromo-3-chloropropane on hepatic heme synthesis

    International Nuclear Information System (INIS)

    Moody, D.E.; Clawson, G.A.; Piper, W.N.; Smuckler, E.A.

    1984-01-01

    Previous studies showed that 1,2-dibromo-3-chloropropane (DBCP) caused a decrease in hepatic microsomal cytochrome P-450 suggesting that hepatic heme metabolism may be affected by DBCP treatment. Various parameters of hepatic heme synthesis were measured at intervals ranging from 0 to 72 hr in male Sprague-Dawley rats given a single oral dose (200 mg/kg) of DBCP. Incorporation of the radiolabeled heme precursor [delta-14C]aminolevulinic acid (14C-ALA) into liver, protein, extracted heme, and subcellular fractions of liver homogenates was significantly decreased to 75, 58, and 81% of controls, respectively, at 24 hr. At 48 and 72 hr after DBCP treatment, the accumulation of 14C-ALA label after 4 hr in liver homogenates and subcellular fractions was significantly increased in comparison to controls. These changes in 14C-ALA uptake were accompanied by decreases in total liver and microsomal heme, but not mitochondrial heme. Decreases were found in the spectral content of two heme proteins, cytochromes P-450 and b5, and the activity of another heme protein, catalase. Heme oxygenase activity increased to 130, 151, 209, and 186% of control values at 12, 24, 48, and 72 hr after DBCP, respectively. A slight, but significant, increase in ALA-synthetase to 112% of controls occurred at 24 hr, and slight, but significant, decreases in ALA-dehydratase to 90 and 80% of control occurred at 12 and 24 hr, respectively. No significant changes in uroporphyrinogen-1-synthetase or ferrochelatase at the time points tested was noted. The porphyrin content of liver was increased to 130% of control, while the serum and urine porphyrin levels were decreased to 30% of the control values at 24 hr. Liver ALA content was not significantly altered through the time period studied, but serum and urine levels were increased at 24 hr to 176 and 130% of the control values, respectively. In conclusion, the decreases in liver heme proteins following a single oral dose of DBCP are accompanied by

  18. Heme oxygenase-1 dependant pathway contributes to protection by tetramethylpyrazine against chronic hypoxic injury on medulla oblongata in rats.

    Science.gov (United States)

    Ding, Yan; Hou, Xuefei; Chen, Li; Zhou, Hua; Gong, Yanju; Dai, Liqun; Zheng, Yu

    2016-02-15

    Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii (Chuan Xiong) has been proved to protect the medulla oblongata from chronic hypoxia injury. However, the underlying mechanism remains unclear. The purpose of this study was to determine whether the protective effects of TMP are associated with the heme oxygenase-1 (HO-1) dependant pathway in adult rats. The morphological changes of neurons in the hypoglossal nucleus (12N), the nucleus ambiguus (Amb), the nucleus tractus solitarius (NTS), and the pre-Bötzinger complex (pre-BötC) were investigated by Nissl staining; the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured to evaluate the anti-oxidant effect; some apoptosis parameters, Bax mRNA and Bcl-2 mRNA, were tested; and the double immunochemistry staining of active caspase-3/NeuN was performed. Meanwhile, the HO-1 protein expression and heme oxygenase (HO) activity were examined. Tin-protoporphyrin (SnPP), a potent inhibitor of HO, was used to further confirm the effect of HO-1. We found that TMP ameliorated the neuron loss in 12N, Amb and NTS, the decrease in SOD activity and the increase in MDA content, the decrease in Bcl-2 mRNA of medulla oblongata (Pmedulla oblongata in the rats. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Selective activation of heme oxygenase-2 by menadione.

    Science.gov (United States)

    Vukomanovic, Dragic; McLaughlin, Brian E; Rahman, Mona N; Szarek, Walter A; Brien, James F; Jia, Zongchao; Nakatsu, Kanji

    2011-11-01

    While substantial progress has been made in elucidating the roles of heme oxygenases-1 (HO-1) and -2 (HO-2) in mammals, our understanding of the functions of these enzymes in health and disease is still incomplete. A significant amount of our knowledge has been garnered through the use of nonselective inhibitors of HOs, and our laboratory has recently described more selective inhibitors for HO-1. In addition, our appreciation of HO-1 has benefitted from the availability of tools for increasing its activity through enzyme induction. By comparison, there is a paucity of information about HO-2 activation, with only a few reports appearing in the literature. This communication describes our observations of the up to 30-fold increase in the in-vitro activation of HO-2 by menadione. This activation was due to an increase in Vmax and was selective, in that menadione did not increase HO-1 activity.

  20. Pilot-scale tests of HEME and HEPA dissolution process

    International Nuclear Information System (INIS)

    Qureshi, Z.H.; Strege, D.K.

    1994-06-01

    A series of pilot-scale demonstration tests for the dissolution of High Efficiency Mist Eliminators (HEME's) and High Efficiency Particulate Airfilters (HEPA) were performed on a 1/5th linear scale. These fiberglass filters are to be used in the Defense Waste Processing Facility (DWPF) to decontaminate the effluents from the off-gases generated during the feed preparation process and vitrification. When removed, these filters will be dissolved in the Decontamination Waste Treatment Tank (DWTT) using 5 wt% NaOH solution. The contaminated fiberglass is converted to an aqueous stream which will be transferred to the waste tanks. The filter metal structure will be rinsed with process water before its disposal as low-level solid waste. The pilot-scale study reported here successfully demonstrated a simple one step process using 5 wt% NaOH solution. The proposed process requires the installation of a new water spray ring with 30 nozzles. In addition to the reduced waste generated, the total process time is reduced to 48 hours only (66% saving in time). The pilot-scale tests clearly demonstrated that the dissolution process of HEMEs has two stages - chemical digestion of the filter and mechanical erosion of the digested filter. The digestion is achieved by a boiling 5 wt% caustic solutions, whereas the mechanical break down of the digested filter is successfully achieved by spraying process water on the digested filter. An alternate method of breaking down the digested filter by increased air sparging of the solution was found to be marginally successful are best. The pilot-scale tests also demonstrated that the products of dissolution are easily pumpable by a centrifugal pump

  1. Peroxide-Dependent Analyte Conversion by the Heme Prosthetic Group, the Heme Peptide “Microperoxidase-11” and Cytochrome c on Chitosan Capped Gold Nanoparticles Modified Electrodes

    Directory of Open Access Journals (Sweden)

    Frieder W. Scheller

    2012-05-01

    Full Text Available In view of the role ascribed to the peroxidatic activity of degradation products of cytochrome c (cyt c in the processes of apoptosis, we investigate the catalytic potential of heme and of the cyt c derived heme peptide MP-11 to catalyse the cathodic reduction of hydrogen peroxide and to oxidize aromatic compounds. In order to check whether cyt c has an enzymatic activity in the native state where the protein matrix should suppress the inherent peroxidatic activity of its heme prosthetic group, we applied a biocompatible immobilization matrix and very low concentrations of the co-substrate H2O2. The biocatalysts were entrapped on the surface of a glassy carbon electrode in a biocompatible chitosan layer which contained gold nanoparticles. The electrochemical signal for the peroxide reduction is generated by the redox conversion of the heme group, whilst a reaction product of the substrate oxidation is cathodically reduced in the substrate indication. The catalytic efficiency of microperoxidase-11 is sufficient for sensors indicating HRP substrates, e.g., p-aminophenol, paracetamol and catechol, but also the hydroxylation of aniline and dehalogenation of 4-fluoroaniline. The lower limit of detection for p-aminophenol is comparable to previously published papers with different enzyme systems. The peroxidatic activity of cyt c immobilized in the chitosan layer for catechol was found to be below 1 per mill and for p-aminophenol about 3% as compared with that of heme or MP-11.

  2. Identification and phylogenetic analysis of heme synthesis genes in trypanosomatids and their bacterial endosymbionts.

    Directory of Open Access Journals (Sweden)

    João M P Alves

    Full Text Available It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellate's cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.

  3. Analysis of the electrochemistry of hemes with Ems spanning 800 mV

    Science.gov (United States)

    Zheng, Zhong; Gunner, M. R.

    2009-01-01

    The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of Ems with a single type of redox cofactor. Proteins containing 141 unique hemes of a-, b-, and c-type, with bis-His, His-Met, and aquo-His ligation were calculated using Multi-Conformation Continuum Electrostatics (MCCE). The experimental Ems range over 800 mV from −350 mV in cytochrome c3 to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the protein backbone, and sidechains. MCCE calculated Ems are in good agreement with measured values. Using no free parameters the slope of the line comparing calculated and experimental Ems is 0.73 (R2 = 0.90), showing the method accounts for 73% of the observed Em range. Adding a +160 mV correction to the His-Met c-type hemes yields a slope of 0.97 (R2 = 0.93). With the correction 65% of the hemes have an absolute error smaller than 60 mV and 92% are within 120 mV. The overview of heme proteins with known structures and Ems shows both the lowest and highest potential hemes are c-type, whereas the b-type hemes are found in the middle Em range. In solution, bis-His ligation lowers the Em by ≈205 mV relative to hemes with His-Met ligands. The bis-His, aquo-His, and His-Met ligated b-type hemes all cluster about Ems which are ≈200 mV more positive in protein than in water. In contrast, the low potential bis-His c-type hemes are shifted little from in solution, whereas the high potential His-Met c-type hemes are raised by ≈300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. For example, the loss of solvation (reaction field) energy, which raises the Em, has been suggested to be a major factor in

  4. Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

    International Nuclear Information System (INIS)

    Ramos-Santana, Brenda J.; López-Garriga, Juan

    2012-01-01

    Highlights: ► H-bonding network loop by PheB10Tyr mutation is proposed. ► The propionate group H-bonding network restricted the flexibility of the heme. ► The hydrogen bonding interaction modulates the electron density of the iron. ► Propionate H-bonding network loop explains the heme-ligand stabilization. -- Abstract: Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. 1 H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00 ppm, GlnE7 N ε1 H/N ε2 H at 10.66 ppm/−3.27 ppm, and PheE11 C δ H at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen

  5. Effects of Zinc Deuteroporphyrin Bis Glycol on Newborn Mice After Heme-Loading

    OpenAIRE

    He, Cynthia X.; Campbell, Claire M.; Zhao, Hui; Kalish, Flora S.; Schulz, Stephanie; Vreman, Hendrik J.; Wong, Ronald J.; Stevenson, David K.

    2011-01-01

    Infants with hemolytic diseases frequently develop hyperbilirubinemia, but standard phototherapy only eliminates bilirubin after its production. A better strategy might be to directly inhibit heme oxygenase (HO), the rate-limiting enzyme in bilirubin production. Metalloporphyrins (Mps) are heme analogs that competitively inhibit HO activity in vitro and in vivo and suppress plasma bilirubin levels in vivo. A promising Mp, zinc deuteroporphyrin bis glycol (ZnBG), is orally absorbed and effecti...

  6. In vitro Activation of heme oxygenase-2 by menadione and its analogs.

    Science.gov (United States)

    Vukomanovic, Dragic; Rahman, Mona N; Bilokin, Yaroslav; Golub, Andriy G; Brien, James F; Szarek, Walter A; Jia, Zongchao; Nakatsu, Kanji

    2014-02-18

    Previously, we reported that menadione activated rat, native heme oxygenase-2 (HO-2) and human recombinant heme oxygenase-2 selectively; it did not activate spleen, microsomal heme oxygenase-1. The purpose of this study was to explore some structure-activity relationships of this activation and the idea that redox properties may be an important aspect of menadione efficacy. Heme oxygenase activity was determined in vitro using rat spleen and brain microsomes as the sources of heme oxygenase-1 and -2, respectively, as well as recombinant, human heme oxygenase-2. Menadione analogs with bulky aliphatic groups at position-3, namely vitamins K1 and K2, were not able to activate HO-2. In contrast, several compounds with similar bulky but less lipophilic moieties at position-2 (and -3) were able to activate HO-2 many fold; these compounds included polar, rigid, furan-containing naphthoquinones, furan-benzoxazine naphthoquinones, 2-(aminophenylphenyl)-3-piperidin-1-yl naphthoquinones. To explore the idea that redox properties might be involved in menadione efficacy, we tested analogs such as 1,4-dimethoxy-2-methylnaphthalene, pentafluoromenadione, monohalogenated naphthoquinones, α-tetralone and 1,4-naphthoquinone. All of these compounds were inactive except for 1,4-naphthoquinone. Menadione activated full-length recombinant human heme oxygenase-2 (FL-hHO-2) as effectively as rat brain enzyme, but it did not activate rat spleen heme oxygenase. These observations are consistent with the idea that naphthoquinones such as menadione bind to a receptor in HO-2 and activate the enzyme through a mechanism that may involve redox properties.

  7. Control of heme synthesis during Friend cell differentiation: role of iron and transferrin

    International Nuclear Information System (INIS)

    Laskey, J.D.; Ponka, P.; Schulman, H.M.

    1986-01-01

    In many types of cells the synthesis of σ-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from this laboratory with reticulocytes suggest that the rate of iron uptake from 125 I-transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels or 59 Fe-Tf (20 μM). Furthermore, in induced Friend cells 100 μM Fe-SIH stimulated 2- 14 C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells

  8. Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state.

    Science.gov (United States)

    Kiger, Laurent; Vasseur, Corinne; Domingues-Hamdi, Elisa; Truan, Gilles; Marden, Michael C; Baudin-Creuza, Véronique

    2014-01-01

    AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7). Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy. AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer. α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis. The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia. © 2013 Elsevier B.V. All rights reserved.

  9. Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

    Directory of Open Access Journals (Sweden)

    Carlo Travaglini-Allocatelli

    2013-01-01

    Full Text Available Cytochromes c (Cyt c are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i heme translocation and delivery, (ii apoCyt thioreductive pathway, and (iii apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria.

  10. Multi-heme Cytochromes in Shewanella oneidensis MR-1: Structures, functions and opportunities

    Energy Technology Data Exchange (ETDEWEB)

    Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen; Butt, Julea N.

    2014-11-05

    Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies.

  11. Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia.

    Science.gov (United States)

    Lunetti, Paola; Damiano, Fabrizio; De Benedetto, Giuseppe; Siculella, Luisa; Pennetta, Antonio; Muto, Luigina; Paradies, Eleonora; Marobbio, Carlo Marya Thomas; Dolce, Vincenza; Capobianco, Loredana

    2016-09-16

    Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Coordination modes of tyrosinate-ligated catalase-type heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states.

    Science.gov (United States)

    Bandara, D M Indika; Sono, Masanori; Bruce, Grant S; Brash, Alan R; Dawson, John H

    2011-12-01

    Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Heme-coordinated histidine residues form non-specific functional "ferritin-heme" peroxidase system: Possible and partial mechanistic relevance to oxidative stress-mediated pathology in neurodegenerative diseases.

    Science.gov (United States)

    Esmaeili, Sajjad; Kooshk, Mohammad Reza Ashrafi; Asghari, Seyyed Mohsen; Khodarahmi, Reza

    2016-10-01

    Ferritin is a giant protein composed of 24 subunits which is able to sequester up to 4500 atoms of iron. We proposed two kinds of heme binding sites in mammalian ferritins and provided direct evidence for peroxidase activity of heme-ferritin, since there is the possibility that "ferritin-heme" systems display unexpected catalytic behavior like heme-containing enzymes. In the current study, peroxidase activity of heme-bound ferritin was studied using TMB(1), l-DOPA, serotonin, and dopamine, in the presence of H2O2, as oxidant substrate. The catalytic oxidation of TMB was consistent with first-order kinetics with respect to ferritin concentration. Perturbation of the binding affinity and catalytic behavior of heme-bound His-modified ferritin were also documented. We also discuss the importance of the peroxidase-/nitrative-mediated oxidation of vital molecules as well as ferritin-induced catalase inhibition using in vitro experimental system. Uncontrollable "heme-ferritin"-based enzyme activity as well as up-regulation of heme and ferritin may inspire that some oxidative stress-mediated cytotoxic effects in AD-affected cells could be correlated to ferritin-heme interaction and/or ferritin-induced catalase inhibition and describe its contribution as an important causative pathogenesis mechanism in some neurodegenerative disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Rapid, convenient method for screening imidazole-containing compounds for heme oxygenase inhibition.

    Science.gov (United States)

    Vlahakis, Jason Z; Rahman, Mona N; Roman, Gheorghe; Jia, Zongchao; Nakatsu, Kanji; Szarek, Walter A

    2011-01-01

    Sensitive assays for measuring heme oxygenase activity have been based on the gas-chromatographic detection of carbon monoxide using elaborate, expensive equipment. The present study describes a rapid and convenient method for screening imidazole-containing candidates for inhibitory activity against heme oxygenase using a plate reader, based on the spectroscopic evaluation of heme degradation. A PowerWave XS plate reader was used to monitor the absorbance (as a function of time) of heme bound to purified truncated human heme oxygenase-1 (hHO-1) in the individual wells of a standard 96-well plate (with or without the addition of a test compound). The degradation of heme by heme oxygenase-1 was initiated using l-ascorbic acid, and the collected relevant absorbance data were analyzed by three different methods to calculate the percent control activity occurring in wells containing test compounds relative to that occurring in control wells with no test compound present. In the cases of wells containing inhibitory compounds, significant shifts in λ(max) from 404 to near 412 nm were observed as well as a decrease in the rate of heme degradation relative to that of the control. Each of the three methods of data processing (overall percent drop in absorbance over 1.5h, initial rate of reaction determined over the first 5 min, and estimated pseudo first-order reaction rate constant determined over 1.5h) gave similar and reproducible results for percent control activity. The fastest and easiest method of data analysis was determined to be that using initial rates, involving data acquisition for only 5 min once reactions have been initiated using l-ascorbic acid. The results of the study demonstrate that this simple assay based on the spectroscopic detection of heme represents a rapid, convenient method to determine the relative inhibitory activity of candidate compounds, and is useful in quickly screening a series or library of compounds for heme oxygenase inhibition

  15. Binding of Pseudomonas aeruginosa Apo-Bacterioferritin Associated Ferredoxin to Bacterioferritin B Promotes Heme Mediation of Electron Delivery and Mobilization of Core Mineral Iron†

    Science.gov (United States)

    Weeratunga, Saroja K.; Gee, Casey E.; Lovell, Scott; Zeng, Yuhong; Woodin, Carrie L.; Rivera, Mario

    2009-01-01

    The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5–10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) Mol. Microbiol. 45, 1277–1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to

  16. Serum heme oxygenase-1 levels in patients with primary dysmenorrhea.

    Science.gov (United States)

    Aksoy, Ayse Nur; Laloglu, Esra; Ozkaya, Alev Lazoglu; Yilmaz, Emsal Pınar Topdagi

    2017-04-01

    Primary dysmenorrhea effects the life-quality of women negatively. The aim of this study was to evaluate heme oxygenase-1 (HO1) activity together with malondialdehyde (MDA) and nitric oxide (NO) levels in patients with primary dysmenorrhea. A total of 28 nulliparous women with the diagnosis of primary dysmenorrhea and 26 healthy controls were included in this study. On the first day of menstruation, all patients underwent ultrasound examination to exclude pelvic pathology and the visual analogue scale was applied to patients. Patient's visual analogue scale (VAS) scores, age, body mass index (BMI), menstrual cycle length (day), length of bleeding (day) were recorded. In the same day, fasting blood samples were taken from each patient for biochemical analysis. Serum MDA, NO and HO1 levels were found to be higher in women with primary dysmenorrhea compared to healthy controls (p = 0.012, p = 0.009, p dysmenorrhea. Antioxidant support might be helpful to reduce pain severity in primary dysmenorrhea.

  17. A product of heme catabolism modulates bacterial function and survival.

    Directory of Open Access Journals (Sweden)

    Christopher L Nobles

    Full Text Available Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC, a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome and Gram-positive bacteria being susceptible to membrane disruption (negative outcome. This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.

  18. Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis.

    Science.gov (United States)

    Chang, Ling-Chu; Chiang, Shih-Kai; Chen, Shuen-Ei; Yu, Yung-Luen; Chou, Ruey-Hwang; Chang, Wei-Chao

    2018-03-01

    Ferroptosis is a form of oxidative cell death and has become a chemotherapeutic target for cancer treatment. BAY 11-7085 (BAY), which is a well-known IκBα inhibitor, suppressed viability in cancer cells via induction of ferroptotic death in an NF-κB-independent manner. Reactive oxygen species scavenging, relief of lipid peroxidation, replenishment of glutathione and thiol-containing agents, as well as iron chelation, rescued BAY-induced cell death. BAY upregulated a variety of Nrf2 target genes related to redox regulation, particularly heme oxygenase-1 (HO-1). Studies with specific inhibitors and shRNA interventions suggested that the hierarchy of induction is Nrf2-SLC7A11-HO-1. SLC7A11 inhibition by erastin, sulfasalazine, or shRNA interference sensitizes BAY-induced cell death. Overexperession of SLC7A11 attenuated BAY-inhibited cell viability. The ferroptotic process induced by hHO-1 overexpression further indicated that HO-1 is a key mediator of BAY-induced ferroptosis that operates through cellular redox regulation and iron accumulation. BAY causes compartmentalization of HO-1 into the nucleus and mitochondrion, and followed mitochondrial dysfunctions, leading to lysosome targeting for mitophagy. In this study, we first discovered that BAY induced ferroptosis via Nrf2-SLC7A11-HO-1 pathway and HO-1 is a key mediator by responding to the cellular redox status. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Increased Plasma Levels of Heme Oxygenase-1 in Human Brucellosis.

    Science.gov (United States)

    Chen, Zhe; Zhang, Yu-Xue; Fu, Dong-Wei; Gao, Qing-Feng; Ge, Feng-Xia; Liu, Wei-Hua

    2016-08-01

    Brucellosis is associated with inflammation and the oxidative stress response. Heme oxygenase-1 (HO-1) is a cytoprotective stress-responsive enzyme that has anti-inflammatory and anti-oxidant effects. Nevertheless, the role of HO-1 in human brucellosis has not yet been studied. The aim of this study was to examine the plasma levels of HO-1 in patients with brucellosis and to evaluate the ability of plasma HO-1 levels as an auxiliary diagnosis, a severity predictor, and a monitor for brucellosis treatments. A total of 75 patients with brucellosis were divided into the acute, subacute, chronic active, and chronic stable groups. An additional 20 volunteers were included as the healthy control group. The plasma HO-1 levels and other laboratory parameters were measured in all groups. Furthermore, the plasma levels of HO-1 in the acute group were compared before and after treatment. The plasma HO-1 levels were considerably increased in the acute (4.97 ± 3.55), subacute (4.98 ± 3.23), and chronic active groups (4.43 ± 3.00) with brucellosis compared to the healthy control group (1.03 ± 0.63) (p brucellosis (r = 0.707, p brucellosis status and may be used as a supplementary plasma marker for diagnosing brucellosis and monitoring its treatment.

  20. Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity.

    Directory of Open Access Journals (Sweden)

    Padmamalini Baskaran

    Full Text Available Nitric oxide signals through activation of soluble guanylyl cyclase (sGC, a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain to the effector domain (catalytic domain, in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105 of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

  1. Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Imperi, Francesco; Coletta, Massimo; Fasano, Mauro

    2008-01-01

    Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k off ) is reported. In the absence of drugs, the value of k off is (1.3 ± 0.2) x 10 -4 s -1 . Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k off value increases to (8.6 ± 0.9) x 10 -4 s -1 . From the dependence of k off on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 ± 0.2) x 10 -3 M and (6.2 ± 0.7) x 10 -5 M, respectively) were determined. The increase of k off values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow's site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors

  2. Optimizing the relaxivity of GdIII complexes appended to InP/ZnS quantum dots by linker tuning.

    Science.gov (United States)

    Stasiuk, Graeme J; Tamang, Sudarsan; Imbert, Daniel; Gateau, Christelle; Reiss, Peter; Fries, Pascal; Mazzanti, Marinella

    2013-06-21

    Three bimodal MRI/optical nanosized contrast agents with high per-nanoparticle relaxivity (up to 2523 mM(-1) s(-1) at 35 MHz and 932 mM(-1) s(-1) at 200 MHz) have been prepared connecting up to 115 tris-aqua Gd(III) complexes to fluorescent non-toxic InP/ZnS quantum dots. The structure of the linker has an important effect on the relaxivity of the final multimeric contrast agent.

  3. Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure

    Czech Academy of Sciences Publication Activity Database

    Yang, F.; Fojtíková, V.; Man, Petr; Stráňava, M.; Martínková, M.; Du, Y.; Huang, D.; Shimizu, T.

    2015-01-01

    Roč. 28, č. 4 (2015), s. 637-652 ISSN 0966-0844 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : Heme * O-2 sensor * Phosphodiesterase Subject RIV: CE - Biochemistry Impact factor: 2.134, year: 2015

  4. Oxidative stability of a heme iron-fortified bakery product: Effectiveness of ascorbyl palmitate and co-spray-drying of heme iron with calcium caseinate.

    Science.gov (United States)

    Alemán, Mercedes; Bou, Ricard; Tres, Alba; Polo, Javier; Codony, Rafael; Guardiola, Francesc

    2016-04-01

    Fortification of food products with iron is a common strategy to prevent or overcome iron deficiency. However, any form of iron is a pro-oxidant and its addition will cause off-flavours and reduce a product's shelf life. A highly bioavailable heme iron ingredient was selected to fortify a chocolate cream used to fill sandwich-type cookies. Two different strategies were assessed for avoiding the heme iron catalytic effect on lipid oxidation: ascorbyl palmitate addition and co-spray-drying of heme iron with calcium caseinate. Oxidation development and sensory acceptability were monitored in the cookies over one-year of storage at room temperature in the dark. The addition of ascorbyl palmitate provided protection against oxidation and loss of tocopherols and tocotrienols during the preparation of cookies. In general, ascorbyl palmitate, either alone or in combination with the co-spray-dried heme iron, prevented primary oxidation and hexanal formation during storage. The combination of both strategies resulted in cookies that were acceptable from a sensory point of view after 1year of storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

    Directory of Open Access Journals (Sweden)

    Flavio Alves Lara

    Full Text Available In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA, a well-known inhibitor of ATP binding cassette (ABC transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may

  6. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells

    Science.gov (United States)

    Lara, Flavio Alves; Pohl, Paula C.; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H. F.; Almeida, Igor C.; Vaz, Itabajara da Silva; Oliveira, Pedro L.

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  7. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

  8. Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ.

    Science.gov (United States)

    Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović-Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G; Oostenbrink, Chris; Obinger, Christian

    2016-09-27

    Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of -205 ± 3 mV for LmHemQ and -207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 10(4) M(-1) s(-1)). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria.

  9. A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans*

    Science.gov (United States)

    Molitor, Bastian; Stassen, Marc; Modi, Anuja; El-Mashtoly, Samir F.; Laurich, Christoph; Lubitz, Wolfgang; Dawson, John H.; Rother, Michael; Frankenberg-Dinkel, Nicole

    2013-01-01

    Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor). PMID:23661702

  10. Histidine at Position 195 is Essential for Association of Heme- b in Lcp1VH2

    Science.gov (United States)

    Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

    2018-05-01

    The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12 , 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly( cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme- b in the central region of Lcp1VH2.

  11. Thioredoxin Cross-Linking by Nitrogen Mustard in Lung Epithelial Cells: Formation of Multimeric Thioredoxin/Thioredoxin Reductase Complexes and Inhibition of Disulfide Reduction

    OpenAIRE

    Jan, Yi-Hua; Heck, Diane E.; Casillas, Robert P.; Laskin, Debra L.; Laskin, Jeffrey D.

    2015-01-01

    The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Tr...

  12. An increase in plasma adiponectin multimeric complexes follows hypocaloric diet-induced weight loss in obese and overweight pre-menopausal women

    Czech Academy of Sciences Publication Activity Database

    Polák, J.; Kováčová, Z.; Jacek, M.; Klimčáková, E.; Kováčiková, M.; Vítková, M.; Kuda, Ondřej; Šebela, M.; Samcová, E.; Štich, V.

    2007-01-01

    Roč. 112, č. 11 (2007), s. 557-565 ISSN 0143-5221 Institutional research plan: CEZ:AV0Z50110509 Keywords : adiponectin * adipose tissue * dietary intervention Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 3.900, year: 2007

  13. Therapeutic Roles of Heme Oxygenase-1 in Metabolic Diseases: Curcumin and Resveratrol Analogues as Possible Inducers of Heme Oxygenase-1

    Directory of Open Access Journals (Sweden)

    Yong Son

    2013-01-01

    Full Text Available Metabolic diseases, such as insulin resistance, type II diabetes, and obesity, are associated with a low-grade chronic inflammation (inflammatory stress, oxidative stress, and endoplasmic reticulum (ER stress. Because the integration of these stresses is critical to the pathogenesis of metabolic diseases, agents and cellular molecules that can modulate these stress responses are emerging as potential targets for intervention and treatment of metabolic diseases. It has been recognized that heme oxygenase-1 (HO-1 plays an important role in cellular protection. Because HO-1 can reduce inflammatory stress, oxidative stress, and ER stress, in part by exerting antioxidant, anti-inflammatory, and antiapoptotic effects, HO-1 has been suggested to play important roles in pathogenesis of metabolic diseases. In the present review, we will explore our current understanding of the protective mechanisms of HO-1 in metabolic diseases and present some emerging therapeutic options for HO-1 expression in treating metabolic diseases, together with the therapeutic potential of curcumin and resveratrol analogues that have their ability to induce HO-1 expression.

  14. Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

    Science.gov (United States)

    Chiabrando, Deborah; Castori, Marco; di Rocco, Maja; Ungelenk, Martin; Gießelmann, Sebastian; Di Capua, Matteo; Madeo, Annalisa; Grammatico, Paola; Bartsch, Sophie; Hübner, Christian A; Altruda, Fiorella; Silengo, Lorenzo; Tolosano, Emanuela; Kurth, Ingo

    2016-12-01

    Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.

  15. Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

    Directory of Open Access Journals (Sweden)

    Deborah Chiabrando

    2016-12-01

    Full Text Available Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs. Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1 gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.

  16. Clinically Important Features of Porphyrin and Heme Metabolism and the Porphyrias

    Directory of Open Access Journals (Sweden)

    Siddesh Besur

    2014-11-01

    Full Text Available Heme, like chlorophyll, is a primordial molecule and is one of the fundamental pigments of life. Disorders of normal heme synthesis may cause human diseases, including certain anemias (X-linked sideroblastic anemias and porphyrias. Porphyrias are classified as hepatic and erythropoietic porphyrias based on the organ system in which heme precursors (5-aminolevulinic acid (ALA, porphobilinogen and porphyrins are chiefly overproduced. The hepatic porphyrias are further subdivided into acute porphyrias and chronic hepatic porphyrias. The acute porphyrias include acute intermittent, hereditary copro-, variegate and ALA dehydratase deficiency porphyria. Chronic hepatic porphyrias include porphyria cutanea tarda and hepatoerythropoietic porphyria. The erythropoietic porphyrias include congenital erythropoietic porphyria (Gűnther’s disease and erythropoietic protoporphyria. In this review, we summarize the key features of normal heme synthesis and its differing regulation in liver versus bone marrow. In both organs, principal regulation is exerted at the level of the first and rate-controlling enzyme, but by different molecules (heme in the liver and iron in the bone marrow. We also describe salient clinical, laboratory and genetic features of the eight types of porphyria.

  17. Proton NMR investigation of heme pocket mobility in hemoglobin via hydrogen isotope exchange kinetics

    International Nuclear Information System (INIS)

    Han, K.

    1985-01-01

    Dynamic mobility of heme cavity, the active site of Hb, was investigated by analyzing the hydrogen isotope exchange kinetics of the proximal histidyl ring NH of various kinds of Hbs with the aid of the high field Fourier Transform 1 H NMR spectroscopy. The exchange reaction occurs faster in oxy or R-state Hb than in deoxy or T-state Hb and there exists a good correlation between the oxygen affinity of Hb and the heme pocket mobility reflected in the hydrogen exchange rate. The effect of pH on the exchange is dramatically different for the two subunits of Hb A. Studying the exchange characteristics of mutant Hbs and chemically modified Hbs not only showed the existence of three well-defined localized paths for transmission of conformational changes between different heme pockets through a 1 b 2 subunit interface, but also indicated that the heme pocket mobility is regulated by the quaternary state of Hb as well as by the ligation state of Hb. Finally, the effect of the quaternary state on the heme pocket mobility is separated from that of the ligation by following the exchange reactions in Hbs where only their quaternary structure transition can be achieved without changing their ligation states by adjusting experimental conditions such as adding inositol hexaphosphate

  18. Generation and characterization of human heme oxygenase-1 transgenic pigs.

    Directory of Open Access Journals (Sweden)

    Hye-Jung Yeom

    Full Text Available Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1, an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs. Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

  19. Generation and characterization of human heme oxygenase-1 transgenic pigs.

    Science.gov (United States)

    Yeom, Hye-Jung; Koo, Ok Jae; Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J; Kim, Hyunil; Surh, Charles D; Lee, Byeong-Chun; Ahn, Curie

    2012-01-01

    Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

  20. Heme oxygenase behavior in ultraviolet-B irradiated soybean plants

    International Nuclear Information System (INIS)

    Yannarelli, G.G.; Noriega, G.O.; Tomaro, M.L.

    2005-01-01

    Full text: Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and leads to the generation of reactive oxygen species (ROS). Heme oxygenase (HO) plays a protective role against oxidative stress in mammals, but little is known about this issue in plants. Here, we report for the first time the response of HO in leaves of soybean plants subjected to UV-B radiation. HO activity, protein and gene expression, as well as stress markers were evaluated. Under lower UV-B doses (7.5 and 15 kJ m -2 ), the production of thiobarbituric acid reactive substances (TBARS) remained unaltered, while quantitative RT-PCR revealed that HO and catalase (CAT) transcripts were increased 40% and 20% after 8 h, respectively. Treatment with 30 kJ m -2 brought about a 90% enhancement in TBARS indicating that an oxidative burst occurred, and a downregulation in gene expression was observed. Immunoblot analysis showed a 4.3 and 3.7-fold increase in HO protein after irradiation with 75 and 15 kJ m -2 , respectively. HO and CAT enzymes activities were enhanced at these doses but diminished at 30 kJ m -2 UV-B. These results indicate that the up regulation of HO and CAT genes at the lower doses occurred as a signal of cell protection against oxidative damage. On the other hand, irradiation with 30 kJ m -2 overcome the cellular antioxidant capacity and repressed the response as a result of ROS overproduction. (author)

  1. Dietary heme injures surface epithelium resulting in hyperproliferation, inhibition of apoptosis and crypt hyperplasia in rat colon

    NARCIS (Netherlands)

    de Vogel, Johan; van-Eck, Wytske Boersma; Sesink, Aloys L. A.; Jonker-Termont, Denise S. M. L.; Kleibeuker, Jan; van der Meer, Roelof

    Epidemiological and animal model studies suggest that a high intake of heme, present in red meat, is associated with an increased risk of colon cancer. The aim of this study was to elucidate the effects of dietary heme on colonic cell homeostasis in rats. Rats were fed a purified, humanized, control

  2. Heme oxygenase is not involved in the anti-proliferative effects of statins on pancreatic cancer cells

    Czech Academy of Sciences Publication Activity Database

    Váňová, K.; Boukalová, Štěpána; Gbelcová, H.; Muchová, L.; Neužil, Jiří; Gürlich, R.; Ruml, T.; Vítek, L.

    2016-01-01

    Roč. 16, May 12 (2016), č. článku 309. ISSN 1471-2407 R&D Projects: GA MZd NT14078; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : Heme * Heme oxygenase * Pancreatic cancer * Statins Subject RIV: FD - Oncology ; Hematology Impact factor: 3.288, year: 2016

  3. Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

    DEFF Research Database (Denmark)

    Farver, Ole; Chen, Ying; Fee, James A

    2006-01-01

    The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and P...

  4. The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen

    DEFF Research Database (Denmark)

    Saederup, Kirstine Lindhardt; Stødkilde-Jørgensen, Kristian; Graversen, Jonas Heilskov

    2016-01-01

    Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd...

  5. Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus.

    Directory of Open Access Journals (Sweden)

    Renata Stiebler

    Full Text Available Hemozoin (Hz is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM. Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE and phosphatidylcholine (uPC, with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

  6. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    Science.gov (United States)

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-09

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

  7. Identification of the heme acquisition system in Vibrio vulnificus M2799.

    Science.gov (United States)

    Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Yamahata, Eri; Yamaguchi, Ryo; Miyaki, Yuta; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Tsujibo, Hiroshi

    2018-04-01

    Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Heme Oxygenase-1/Carbon Monoxide System and Embryonic Stem Cell Differentiation and Maturation into Cardiomyocytes

    Science.gov (United States)

    Suliman, Hagir B.; Zobi, Fabio

    2016-01-01

    Abstract Aims: The differentiation of embryonic stem (ES) cells into energetically efficient cardiomyocytes contributes to functional cardiac repair and is envisioned to ameliorate progressive degenerative cardiac diseases. Advanced cell maturation strategies are therefore needed to create abundant mature cardiomyocytes. In this study, we tested whether the redox-sensitive heme oxygenase-1/carbon monoxide (HO-1/CO) system, operating through mitochondrial biogenesis, acts as a mechanism for ES cell differentiation and cardiomyocyte maturation. Results: Manipulation of HO-1/CO to enhance mitochondrial biogenesis demonstrates a direct pathway to ES cell differentiation and maturation into beating cardiomyocytes that express adult structural markers. Targeted HO-1/CO interventions up- and downregulate specific cardiogenic transcription factors, transcription factor Gata4, homeobox protein Nkx-2.5, heart- and neural crest derivatives-expressed protein 1, and MEF2C. HO-1/CO overexpression increases cardiac gene expression for myosin regulatory light chain 2, atrial isoform, MLC2v, ANP, MHC-β, and sarcomere α-actinin and the major mitochondrial fusion regulators, mitofusin 2 and MICOS complex subunit Mic60. This promotes structural mitochondrial network expansion and maturation, thereby supporting energy provision for beating embryoid bodies. These effects are prevented by silencing HO-1 and by mitochondrial reactive oxygen species scavenging, while disruption of mitochondrial biogenesis and mitochondrial DNA depletion by loss of mitochondrial transcription factor A compromise infrastructure. This leads to failure of cardiomyocyte differentiation and maturation and contractile dysfunction. Innovation: The capacity to augment cardiomyogenesis via a defined mitochondrial pathway has unique therapeutic potential for targeting ES cell maturation in cardiac disease. Conclusion: Our findings establish the HO-1/CO system and redox regulation of mitochondrial biogenesis as

  9. Heme oxygenase-1 modulates degeneration of the intervertebral disc after puncture in Bach 1 deficient mice.

    Science.gov (United States)

    Ohta, Ryo; Tanaka, Nobuhiro; Nakanishi, Kazuyoshi; Kamei, Naosuke; Nakamae, Toshio; Izumi, Bunichiro; Fujioka, Yuki; Ochi, Mitsuo

    2012-09-01

    Intervertebral disc degeneration is considered to be a major feature of low back pain. Furthermore, oxidative stress has been shown to be an important factor in degenerative diseases such as osteoarthritis and is considered a cause of intervertebral disc degeneration. The purpose of this study was to clarify the correlation between oxidative stress and intervertebral disc degeneration using Broad complex-Tramtrack-Bric-a-brac and cap'n'collar homology 1 deficient (Bach 1-/-) mice which highly express heme oxygenase-1 (HO-1). HO-1 protects cells from oxidative stress. Caudal discs of 12-week-old and 1-year-old mice were evaluated as age-related models. Each group and period, 5 mice (a total of 20 mice, a total of 20 discs) were evaluated as age-related model. C9-C10 caudal discs in 12-week-old Bach 1-/- and wild-type mice were punctured using a 29-gauge needle as annulus puncture model. Each group and period, 5 mice (a total of 60 mice, a total of 60 discs) were evaluated. The progress of disc degeneration was evaluated at pre-puncture, 1, 2, 4, 8 and 12 weeks post-puncture. Radiographic, histologic and immunohistologic analysis were performed to compare between Bach 1-/- and wild-type mice. In the age-related model, there were no significant differences between Bach 1-/- and wild-type mice radiologically and histologically. However, in the annulus puncture model, histological scoring revealed significant difference at 8 and 12 weeks post-puncture. The number of HO-1 positive cells was significantly greater in Bach 1-/- mice at every period. The apoptosis rate was significantly lower at 1 and 2 weeks post-puncture in Bach 1-/- mice. Oxidative stress prevention may avoid the degenerative process of the intervertebral disc after puncture, reducing the number of apoptosis cells. High HO-1 expression may also inhibit oxidative stress and delay the process of intervertebral disc degeneration.

  10. Unraveling the role of animal heme peroxidases in superoxide mediated Mn oxide formation

    Science.gov (United States)

    Learman, D. R.; Hansel, C. M.

    2013-12-01

    Manganese(III,IV) oxides are important in the environment as they can impact the fate of a broad range of nutrients (e.g. carbon and phosphate) and contaminates (e.g. lead and chromium). Bacteria play a valuable role in the production of Mn oxides, yet the mechanisms and physiological reasons remain unclear. Roseobacter sp. AzwK-3b, an organism within the abundant and ubiquitous Roseobacter clade, has recently been shown to oxidize Mn(II) via a novel pathway that involves enzymatic extracellular superoxide production. However, in reactions with only Mn(II) and abiotically generated superoxide, we find superoxide alone is not enough to produce Mn(III,IV) oxides. Scavenging of the byproduct hydrogen peroxide (via the addition of catalase) is required to generate Mn oxides via abiotic reaction of Mn(II) with superoxide. Thus, R. AzwK-3b must produce superoxide and also scavenge hydrogen peroxide to form Mn oxides. Further, in-gel Mn(II) oxidation assay revealed a protein band that could generate Mn oxides in the presence of soluble Mn(II). This Mn(II)-oxidizing protein band was excised from the gel and the peptides identified via mass spectrometry. An animal heme peroxidase (AHP) was the predominant protein found in this band. This protein is homologous to the AHPs previously implicated as a Mn(II)-oxidizing enzyme within the Alphaproteobacteria, Erythrobacter SD-21 and Aurantimonas manganoxydans strain SI85-9A1. Currently, protein expression of the AHPs in R. AzwK-3b is being examined to determine if expression is correlated with Mn(II) concentration or oxidative stress. Our data suggests that AHPs do not directly oxidize Mn(II) but rather plays a role in scavenging hydrogen peroxide and/or producing an organic Mn(III) ligand that complexes Mn(III) and likely aids in Mn oxide precipitation.

  11. Phenol degradation catalyzed by a peroxidase mimic constructed through the grafting of heme onto metal-organic frameworks.

    Science.gov (United States)

    Jiang, Wei; Yang, Jiebing; Wang, Xinghuo; Han, Haobo; Yang, Yan; Tang, Jun; Li, Quanshun

    2018-01-01

    The aim of this work was to construct a peroxidase mimic for achieving the phenol degradation through Fenton reaction. The enzyme mimic was synthesized through the conjugation of heme with the amino group of 2-amino-1,4-benzene dicarboxylate in UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. Compared to free heme, the composite Heme-ZrMOF exhibited an obviously enhanced ability for phenol degradation with up to 97.3% of phenol removal after 2h. Meanwhile, it could achieve the easy separation of catalyst from the system and the elimination of iron residues in the process of phenol degradation. Finally, the catalyst Heme-ZrMOF was observed to possess good recyclability in the phenol degradation with still 76.2% of phenol removal after 4 cycles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Factors for the bioavailability of heme iron preparation in female rats

    OpenAIRE

    村上, 亜由美; 岸本, 三香子; 川口, 真規子; 松浦, 寿喜; 市川, 富夫; Ayumi, Murakami; Mikako, Kishimoto; Makiko, Kawaguchi; Toshiki, Matsuura; Tomio, Ichikawa

    1998-01-01

    Factors for iron absorption in small intestine using heme iron preparation (HIP) and ferric citrate (FC) were investigated. We measured the solubility of iron of experimental diets (FC-normal, FC-overload, HIP-normal, HIP-overload) in water (adjusted pH6.8) and the diffusibility of dietary iron after digestion in vitro. The results did not show significantly differences between FC and HIP. Also, we measured microsomal heme oxygenase (HO) activity in intestinal mucosa of female rats fed experi...

  13. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Directory of Open Access Journals (Sweden)

    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  14. Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis.

    Directory of Open Access Journals (Sweden)

    Halina Wójtowicz

    2009-05-01

    Full Text Available Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-beta fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.

  15. Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

    Science.gov (United States)

    Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

    2014-01-01

    The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

  16. A non-heme iron-mediated chemical demethylation in DNA and RNA.

    Science.gov (United States)

    Yi, Chengqi; Yang, Cai-Guang; He, Chuan

    2009-04-21

    DNA methylation is arguably one of the most important chemical signals in biology. However, aberrant DNA methylation can lead to cytotoxic or mutagenic consequences. A DNA repair protein in Escherichia coli, AlkB, corrects some of the unwanted methylations of DNA bases by a unique oxidative demethylation in which the methyl carbon is liberated as formaldehyde. The enzyme also repairs exocyclic DNA lesions--that is, derivatives in which the base is augmented with an additional heterocyclic subunit--by a similar mechanism. Two proteins in humans that are homologous to AlkB, ABH2 and ABH3, repair the same spectrum of lesions; another human homologue of AlkB, FTO, is linked to obesity. In this Account, we describe our studies of AlkB, ABH2, and ABH3, including our development of a general strategy to trap homogeneous protein-DNA complexes through active-site disulfide cross-linking. AlkB uses a non-heme mononuclear iron(II) and the cofactors 2-ketoglutarate (2KG) and dioxygen to effect oxidative demethylation of the DNA base lesions 1-methyladenine (1-meA), 3-methylcytosine (3-meC), 1-methylguanine (1-meG), and 3-methylthymine (3-meT). ABH3, like AlkB, works better on single-stranded DNA (ssDNA) and is capable of repairing damaged bases in RNA. Conversely, ABH2 primarily repairs lesions in double-stranded DNA (dsDNA); it is the main housekeeping enzyme that protects the mammalian genome from 1-meA base damage. The AlkB-family proteins have moderate affinities for their substrates and bind DNA in a non-sequence-specific manner. Knowing that these proteins flip the damaged base out from the duplex DNA and insert it into the active site for further processing, we first engineered a disulfide cross-link in the active site to stabilize the Michaelis complex. Based on the detailed structural information afforded by the active-site cross-linked structures, we can readily install a cross-link away from the active site to obtain the native-like structures of these complexes

  17. Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

    Science.gov (United States)

    Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

    2011-08-05

    We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance

    Science.gov (United States)

    Liu, Meilian; Xiang, Ruihua; Wilk, Sarah Ann; Zhang, Ning; Sloane, Lauren B.; Azarnoush, Kian; Zhou, Lijun; Chen, Hongzhi; Xiang, Guangda; Walter, Christi A.; Austad, Steven N.; Musi, Nicolas; DeFronzo, Ralph A.; Asmis, Reto; Scherer, Philipp E.; Dong, Lily Q.; Liu, Feng

    2012-01-01

    The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad−/−). In addition, the fDsbA-L/Ad−/− mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders. PMID:22807031

  19. Iron(III) complexes of certain tetradentate phenolate ligands as ...

    Indian Academy of Sciences (India)

    non-heme iron enzymes, which catalyse the oxidative cleavage of catechols to cis, cis-muconic acids with the incorporation of ... nature of heterocyclic rings of the ligands and the methyl substituents on them regulate the electronic spectral features .... and simple substitution reactions.19,21 The complexes of [H2(L5)] and ...

  20. 14 CFR 135.271 - Helicopter hospital emergency medical evacuation service (HEMES).

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Helicopter hospital emergency medical....271 Helicopter hospital emergency medical evacuation service (HEMES). (a) No certificate holder may... certificate holder may assign a helicopter flight crewmember, and no flight crewmember may accept an...

  1. Microbiota facilitates dietary heme-induced epithelial hyperproliferation and hyperplasia by breaking the mucus barrier

    NARCIS (Netherlands)

    IJssennagger, Noortje; Belzer, Clara; Hooiveld, Guido; Dekker, Jan; Muller, Michael; Kleerebezem, Michiel; Meer, van der Roelof

    2015-01-01

    Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in

  2. Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon

    NARCIS (Netherlands)

    Ijssennagger, Noortje; Belzer, Clara; Hooiveld, Guido J; Dekker, Jan; van Mil, Saskia W C; Müller, Michael; Kleerebezem, Michiel; van der Meer, Roelof; van Mil, SWC

    2015-01-01

    Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in

  3. Interaction between Mitochondrial Reactive Oxygen Species, Heme Oxygenase, and Nitric Oxide Synthase Stimulates Phagocytosis in Macrophages

    Directory of Open Access Journals (Sweden)

    Andrea Müllebner

    2018-01-01

    Full Text Available BackgroundMacrophages are cells of the innate immune system that populate every organ. They are required not only for defense against invading pathogens and tissue repair but also for maintenance of tissue homeostasis and iron homeostasis.AimThe aim of this study is to understand whether heme oxygenase (HO and nitric oxide synthase (NOS contribute to the regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX activity and phagocytosis, two key components of macrophage function.MethodsThis study was carried out using resting J774A.1 macrophages treated with hemin or vehicle. Activity of NOS, HO, or NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS formation was determined by Amplex® red assay, and phagocytosis was measured using fluorescein isothiocyanate-labeled bacteria. In addition, we analyzed the fate of the intracellular heme by using electron spin resonance.ResultsWe show that both enzymes NOS and HO are essential for phagocytic activity of macrophages. NOS does not directly affect phagocytosis, but stimulates NOX activity via nitric oxide-triggered ROS production of mitochondria. Treatment of macrophages with hemin results in intracellular accumulation of ferrous heme and an inhibition of phagocytosis. In contrast to NOS, HO products, including carbon monoxide, neither clearly affect NOX activity nor clearly affect phagocytosis, but phagocytosis is accelerated by HO-mediated degradation of heme.ConclusionBoth enzymes contribute to the bactericidal activity of macrophages independently, by controlling different pathways.

  4. In vitro studies on heme oxygenase-1 and P24 antigen HIV-1 level ...

    African Journals Online (AJOL)

    Background: Heme oxygenase-1 (HO-1) is a protein secreted by immune cells as a part of immune response mechanism.HO-1 can be induced by variety agents that causingoxidative stress, such as exposure to 100% oxygenat2,4 ATA pressure.It plays a vital role in maintaining cellular homeostasis.This study was ...

  5. Heme-Protein Active Site Models via Self-Assembly in Water

    NARCIS (Netherlands)

    Fiammengo, R.; Wojciechowski, Kamil; Crego Calama, Mercedes; Figoli, A.; Wessling, Matthias; Reinhoudt, David; Timmerman, P.

    2003-01-01

    Water-soluble models of heme-protein active sites are obtained via the self-assembly of cationic porphyrins 1 and tetrasulfonato calix[4]arene 2 (K1·2 = 105 M-1). Selective binding of ligands either outside or inside the cavity of assemblies 1·2 via coordination to the zinc center has been observed.

  6. Red meat and colon cancer : The cytotoxic and hyperproliferative effects of dietary heme

    NARCIS (Netherlands)

    Sesink, ALA; Termont, DSML; Kleibeuker, JH; Van der Meer, R

    1999-01-01

    The intake of a Western diet with a high amount of red meat is associated with a high risk for colon cancer. We hypothesize that heme, the iron carrier of red meat, is involved in diet-induced colonic epithelial damage, resulting in increased epithelial proliferation. Rats were fed purified control

  7. Significance of heme-based respiration in meat spoilage caused by Leuconostoc gasicomitatum.

    Science.gov (United States)

    Jääskeläinen, Elina; Johansson, Per; Kostiainen, Olli; Nieminen, Timo; Schmidt, Georg; Somervuo, Panu; Mohsina, Marzia; Vanninen, Paula; Auvinen, Petri; Björkroth, Johanna

    2013-02-01

    Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) which causes spoilage in cold-stored modified-atmosphere-packaged (MAP) meat products. In addition to the fermentative metabolism, L. gasicomitatum is able to respire when exogenous heme and oxygen are available. In this study, we investigated the respiration effects on growth rate, biomass, gene expression, and volatile organic compound (VOC) production in laboratory media and pork loin. The meat samples were evaluated by a sensory panel every second or third day for 29 days. We observed that functional respiration increased the growth (rate and yield) of L. gasicomitatum in laboratory media with added heme and in situ meat with endogenous heme. Respiration increased enormously (up to 2,600-fold) the accumulation of acetoin and diacetyl, which are buttery off-odor compounds in meat. Our transcriptome analyses showed that the gene expression patterns were quite similar, irrespective of whether respiration was turned off by excluding heme from the medium or mutating the cydB gene, which is essential in the respiratory chain. The respiration-based growth of L. gasicomitatum in meat was obtained in terms of population development and subsequent development of sensory characteristics. Respiration is thus a key factor explaining why L. gasicomitatum is so well adapted in high-oxygen packed meat.

  8. Effects of Zinc Deuteroporphyrin Bis Glycol on Newborn Mice After Heme-Loading

    Science.gov (United States)

    He, Cynthia X.; Campbell, Claire M.; Zhao, Hui; Kalish, Flora S.; Schulz, Stephanie; Vreman, Hendrik J.; Wong, Ronald J.; Stevenson, David K.

    2011-01-01

    Infants with hemolytic diseases frequently develop hyperbilirubinemia, but standard phototherapy only eliminates bilirubin after its production. A better strategy might be to directly inhibit heme oxygenase (HO), the rate-limiting enzyme in bilirubin production. Metalloporphyrins (Mps) are heme analogs that competitively inhibit HO activity in vitro and in vivo and suppress plasma bilirubin levels in vivo. A promising Mp, zinc deuteroporphyrin bis glycol (ZnBG), is orally absorbed and effectively inhibits HO activity at relatively low doses. We determined the I50 (the dose needed to inhibit HO activity by 50%) of orally administered ZnBG in vivo and then evaluated ZnBG’s effects on in vivo bilirubin production, HO activity, HO protein levels, and HO-1 gene expression in newborn mice following heme-loading, a model analogous to a hemolytic infant. The I50 of ZnBG was found to be 4.0 μmol/kg body weight (BW). At a dose of 15-μmol/kg BW, ZnBG reduced in vivo bilirubin production, inhibited heme-induced liver HO activity and spleen HO activity to and below baseline, respectively, transiently induced liver and spleen HO-1 gene transcription, and induced liver and spleen HO-1 protein levels. We conclude that ZnBG may be an attractive compound for treating severe neonatal hyperbilirubinemia caused by hemolytic disease. PMID:21785387

  9. Rapamycin Induces Heme Oxygenase-1 in Liver but Inhibits Bile Flow Recovery after Ischemia

    NARCIS (Netherlands)

    Kist, Alwine; Wakkie, Joris; Madu, Max; Versteeg, Ruth; ten Berge, Judith; Nikolic, Andrej; Nieuwenhuijs, Vincent B.; Porte, Robert J.; Padbury, Robert T. A.; Barritt, Greg J.

    Background/Aims. Rapamycin, which is employed in the management of patients undergoing liver surgery, induces the synthesis of heme oxygenase-1 (HO-1) in some non-liver cell types. The aim was to investigate whether rapamycin can induce HO-1 expression in the liver, and to test the effects of

  10. Evidence for dynamic behavior of O2 in oxy-heme model compounds

    International Nuclear Information System (INIS)

    Montiel-Montoya, R.; Bill, E.; Trautwein, A.X.; Winkler, H.

    1986-01-01

    The authors have performed Moessbauer studies on several oxy-heme model compounds, and for two of them they have also derived the three dimensional structure from X-ray studies. The X-ray structure analysis of these model compounds provides the information that O 2 occupies three different sites in one and only two sites in the other. (Auth.)

  11. PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis.

    Science.gov (United States)

    Ryu, Moon-Suhn; Zhang, Deliang; Protchenko, Olga; Shakoury-Elizeh, Minoo; Philpott, Caroline C

    2017-05-01

    Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC-binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.

  12. Novel Insights in Mammalian Catalase Heme Maturation: Effect of NO and Thioredoxin-1

    Science.gov (United States)

    Chakravarti, Ritu; Gupta, Karishma; Majors, Alana; Ruple, Lisa; Aronica, Mark; Stuehr, Dennis J.

    2016-01-01

    Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorporation was associated with defective oligomerization of catalase, such that inactive catalase monomers and dimers accumulated in place of the mature tetrameric enzyme. We also found that GAPDH plays a key role in mediating these NO effects on the structure and activity of catalase. Moreover, the NO sensitivity of catalase maturation could be altered up or down by manipulating the cellular expression level or activity of thioredoxin-1, a known protein-SNO denitrosylase enzyme. In a mouse model of allergic inflammatory asthma, we found that lungs from allergen-challenged mice contained a greater percentage of dimeric catalase relative to tetrameric catalase in the unchallenged control, suggesting that the mechanisms described here are in play in the allergic asthma model. Together, our study shows how maturation of active catalase can be influenced by NO, S-nitrosylated GAPDH, and thioredoxin-1, and how maturation may become compromised in inflammatory conditions such as asthma. PMID:25659933

  13. Dry powder inhalation of hemin to induce heme oxygenase expression in the lung

    NARCIS (Netherlands)

    Zijlstra, G.S.; Brandsma, C.; Harpe, M.F.H.; Van Dam, G.M.; Slebos, D.J.; Kerstjens, H.A.M.; de Boer, Anne; Frijlink, H.W.

    2007-01-01

    The purpose of this study was to formulate hemin as a powder for inhalation and to show proof of concept of heme oxygenase 1 (HO-1) expression in the lungs of mice by inhalation of hemin. Hemin was spray dried from a neutralized sodium hydroxide solution. The particle size distribution of the powder

  14. Novel insights in mammalian catalase heme maturation: effect of NO and thioredoxin-1.

    Science.gov (United States)

    Chakravarti, Ritu; Gupta, Karishma; Majors, Alana; Ruple, Lisa; Aronica, Mark; Stuehr, Dennis J

    2015-05-01

    Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorporation was associated with defective oligomerization of catalase, such that inactive catalase monomers and dimers accumulated in place of the mature tetrameric enzyme. We also found that GAPDH plays a key role in mediating these NO effects on the structure and activity of catalase. Moreover, the NO sensitivity of catalase maturation could be altered up or down by manipulating the cellular expression level or activity of thioredoxin-1, a known protein-SNO denitrosylase enzyme. In a mouse model of allergic inflammatory asthma, we found that lungs from allergen-challenged mice contained a greater percentage of dimeric catalase relative to tetrameric catalase in the unchallenged control, suggesting that the mechanisms described here are in play in the allergic asthma model. Together, our study shows how maturation of active catalase can be influenced by NO, S-nitrosylated GAPDH, and thioredoxin-1, and how maturation may become compromised in inflammatory conditions such as asthma. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. ["Kuhu me siis läheme? - Eks ikka koju."] / M. J.

    Index Scriptorium Estoniae

    Jõgi, Mall, 1947-

    2016-01-01

    Tutvustus: „Kuhu me siis läheme? - Eks ikka koju.“ : sajandivahetus saksa kirjanduses 200 aastat tagasi : Goethe, Schiller, Tieck, Kleist, Hoffmann, Eichendorff, Büchner, Novalis, Hegel (Schelling? Hölderlin?), Schlegel / saksa keelest valinud ja tõlkinud Mati Sirkel. Tallinn : Eesti Keele Sihtasutus, 2015

  16. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy

  17. Heme oxygenase-1 expression protects melanocytes from stress-induced cell death: implications for vitiligo

    NARCIS (Netherlands)

    Elassiuty, Yasser E.; Klarquist, Jared; Speiser, Jodi; Yousef, Randa M.; El Refaee, Abdelaziz A.; Hunter, Nahla S.; Shaker, Olfat G.; Gundeti, Mohan; Nieuweboer-Krobotova, Ludmila; Caroline Le Poole, I.

    2011-01-01

    To study protection of melanocytes from stress-induced cell death by heme oxygenases during depigmentation and repigmentation in vitiligo, expression of isoforms 1 and 2 was studied in cultured control and patient melanocytes and normal skin explants exposed to UV or bleaching agent 4-TBP.

  18. Dietary heme induces acute oxidative stress, but delayed cytotoxicity and compensatory hyperproliferation in mouse colon

    NARCIS (Netherlands)

    IJssenagger, N.; Rijnierse, A.; Wit, de N.J.W.; Boekschoten, M.V.; Dekker, J.; Schonewille, A.; Müller, M.R.; Meer, van der M.

    2013-01-01

    Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by generating cytotoxic and oxidative stress. Recently, we found that this surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which

  19. Dietary heme induces instantaneous oxidative stress but delayed cytotoxicity and compensatory hyperproliferation in mouse colon

    NARCIS (Netherlands)

    IJssennagger, Noortje; Rijnierse, A.; Wit, de Nicole; Boekschoten, Mark; Dekker, Jan; Schonewille, Arjan; Muller, Michael; Meer, van der Roelof

    2013-01-01

    Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a

  20. Nuclear resonance vibrational spectroscopy applied to [Fe(OEP)(NO)]: the vibrational assignments of five-coordinate ferrous heme-nitrosyls and implications for electronic structure.

    Science.gov (United States)

    Lehnert, Nicolai; Galinato, Mary Grace I; Paulat, Florian; Richter-Addo, George B; Sturhahn, Wolfgang; Xu, Nan; Zhao, Jiyong

    2010-05-03

    This study presents Nuclear Resonance Vibrational Spectroscopy (NRVS) data on the five-coordinate (5C) ferrous heme-nitrosyl complex [Fe(OEP)(NO)] (1, OEP(2-) = octaethylporphyrinato dianion) and the corresponding (15)N(18)O labeled complex. The obtained spectra identify two isotope sensitive features at 522 and 388 cm(-1), which shift to 508 and 381 cm(-1), respectively, upon isotope labeling. These features are assigned to the Fe-NO stretch nu(Fe-NO) and the in-plane Fe-N-O bending mode delta(ip)(Fe-N-O), the latter has been unambiguously assigned for the first time for 1. The obtained NRVS data were simulated using our quantum chemistry centered normal coordinate analysis (QCC-NCA). Since complex 1 can potentially exist in 12 different conformations involving the FeNO and peripheral ethyl orientations, extended density functional theory (DFT) calculations and QCC-NCA simulations were performed to determine how these conformations affect the NRVS properties of [Fe(OEP)NO]. These results show that the properties and force constants of the FeNO unit are hardly affected by the conformational changes involving the ethyl substituents. On the other hand, the NRVS-active porphyrin-based vibrations around 340-360, 300-320, and 250-270 cm(-1) are sensitive to the conformational changes. The spectroscopic changes observed in these regions are due to selective mechanical couplings of one component of E(u)-type (in ideal D(4h) symmetry) porphyrin-based vibrations with the in-plane Fe-N-O bending mode. This leads to the observed variations in Fe(OEP) core mode energies and NRVS intensities without affecting the properties of the FeNO unit. The QCC-NCA simulated NRVS spectra of 1 show excellent agreement with experiment, and indicate that conformer F is likely present in the samples of this complex investigated here. The observed porphyrin-based vibrations in the NRVS spectra of 1 are also assigned based on the QCC-NCA results. The obtained force constants of the Fe-NO and N

  1. Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells

    Directory of Open Access Journals (Sweden)

    Ringhieri P

    2017-01-01

    Full Text Available Paola Ringhieri,1 Silvia Mannucci,2 Giamaica Conti,2 Elena Nicolato,2 Giulio Fracasso,3 Pasquina Marzola,4 Giancarlo Morelli,1 Antonella Accardo1 1Department of Pharmacy and Interuniversity Research Centre on Bioactive Peptides (CIRPeB, University of Naples “Federico II”, Napoli, 2Department of Neurological Biomedical and Movement Sciences, 3Section of Immunology, Department of Medicine, 4Department of Informatics, University of Verona, Verona, Italy Abstract: Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd] have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2 receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents. Keywords: anti-HER2 liposomes, target peptide, KCCYSL peptide, breast cancer, click chemistry, branched peptides 

  2. Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization

    International Nuclear Information System (INIS)

    Moreau, Karen; Faure, Claudine; Violot, Sebastien; Gouet, Patrice; Verdier, Gerard; Ronfort, Corinne

    2004-01-01

    During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme

  3. Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras.

    Science.gov (United States)

    Braeuning, Albert; Schwarz, Michael

    2010-11-01

    Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.

  4. A peroxidase mimic with atom transfer radical polymerization activity constructed through the grafting of heme onto metal-organic frameworks.

    Science.gov (United States)

    Jiang, Wei; Pan, Yue; Yang, Jiebing; Liu, Yong; Yang, Yan; Tang, Jun; Li, Quanshun

    2018-07-01

    Atom transfer radical polymerization (ATRP) has been considered to be an efficient strategy for constructing functional macromolecules owing to its simple operation and versatile monomers, and thus it is of great significance to develop ideal catalysts with higher activity and perfect reusability. We constructed a peroxidase mimic through the grafting of heme onto metal-organic frameworks UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. After the systematic characterization of structure, the composite Heme-ZrMOF was demonstrated to possess high peroxidase activity using 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) and 3,3',5,5'-tetramethylbenzidine as substrates. The enzyme mimic was then used as catalysts in the ATRP reactions of different monomers, in which favorable monomer conversion (44.6-98.0%) and product molecular weight (8600-25,600 g/mol) could be obtained. Compared to free heme, Heme-ZrMOF could efficiently achieve the easy separation of heme from the catalytic system and facilitate the ATRP reaction in an aqueous environment to avoid the utilization of organic solvents. In conclusion, the enzyme mimic Heme-ZrMOF could be potentially used as an effective catalyst for preparing well-defined polymers with biomedical applications. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. A central role for heme iron in colon carcinogenesis associated with red meat intake.

    Science.gov (United States)

    Bastide, Nadia M; Chenni, Fatima; Audebert, Marc; Santarelli, Raphaelle L; Taché, Sylviane; Naud, Nathalie; Baradat, Maryse; Jouanin, Isabelle; Surya, Reggie; Hobbs, Ditte A; Kuhnle, Gunter G; Raymond-Letron, Isabelle; Gueraud, Françoise; Corpet, Denis E; Pierre, Fabrice H F

    2015-03-01

    Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines, and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP + MeIQx, 50 + 25 μg/kg in diet), and NOC (induced by NaNO₂+ NaNO₂; 0.17 + 0.23 g/L of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc-deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions, but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67 ± 39 mm²; 2.5% hemoglobin diet: 114 ± 47 mm², P = 0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation. . ©2015 American Association for Cancer Research.

  6. No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

    Science.gov (United States)

    Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

    2017-07-20

    Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

    Science.gov (United States)

    Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

    2004-08-15

    To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

  8. A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus.

    Science.gov (United States)

    Laakso, Holly A; Marolda, Cristina L; Pinter, Tyler B; Stillman, Martin J; Heinrichs, David E

    2016-01-01

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

    Science.gov (United States)

    Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

    2016-01-01

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

  10. Design and synthesis of a tetradentate '3-amine-1-carboxylate' ligand to mimic the metal binding environment at the non-heme iron(II) oxidase active site.

    Science.gov (United States)

    Dungan, Victoria J; Ortin, Yannick; Mueller-Bunz, Helge; Rutledge, Peter J

    2010-04-07

    Non-heme iron(II) oxidases (NHIOs) catalyse a diverse array of oxidative chemistry in Nature. As part of ongoing efforts to realize biomimetic, iron-mediated C-H activation, we report the synthesis of a new 'three-amine-one-carboxylate' ligand designed to complex with iron(II) and mimic the NHIO active site. The tetradentate ligand has been prepared as a single enantiomer in nine synthetic steps from N-Cbz-L-alanine, pyridine-2,6-dimethanol and diphenylamine, using Seebach oxazolidinone chemistry to control the stereochemistry. X-Ray crystal structures are reported for two important intermediates, along with variable temperature NMR experiments to probe the hindered interconversion of conformational isomers of several key intermediates, 2,6-disubstituted pyridine derivatives. The target ligand and an N-Cbz-protected precursor were each then complexed with iron(II) and tested for their ability to promote alkene dihydroxylation, using hydrogen peroxide as the oxidant.

  11. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.

    Science.gov (United States)

    Parhad, Swapnil S; Jaiswal, Deepa; Ray, Krishanu; Mazumdar, Shyamalava

    2016-03-25

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. 3D complex: a structural classification of protein complexes.

    Directory of Open Access Journals (Sweden)

    Emmanuel D Levy

    2006-11-01

    Full Text Available Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

  13. Modeling and computations of the intramolecular electron transfer process in the two-heme protein cytochrome em>c>4

    DEFF Research Database (Denmark)

    Natzmutdinov, Renat R.; Bronshtein, Michael D.; Zinkicheva, Tamara T.

    2012-01-01

    force were determined using dielectric continuum models. We then calculated the electronic transmission coefficient of the intramolecular ET rate using perturbation theory combined with the electronic wave functions determined by the DFT calculations for different heme group orientations and Fe...

  14. Voltammetry and In Situ Scanning Tunnelling Microscopy of De Novo Designed Heme Protein Monolayers on Au(111)-Electrode Surfaces

    DEFF Research Database (Denmark)

    Albrecht, Tim; Li, Wu; Haehnel, Wolfgang

    2006-01-01

    to the tunnelling current, apparently due to slow electron transfer kinetics. As a consequence, STM images of heme-containing and heme-free MOP-C did not reveal any notable differences in apparent height or physical extension. The apparent height of heme-containing MOP-C did not show any dependence on the substrate...... potential being varied around the redox potential of the protein. The mere presence of an accessible molecular energy level is not sufficient to result in detectable tunnelling current modulation. (c) 2006 Elsevier B.V. All rights reserved.......In the present work, we report the electrochemical characterization and in situ scanning tunnelling microscopy (STM) studies of monolayers of an artificial de novo designed heme protein MOP-C, covalently immobilized on modified Au(111) surfaces. The protein forms closely packed monolayers, which...

  15. Relationship of Heme Oxygenase-1 (HO-1 Level with Onset and Severity in Normotensive Pregnancy and Severe Preeclampsia

    Directory of Open Access Journals (Sweden)

    John Johannes Wantania

    2016-08-01

    Full Text Available Background: Preeclampsia still becomes a major problem in pregnancies. Various evidences showed that heme oxygenase-1 (HO-1 is very important in pregnancy. This study aims to understand the relationship of heme oxygenase-1 level with onset and severity in normotensive pregnancy and severe preeclampsia. Methods: This is a cross sectional analytic comparative study, the subjects consisted of 26 patients with normotensive pregnancies and 26 patients with severe preeclampsia. Blood samples from women with < 34 / ≥ 34 weeks’ normotensive pregnancies and women with severe preeclampsia were taken. HO-1 ELISA kit used to quantitate heme oxygenase-1 level in samples. Results: The level of heme oxygenase-1 in normotensive pregnant women < 34 weeks lower than severe preeclampsia pregnant women < 34 weeks (3.28 ± 0.46 ng/mL vs 4.20 ± 0.64 ng/mL, p=0.003, respectively. The median level of heme oxygenase-1 in normotensive pregnant women ≥ 34 weeks was 2.96 (2.41–4.39 ng/mL, while severe preeclampsia pregnant women ≥ 34 weeks was 3.52 (2.88–5.43 ng/mL, (p=0.040. The median level of heme oxygenase-1 in normotensive pregnant women was 3.04 (2.41–4.39 ng/mL, while severe preeclampsia pregnant women was 3.68 (2.88–5.67 ng/mL, (p=0.001. Conclusions: There is correlation between the incidence of severe preeclampsia with heme oxygenase-1 level in < 34 and ≥ 34 weeks of pregnancy. There is a significant difference between the level of heme oxygenase-1 in pregnant women with severe preeclampsia and in women with normotensive pregnancy. 

  16. The Heme Biosynthesis Pathway Is Essential for Plasmodium falciparum Development in Mosquito Stage but Not in Blood Stages*

    Science.gov (United States)

    Ke, Hangjun; Sigala, Paul A.; Miura, Kazutoyo; Morrisey, Joanne M.; Mather, Michael W.; Crowley, Jan R.; Henderson, Jeffrey P.; Goldberg, Daniel E.; Long, Carole A.; Vaidya, Akhil B.

    2014-01-01

    Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[13C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs. PMID:25352601

  17. Comparative study of enzyme activity and heme reactivity in Drosophila melanogaster and Homo sapiens cystathionine β-synthases.

    Science.gov (United States)

    Su, Yang; Majtan, Tomas; Freeman, Katherine M; Linck, Rachel; Ponter, Sarah; Kraus, Jan P; Burstyn, Judith N

    2013-01-29

    Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5'-phosphate (PLP) for catalysis, and S-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear. This paper reports biochemical and spectroscopic characterization of CBS from fruit fly Drosophila melanogaster (DmCBS) and the CO/NO gas binding reactions of DmCBS and human CBS. Like CBS enzymes from lower organisms (e.g., yeast), DmCBS is intrinsically highly active and is not regulated by AdoMet. The DmCBS heme coordination environment, the reactivity, and the accompanying effects on enzyme activity are similar to those of human CBS. The DmCBS heme bears histidine and cysteine axial ligands, and the enzyme becomes inactive when the cysteine ligand is replaced. The Fe(II) heme in DmCBS is less stable than that in human CBS, undergoing more facile reoxidation and ligand exchange. In both CBS proteins, the overall stability of the protein is correlated with the heme oxidation state. Human and DmCBS Fe(II) hemes react relatively slowly with CO and NO, and the rate of the CO binding reaction is faster at low pH than at high pH. Together, the results suggest that heme incorporation and AdoMet regulation in CBS are not correlated, possibly providing two independent means for regulating the enzyme.

  18. Abscisic Acid Participates in the Control of Cell Cycle Initiation Through Heme Homeostasis in the Unicellular Red Alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Kobayashi, Yuki; Ando, Hiroyuki; Hanaoka, Mitsumasa; Tanaka, Kan

    2016-05-01

    ABA is a phytohormone that is synthesized in response to abiotic stresses and other environmental changes, inducing various physiological responses. While ABA has been found in unicellular photosynthetic organisms, such as cyanobacteria and eukaryotic algae, its function in these organisms is poorly understood. Here, we found that ABA accumulated in the unicellular red alga Cyanidioschyzon merolae under conditions of salt stress and that the cell cycle G1/S transition was inhibited when ABA was added to the culture medium. A gene encoding heme-scavenging tryptophan-rich sensory protein-related protein (CmTSPO; CMS231C) was positively regulated by ABA, as in Arabidopsis, and CmTSPO bound heme in vitro. The intracellular content of total heme was increased by addition of ABA, but unfettered heme decreased, presumably due to scavenging by CmTSPO. The inhibition of DNA replication by ABA was negated by addition of heme to the culture medium. Thus, we propose a regulatory role for ABA and heme in algal cell cycle initiation. Finally, we found that a C. merolae mutant that is defective in ABA production was more susceptible to salt stress, indicating the importance of ABA to stress resistance in red algae. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. A Heme Oxygenase-1 Transducer Model of Degenerative and Developmental Brain Disorders

    Directory of Open Access Journals (Sweden)

    Hyman M. Schipper

    2015-03-01

    Full Text Available Heme oxygenase-1 (HO-1 is a 32 kDa protein which catalyzes the breakdown of heme to free iron, carbon monoxide and biliverdin. The Hmox1 promoter contains numerous consensus sequences that render the gene exquisitely sensitive to induction by diverse pro-oxidant and inflammatory stimuli. In “stressed” astroglia, HO-1 hyperactivity promotes mitochondrial iron sequestration and macroautophagy and may thereby contribute to the pathological iron deposition and bioenergetic failure documented in Alzheimer disease, Parkinson disease and certain neurodevelopmental conditions. Glial HO-1 expression may also impact neuroplasticity and cell survival by modulating brain sterol metabolism and the proteasomal degradation of neurotoxic proteins. The glial HO-1 response may represent a pivotal transducer of noxious environmental and endogenous stressors into patterns of neural damage and repair characteristic of many human degenerative and developmental CNS disorders.

  20. Effects of illumination and packaging on non-heme iron and color attributes of sliced ham.

    Science.gov (United States)

    Li, H; Li, C B; Xu, X L; Zhou, G H

    2012-08-01

    This study was designed to investigate effects of illumination and packaging on color of cooked cured sliced ham during refrigeration, and the possibility of decomposition of nitrosylheme under light and oxygen exposure. Three illumination levels and three packaging films with different oxygen transmission rates (OTRs) were used in two separate experiments during 35 days storage, and pH value, a* value, nitrosylheme, residual nitrite and non-heme iron were evaluated. Packaging OTRs had significant effects (P0.05) nitrosylheme concentration during storage. For both groups, storage time had a significant effect (P<0.01) on a* value and nitrosylheme. Negative relationships between nitrosylheme and nitrite in the illumination group, and between nitrosylheme and non-heme iron in the packaging group were observed. Therefore, illumination level and packaging OTR had limited effects on overall pigment stability, but more discoloration and loss of redness occurred on the surface of products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Tick iron and heme metabolism – New target for an anti-tick intervention

    Czech Academy of Sciences Publication Activity Database

    Hajdušek, Ondřej; Šíma, Radek; Perner, Jan; Loosová, Gabriela; Harcubová, Adéla; Kopáček, Petr

    2016-01-01

    Roč. 7, č. 4 (2016), s. 565-572 ISSN 1877-959X R&D Projects: GA ČR GA13-11043S; GA ČR GP13-27630P; GA ČR GP13-12816P EU Projects: European Commission(XE) 316304 - MODBIOLIN Institutional support: RVO:60077344 Keywords : tick * iron * heme * RNAi * vaccine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.230, year: 2016

  2. Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer.

    Science.gov (United States)

    Pintscher, Sebastian; Kuleta, Patryk; Cieluch, Ewelina; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2016-03-25

    In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemesb The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on hemebligand mutants of cytochromebc1in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functionalin vivo This reveals that cytochromebc1can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemesbin this cytochrome and in other membranous cytochromesbact as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Impairment of heme synthesis in myelin as potential trigger of multiple sclerosis.

    Science.gov (United States)

    Morelli, Alessandro; Ravera, Silvia; Calzia, Daniela; Panfoli, Isabella

    2012-06-01

    The pathogenesis of multiple sclerosis (MS), a disease characterized by demyelination and subsequent axonal degeneration, is as yet unknown. Also, the nature of the disease is as yet not established, since doubts have been cast on its autoimmune origin. Genetic and environmental factors have been implied in MS, leading to the idea of an overall multifactorial origin. An unexpected role in energizing the axon has been reported for myelin, supposed to be the site of consumption of most of oxygen in brain. Myelin would be able to perform oxidative phosphorylation to supply the axons with ATP, thanks to the expression therein of mitochondrial F(o)F(1)-ATP synthase, and respiratory chains. Interestingly, myelin expresses the pathway of heme synthesis, hence of cytochromes, that rely on heme group, in turn depending on Fe availability. Poisoning by these pollutants shares the common characteristic to bring about demyelination both in animal models and in man. Carbon monoxide (CO) and lead poisoning which cause functional imbalance of the heme group, as well as of heme synthesis, cause myelin damage. On the other hand, a lack of essential metals such as iron and copper, produces dramatic myelin decrease. Myelin is a primary target, of iron shortage, indicating that in myelin Fe-dependent processes are more active than in other tissues. The predominant spread of MS in industrialized countries where pollution by heavy metals, and CO poisoning is widespread, suggests a relationship among toxic action of metal pollutants and MS. According to the present hypothesis, MS can be primarily triggered by environmental factors acting on a genetic susceptibility, while the immune response may be a consequence of a primary oxidative damage due to reactive oxygen species produced consequently to an imbalance of cytochromes and respiratory chains in the sheath. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis.

    Science.gov (United States)

    Perry, Christopher; de Los Santos, Emmanuel L C; Alkhalaf, Lona M; Challis, Gregory L

    2018-04-13

    Covering: up to the end of 2017The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on experimentally characterised examples. Enzymes belonging to this class are known to catalyse a range of transformations, including oxidative carbocyclisation, N-oxygenation, C-hydroxylation and C-C desaturation. Examples of such enzymes that have yet to be experimentally investigated are also briefly described and their likely functions are discussed.

  5. EPR spectral changes of nitrosil hemes and their relation to the hemoglobin T-R transition

    International Nuclear Information System (INIS)

    Louro, S.R.W.; Ribeiro, P.C.; Bemski, G.

    1980-09-01

    EPR spectra of nitrosil-hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25 0 C. After the equilibration of NO among the α and β subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO - β hemes and the other two arising from NO - α hemes of molecules in the high and low affinity conformations. The fractional amounts of α subunits exhibiting the high affinity spectrum fitted the two-state model with L = 7 x 10 6 , and csup(α) sub(NO) and csup(β) sub(NO) approximately 0.01. Hemoglobin has been marked with nitric oxide at one chain using low-saturation amounts of nitric oxide. The EPR spectra were studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high affinity spectrum fitted the two-state model with L = 7 x 10 6 , csub(O 2 ) = 0.0033 and csup(α) sub(NO) = 0.08, instead of csup(α) sub(NO) = 0.01.Thus, the two state model is not adequate to describe the conformational transition of these hybrids. The results are evidence of the nonequivalence between oxygen and nitric oxide as ligands. (Author) [pt

  6. Diamond Blackfan Anemia at the Crossroad between Ribosome Biogenesis and Heme Metabolism

    Directory of Open Access Journals (Sweden)

    Deborah Chiabrando

    2010-01-01

    Full Text Available Diamond-Blackfan anemia (DBA is a rare, pure red-cell aplasia that presents during infancy. Approximately 40% of cases are associated with other congenital defects, particularly malformations of the upper limb or craniofacial region. Mutations in the gene coding for the ribosomal protein RPS19 have been identified in 25% of patients with DBA, with resulting impairment of 18S rRNA processing and 40S ribosomal subunit formation. Moreover, mutations in other ribosomal protein coding genes account for about 25% of other DBA cases. Recently, the analysis of mice from which the gene coding for the heme exporter Feline Leukemia Virus subgroup C Receptor (FLVCR1 is deleted suggested that this gene may be involved in the pathogenesis of DBA. FLVCR1-null mice show a phenotype resembling that of DBA patients, including erythroid failure and malformations. Interestingly, some DBA patients have disease linkage to chromosome 1q31, where FLVCR1 is mapped. Moreover, it has been reported that cells from DBA patients express alternatively spliced isoforms of FLVCR1 which encode non-functional proteins. Herein, we review the known roles of RPS19 and FLVCR1 in ribosome function and heme metabolism respectively, and discuss how the deficiency of a ribosomal protein or of a heme exporter may result in the same phenotype.

  7. A Lactobacillus rhamnosus strain induces a heme oxygenase dependent increase in Foxp3+ regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Khalil Karimi

    Full Text Available We investigated the consequences of feeding with a Lactobacillus species on the immune environment in GALT, and the role of dendritic cells and heme oxygenase-1 in mediating these responses. Feeding with a specific strain of Lactobacillus rhamnosus induced a significant increase in CD4+CD25+Foxp3+ functional regulatory T cells in GALT. This increase was greatest in the mesenteric lymph nodes and associated with a marked decrease in TNF and IFNγ production. Dendritic cell regulatory function and HO-1 expression was also increased. The increase in Foxp3+ T cells could be prevented by treatment with a heme oxygenase inhibitor. However, neither inhibition of heme oxygenase nor blockade of IL-10 and TGFβ prevented the inhibition of inflammatory cytokine production. In conclusion Lactobacillus feeding induced a tolerogenic environment in GALT. HO-1 was critical to the enhancement of Foxp3+ regulatory T cells while additional, as yet unknown, pathways were involved in the down-regulation of inflammatory cytokine production by T cells.

  8. Human heme oxygenase-1 gene transfer lowers blood pressure and promotes growth in spontaneously hypertensive rats.

    Science.gov (United States)

    Sabaawy, H E; Zhang, F; Nguyen, X; ElHosseiny, A; Nasjletti, A; Schwartzman, M; Dennery, P; Kappas, A; Abraham, N G

    2001-08-01

    Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, with release of free iron and carbon monoxide. Both heme and carbon monoxide have been implicated in the regulation of vascular tone. A retroviral vector containing human HO-1 cDNA (LSN-HHO-1) was constructed and subjected to purification and concentration of the viral particles to achieve 5x10(9) to 1x10(10) colony-forming units per milliliter. The ability of concentrated infectious viral particles to express human HO-1 (HHO-1) in vivo was tested. A single intracardiac injection of the concentrated infectious viral particles (expressing HHO-1) to 5-day-old spontaneously hypertensive rats resulted in functional expression of the HHO-1 gene and attenuation of the development of hypertension. Rats expressing HHO-1 showed a significant decrease in urinary excretion of a vasoconstrictor arachidonic acid metabolite and a reduction in myogenic responses to increased intraluminal pressure in isolated arterioles. Unexpectedly, HHO-1 chimeric rats showed a simultaneous significant proportionate increase in somatic growth. Thus, delivery of HHO-1 gene by retroviral vector attenuates the development of hypertension and promotes body growth in spontaneously hypertensive rats.

  9. Do pH and flavonoids influence hypochlorous acid-induced catalase inhibition and heme modification?

    Science.gov (United States)

    Krych-Madej, Justyna; Gebicka, Lidia

    2015-09-01

    Hypochlorous acid (HOCl), highly reactive oxidizing and chlorinating species, is formed in the immune response to invading pathogens by the reaction of hydrogen peroxide with chloride catalyzed by the enzyme myeloperoxidase. Catalase, an important antioxidant enzyme, catalyzing decomposition of hydrogen peroxide to water and molecular oxygen, hampers in vitro HOCl formation, but is also one of the main targets for HOCl. In this work we have investigated HOCl-induced catalase inhibition at different pH, and the influence of flavonoids (catechin, epigallocatechin gallate and quercetin) on this process. It has been shown that HOCl-induced catalase inhibition is independent on pH in the range 6.0-7.4. Preincubation of catalase with epigallocatechin gallate and quercetin before HOCl treatment enhances the degree of catalase inhibition, whereas catechin does not affect this process. Our rapid kinetic measurements of absorption changes around the heme group have revealed that heme modification by HOCl is mainly due to secondary, intramolecular processes. The presence of flavonoids, which reduce active catalase intermediate, Compound I to inactive Compound II have not influenced the kinetics of HOCl-induced heme modification. Possible mechanisms of the reaction of hypochlorous acid with catalase are proposed and the biological consequences are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Heterologous expression and characterization of a new heme-catalase in Bacillus subtilis 168.

    Science.gov (United States)

    Philibert, Tuyishime; Rao, Zhiming; Yang, Taowei; Zhou, Junping; Huang, Genshu; Irene, Komera; Samuel, Niyomukiza

    2016-06-01

    Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent K m of catalytic activity was 59.6 ± 13 mM with higher turnover rate (K cat = 322.651 × 10(3) s(-1)). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe(2+) preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

  11. Efficiency and Loading Evaluation of High Efficiency Mist Eliminators (HEME) - 12003

    Energy Technology Data Exchange (ETDEWEB)

    Giffin, Paxton K.; Parsons, Michael S.; Waggoner, Charles A. [Institute for Clean Energy Technology, Mississippi State University, 205 Research Blvd Starkville, MS 39759 (United States)

    2012-07-01

    High efficiency mist eliminators (HEME) are filters primarily used to remove moisture and/or liquid aerosols from an air stream. HEME elements are designed to reduce aerosol and particulate load on primary High Efficiency Particulate Air (HEPA) filters and to have a liquid particle removal efficiency of approximately 99.5% for aerosols down to sub-micron size particulates. The investigation presented here evaluates the loading capacity of the element in the absence of a water spray cleaning system. The theory is that without the cleaning system, the HEME element will suffer rapid buildup of solid aerosols, greatly reducing the particle loading capacity. Evaluation consists of challenging the element with a waste surrogate dry aerosol and di-octyl phthalate (DOP) at varying intervals of differential pressure to examine the filtering efficiency of three different element designs at three different media velocities. Also, the elements are challenged with a liquid waste surrogate using Laskin nozzles and large dispersion nozzles. These tests allow the loading capacity of the unit to be determined and the effectiveness of washing down the interior of the elements to be evaluated. (authors)

  12. Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes.

    Science.gov (United States)

    Jian, Chongshu; Xu, Fengli; Hou, Tingting; Sun, Tao; Li, Jinghang; Cheng, Heping; Wang, Xianhua

    2017-08-01

    Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis. © 2017. Published by The Company of Biologists Ltd.

  13. Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

    OpenAIRE

    Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

    2013-01-01

    Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experi...

  14. Delayed globin synthesis leads to excess heme and the macrocytic anemia of Diamond Blackfan anemia and del(5q) myelodysplastic syndrome.

    Science.gov (United States)

    Yang, Zhantao; Keel, Siobán B; Shimamura, Akiko; Liu, Li; Gerds, Aaron T; Li, Henry Y; Wood, Brent L; Scott, Bart L; Abkowitz, Janis L

    2016-05-11

    Diamond Blackfan anemia (DBA) and myelodysplastic syndrome (MDS) with isolated del(5q) are severe macrocytic anemias; although both are associated with impaired ribosome assembly, why the anemia occurs is not known. We cultured marrow cells from DBA (n = 3) and del(5q) MDS (n = 6) patients and determined how heme (a toxic chemical) and globin (a protein) are coordinated. We show that globin translation initiates slowly, whereas heme synthesis proceeds normally. This results in insufficient globin protein, excess heme and excess reactive oxygen species in early erythroid precursors, and CFU-E (colony-forming unit-erythroid)/proerythroblast cell death. The cells that can more rapidly and effectively export heme or can slow heme synthesis preferentially survive and appropriately mature. Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS marrow cultures by 68 to 95% (P = 0.03 to 0.05), whereas the erythroid cell output of concurrent control marrow cultures decreased by 4 to 13%. Our studies demonstrate that erythropoiesis fails when heme exceeds globin. Our data further suggest that therapies that decrease heme synthesis (or facilitate heme export) could improve the red blood cell production of persons with DBA, del(5q) MDS, and perhaps other macrocytic anemias. Copyright © 2016, American Association for the Advancement of Science.

  15. Histidine at Position 195 is Essential for Association of Heme-b in Lcp1VH2

    Science.gov (United States)

    Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

    2018-03-01

    The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12, 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly(cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme-b in the central region of Lcp1VH2.

  16. Aldoxime dehydratase: probing the heme environment involved in the synthesis of the carbon-nitrogen triple bond.

    Science.gov (United States)

    Pinakoulaki, Eftychia; Koutsoupakis, Constantinos; Sawai, Hitomi; Pavlou, Andrea; Kato, Yasuo; Asano, Yasuhisa; Aono, Shigetoshi

    2011-11-10

    Fourier transform infrared (FTIR) spectra, "light" minus "dark" difference FTIR spectra, and time-resolved step-scan (TRS(2)) FTIR spectra are reported for carbonmonoxy aldoxime dehydratase. Two C-O modes of heme at 1945 and 1964 cm(-1) have been identified and remained unchanged in H(2)O/D(2)O exchange and in the pH 5.6-8.5 range, suggesting the presence of two conformations at the active site. The observed C-O frequencies are 5 and 16 cm(-1) lower and higher, respectively, than that obtained previously (Oinuma, K.-I.; et al. FEBS Lett.2004, 568, 44-48). We suggest that the strength of the Fe-His bond and the neutralization of the negatively charged propionate groups modulate the ν(Fe-CO)/ν(CO) back-bonding correlation. The "light" minus "dark" difference FTIR spectra indicate that the heme propionates are in both the protonated and deprotonated forms, and the photolyzed CO becomes trapped within a ligand docking site (ν(CO) = 2138 cm(-1)). The TRS(2)-FTIR spectra show that the rate of recombination of CO to the heme is k(1945 cm(-1)) = 126 ± 20 s(-1) and k(1964 cm(-1)) = 122 ± 20 s(-1) at pH 5.6, and k(1945 cm(-1)) = 148 ± 30 s(-1) and k(1964 cm(-1)) = 158 ± 32 s(-1) at pH 8.5. The rate of decay of the heme propionate vibrations is on a time scale coincident with the rate of rebinding, suggesting that there is a coupling between ligation dynamics in the distal heme environment and the environment sensed by the heme propionates. The implications of these results with respect to the proximal His-Fe heme environment including the propionates and the positively charged or proton-donating residues in the distal pocket which are crucial for the synthesis of nitriles are discussed.

  17. Differences and Comparisons of the Properties and Reactivities of Iron(III)–hydroperoxo Complexes with Saturated Coordination Sphere

    Science.gov (United States)

    Faponle, Abayomi S; Quesne, Matthew G; Sastri, Chivukula V; Banse, Frédéric; de Visser, Sam P

    2015-01-01

    Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high-valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O–O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L52). Direct C–O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L52)FeIII(OOH)]2+ should be able to initiate an aromatic hydroxylation process, although a low-energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O–O bond, whereas a heterolytic O–O bond breaking in heme iron(III)–hydroperoxo is found. PMID:25399782

  18. Density functional theory calculations of the lowest energy quintet and triplet states of model hemes: role of functional, basis set, and zero-point energy corrections.

    Science.gov (United States)

    Khvostichenko, Daria; Choi, Andrew; Boulatov, Roman

    2008-04-24

    We investigated the effect of several computational variables, including the choice of the basis set, application of symmetry constraints, and zero-point energy (ZPE) corrections, on the structural parameters and predicted ground electronic state of model 5-coordinate hemes (iron(II) porphines axially coordinated by a single imidazole or 2-methylimidazole). We studied the performance of B3LYP and B3PW91 with eight Pople-style basis sets (up to 6-311+G*) and B97-1, OLYP, and TPSS functionals with 6-31G and 6-31G* basis sets. Only hybrid functionals B3LYP, B3PW91, and B97-1 reproduced the quintet ground state of the model hemes. With a given functional, the choice of the basis set caused up to 2.7 kcal/mol variation of the quintet-triplet electronic energy gap (DeltaEel), in several cases, resulting in the inversion of the sign of DeltaEel. Single-point energy calculations with triple-zeta basis sets of the Pople (up to 6-311G++(2d,2p)), Ahlrichs (TZVP and TZVPP), and Dunning (cc-pVTZ) families showed the same trend. The zero-point energy of the quintet state was approximately 1 kcal/mol lower than that of the triplet, and accounting for ZPE corrections was crucial for establishing the ground state if the electronic energy of the triplet state was approximately 1 kcal/mol less than that of the quintet. Within a given model chemistry, effects of symmetry constraints and of a "tense" structure of the iron porphine fragment coordinated to 2-methylimidazole on DeltaEel were limited to 0.3 kcal/mol. For both model hemes the best agreement with crystallographic structural data was achieved with small 6-31G and 6-31G* basis sets. Deviation of the computed frequency of the Fe-Im stretching mode from the experimental value with the basis set decreased in the order: nonaugmented basis sets, basis sets with polarization functions, and basis sets with polarization and diffuse functions. Contraction of Pople-style basis sets (double-zeta or triple-zeta) affected the results

  19. Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

    Science.gov (United States)

    Assunção-Miranda, I; Cruz-Oliveira, C; Neris, R L S; Figueiredo, C M; Pereira, L P S; Rodrigues, D; Araujo, D F F; Da Poian, A T; Bozza, M T

    2016-03-01

    To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV. © 2016 The Society for Applied Microbiology.

  20. On the molecular basis of the activity of the antimalarial drug chloroquine: EXAFS-assisted DFT evidence of a direct Fe–N bond with free heme in solution

    International Nuclear Information System (INIS)

    Macetti, Giovanni; Rizzato, Silvia; Beghi, Fabio; Presti, Leonardo Lo; Silvestrini, Lucia

    2016-01-01

    4-aminoquinoline antiplasmodials interfere with the biocrystallization of the malaria pigment, a key step of the malaria parasite metabolism. It is commonly believed that these drugs set stacking π···π interactions with the Fe-protoporphyrin scaffold of the free heme, even though the details of the heme:drug recognition process remain elusive. In this work, the local coordination of Fe(III) ions in acidic solutions of hematin at room temperature was investigated by extended x-ray absorption fine structure (EXAFS) spectroscopy in the 4.0–5.5 pH range, both in the presence and in the absence of the antimalarial drug chloroquine. EXAFS results were complemented by DFT simulations in polarizable continuum media to model solvent effects. We found evidence that a complex where the drug quinoline nitrogen is coordinated with the iron center might coexist with formerly proposed adduct geometries, based on stacking interactions. Charge-assisted hydrogen bonds among lateral chains of the two molecules play a crucial role in stabilizing this complex, whose formation is favored by the presence of lipid micelles. The direct Fe–N bond could reversibly block the axial position in the Fe 1st coordination shell in free heme, acting as an inhibitor for the crystallization of the malaria pigment without permanently hampering the catalytic activity of the redox center. These findings are discussed in the light of possible implications on the engineering of drugs able to thwart the adaptability of the malaria parasite against classical aminoquinoline-based therapies. (invited comment)

  1. On the molecular basis of the activity of the antimalarial drug chloroquine: EXAFS-assisted DFT evidence of a direct Fe-N bond with free heme in solution

    Science.gov (United States)

    Macetti, Giovanni; Rizzato, Silvia; Beghi, Fabio; Silvestrini, Lucia; Lo Presti, Leonardo

    2016-02-01

    4-aminoquinoline antiplasmodials interfere with the biocrystallization of the malaria pigment, a key step of the malaria parasite metabolism. It is commonly believed that these drugs set stacking π···π interactions with the Fe-protoporphyrin scaffold of the free heme, even though the details of the heme:drug recognition process remain elusive. In this work, the local coordination of Fe(III) ions in acidic solutions of hematin at room temperature was investigated by extended x-ray absorption fine structure (EXAFS) spectroscopy in the 4.0-5.5 pH range, both in the presence and in the absence of the antimalarial drug chloroquine. EXAFS results were complemented by DFT simulations in polarizable continuum media to model solvent effects. We found evidence that a complex where the drug quinoline nitrogen is coordinated with the iron center might coexist with formerly proposed adduct geometries, based on stacking interactions. Charge-assisted hydrogen bonds among lateral chains of the two molecules play a crucial role in stabilizing this complex, whose formation is favored by the presence of lipid micelles. The direct Fe-N bond could reversibly block the axial position in the Fe 1st coordination shell in free heme, acting as an inhibitor for the crystallization of the malaria pigment without permanently hampering the catalytic activity of the redox center. These findings are discussed in the light of possible implications on the engineering of drugs able to thwart the adaptability of the malaria parasite against classical aminoquinoline-based therapies.

  2. Copper(II) complex as a precursor for formation of cyano-bridged ...

    Indian Academy of Sciences (India)

    chemsci

    structural similarities to the natural hexaazamacrocyclic complexes such as heme in human red blood cell, chlorophyll A and B, and vitamin B12.1 ...... and phenolic contents of medicinal plants used by the. Venda people for conditions related ...

  3. Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

    Science.gov (United States)

    Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

    2014-01-01

    Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

  4. Effects of Metalloporphyrins on Heme Oxygenase-1 Transcription: Correlative Cell Culture Assays Guide in Vivo Imaging

    Directory of Open Access Journals (Sweden)

    Monica Hajdena-Dawson

    2003-07-01

    Full Text Available Heme oxygenase (HO is the rate-limiting step in the heme degradation pathway and is a potential target for the control, or prevention, of pathologic jaundice in neonates. Metalloporphyrins (Mps, a diverse set of synthetic derivatives of heme, can competitively inhibit the HO enzymes. However, certain Mps are phototoxic and some increase transcription of HO-1, the inducible HO isozyme. Therefore, effective development of this class of compounds as therapeutics for treating pathologic jaundice will require rapid and integrated biological screens to identify the most efficacious and safe Mps. To study the safety of these compounds, we assessed their cytotoxic effects and measured luciferase activity by bioluminescent imaging (BLI as an index of HO-1 transcription, first in live cell cultures and then in living transgenic reporter mice. A total of 12 Mps were first evaluated in the correlative cell culture assay. Based on results from this study, 2 Mps, zinc protoporphyrin (ZnPP and zinc bis glycol porphyrin (ZnBG, were selected for further studies in the live animal model. In vitro BLI showed ZnPP to be a strong inducer of HO-1 transcription in comparison to ZnBG, which showed minimal induction. Cytotoxicity studies revealed that ZnPP was phototoxic, whereas ZnBG had no effect on cell viability. In vivo BLI showed that both ZnPP and ZnBG had minimal effects on the levels of HO-1 transcription in the animals. Furthermore, serum enzyme assays indicated that neither caused detectable liver toxicity. These findings, and especially those with ZnBG, support the use of selected Mps as therapies for pathologic jaundice. Coupling the high throughput advantage of cell culture with the capability of imaging for whole-body temporal analyses could accelerate and refine the preclinical phases of drug development. Thus, this study serves as a model for understanding the effects of specific compounds in relation to defined targets using an integrated approach.

  5. Meat, Dietary Heme Iron, and Risk of Type 2 Diabetes Mellitus: The Singapore Chinese Health Study.

    Science.gov (United States)

    Talaei, Mohammad; Wang, Ye-Li; Yuan, Jian-Min; Pan, An; Koh, Woon-Puay

    2017-10-01

    We evaluated the relationships of red meat, poultry, fish, and shellfish intakes, as well as heme iron intake, with the risk of type 2 diabetes mellitus (T2D).The Singapore Chinese Health Study is a population-based cohort study that recruited 63,257 Chinese adults aged 45-74 years from 1993 to 1998. Usual diet was evaluated using a validated 165-item semiquantitative food frequency questionnaire at recruitment. Physician-diagnosed T2D was self-reported during 2 follow-up interviews in 1999-2004 and 2006-2010. During a mean follow-up of 10.9 years, 5,207 incident cases of T2D were reported. When comparing persons in the highest intake quartiles with those in the lowest, the multivariate-adjusted hazard ratio for T2D was 1.23 (95% confidence interval (CI): 1.14, 1.33) for red meat intake (P for trend meat intake remained significantly associated with T2D risk (multivariate-adjusted hazard ratio = 1.13, 95% CI: 1.01, 1.25; P for trend = 0.02). Heme iron was associated with a higher risk of T2D even after additional adjustment for red meat intake (multivariate-adjusted hazard ratio = 1.14, 95% CI: 1.02, 1.28; P for trend = 0.03). In conclusion, red meat and poultry intakes were associated with a higher risk of T2D. These associations were mediated completely for poultry and partially for red meat by heme iron intake. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Genetic analyses of heme oxygenase 1 (HMOX1 in different forms of pancreatitis.

    Directory of Open Access Journals (Sweden)

    Sebastian Weis

    Full Text Available Heme oxygenase 1 (HMOX1 is the rate limiting enzyme in heme degradation and a key regulator of inflammatory processes. In animal models the course of pancreatitis was ameliorated by up-regulation of HMOX1 expression. Additionally, carbon monoxide released during heme breakdown inhibited proliferation of pancreatic stellate cells and might thereby prevent the development of chronic pancreatitis (CP. Transcription of HMOX1 in humans is influenced by a GT-repeat located in the promoter. As such, HMOX1 variants might be of importance in the pathogenesis of pancreatitis.The GT-repeat and SNP rs2071746 were investigated with fluorescence labelled primers and by melting curve analysis in 285 patients with acute pancreatitis, 208 patients with alcoholic CP, 207 patients with idiopathic/hereditary CP, 147 patients with alcoholic liver cirrhosis, and in 289 controls, respectively. GT-repeat analysis was extended to a total of 446 alcoholic CP patients. In addition, we performed DNA sequencing in 145 patients with alcoholic CP, 138 patients with idiopathic/hereditary CP, 147 patients with alcoholic liver cirrhosis, and 151 controls. Exon 3 screening was extended to additional patients and controls.S- and L-alleles of the GT-repeat, genotypes and alleles of SNP rs2071746 and non-synonymous variants detected by sequencing were found with similar frequencies in all groups.Although functional data implicate a potential influence of HMOX1 variants on the pathogenesis of pancreatitis, we did not find any association. As rare non-synonymous HMOX1 variants were found in patients and controls, it is rather unlikely that they will have functional consequences essential for pancreatitis development.

  7. Cloning and characterization of a heme oxygenase-2 gene from alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Fu, Guang-Qing; Jin, Qi-Jiang; Lin, Yu-Ting; Feng, Jian-Fei; Nie, Li; Shen, Wen-Biao; Zheng, Tian-Qing

    2011-11-01

    Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H(2)O(2) (especially) treatment, respectively.

  8. Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells

    Directory of Open Access Journals (Sweden)

    Kaori Yama

    2015-04-01

    Full Text Available Epalrestat (EPS is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs, an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

  9. Altered activity of heme biosynthesis pathway enzymes in individuals chronically exposed to arsenic in Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Zavala, A.; Del Razo, L.M.; Garcia-Vargas, G.G.; Aguilar, C.; Borja, V.H.; Albores, A.; Cebrian, M.E. [CINVESTAV-IPN, Mexico (Mexico). Dept. de Farmacologia y Toxicologica

    1999-03-01

    Our objective was to evaluate the activities of some enzymes of the heme biosynthesis pathway and their relationship with the profile of urinary porphyrin excretion in individuals exposed chronically to arsenic (As) via drinking water in Region Lagunera, Mexico. We selected 17 individuals from each village studied: Benito Juarez, which has current exposure to 0.3 mg As/l; Santa Ana, where individuals have been exposed for more than 35 years to 0.4 mg As/l, but due to changes in the water supply (in 1992) exposure was reduced to its current level (0.1 mg As/l), and Nazareno, with 0.014 mg As/l. Average arsenic concentrations in urine were 2058, 398, and 88 {mu}g As/g creatinine, respectively. The more evident alterations in heme metabolism observed in the highly exposed individuals were: (1) small but significant increases in porphobilinogen deaminase (PBG-D) and uroporphyrinogen decarboxylase (URO-D) activities in peripheral blood erythrocytes; (2) increases in the urinary excretion of total porphyrins, mainly due to coproporphyrin III (COPROIII) and uroporphyrin III (UROIII); and (3) increases in the COPRO/URO and COPROIII/COPROI ratios. No significant changes were observed in uroporphyrinogen III synthetase (UROIII-S) activity. The direct relationships between enzyme activities and urinary porphyrins, suggest that the increased porphyrin excretion was related to PBG-D, whereas the increased URO-D activity would enhance coproporphyrin synthesis and excretion at the expense of uroporphyrin. None of the human studies available have reported the marked porphyric response and enzyme inhibition observed in rodents. In conclusion, chronic As exposure alters human heme metabolism; however the severity of the effects appears to depend on characteristics of exposure not yet fully characterized. (orig.) With 1 fig., 3 tabs., 20 refs.

  10. Heme oxygenase-1: A new druggable target in the management of chronic and acute myeloid leukemia.

    Science.gov (United States)

    Salerno, Loredana; Romeo, Giuseppe; Modica, Maria N; Amata, Emanuele; Sorrenti, Valeria; Barbagallo, Ignazio; Pittalà, Valeria

    2017-12-15

    Heme oxygenase-1 (HO-1) is the enzyme catalyzing the rate-limiting oxidative degradation of cellular heme into free iron, carbon monoxide (CO), and biliverdin, which is then rapidly converted into bilirubin. By means of these catabolic end-products and by removal of pro-oxidant heme, HO-1 exerts antioxidant, antiapoptotic, and immune-modulating effects, leading to overall cytoprotective and beneficial functions in mammalian cells. Therefore, HO-1 is considered a survival molecule in various stress-related conditions. By contrast, growing evidence suggests that HO-1 is a survival-enhancing molecule also in various solid and blood cancers, such as various types of leukemia, promoting carcinogenesis, tumor progression, and chemo-resistance. Among leukemias, chronic myeloid leukemia (CML) is currently therapeutically well treated with tyrosine kinase inhibitors (TKIs) such as Imatinib (IM) and its congeners; nevertheless, resistance to all kinds of current drugs persist in a number of patients. Moreover, treatment outcomes for acute myeloid leukemia (AML) remain unsatisfactory, despite progress in chemotherapy and hematopoietic stem cell transplantation. Therefore, identification of new eligible targets that may improve leukemias therapy is of general interest. Several recent papers prove that inhibition of HO-1 through HO-1 inhibitors as well as modulation of other pathways involving HO-1 by a number of different new or known molecules, are critical for leukemia treatment. This review summarizes the current understanding of the pro-tumorigenic role of HO-1 and its potential as a molecular target for the treatment of leukemias. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. The Effect of Distal Interactions on O2-Binding to Heme

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Dasmeh, Pouria

    2013-01-01

    This paper reports DFT-computed electronic ground states, Mössbauer isomer shifts, O-O and Fe-O vibration frequencies, and thermodynamics of O2-binding of heme models representing different distal (position E7) interactions, strictly validated against experimental data. Based on the results...... hydrogen bonds can further reduce isomer shifts up to 0.07 mm/s. The O-O stretch vibration, the O-O distance, and the isomer shift possess substantial heuristic value in interpreting electronic structure, whereas other properties are less effective, based on computed correlation coefficients. Shorter Fe...

  12. Deformations of the Heme Group of Different Ferrocytochrome c Proteins Probed by Resonance Raman Spectroscopy

    International Nuclear Information System (INIS)

    Hagarman, Andrew; Schweitzer-Stenner, Reinhard; Wallace, Carmichael; Laberge, Monique

    2008-01-01

    We measured the low-frequency polarized resonance Raman spectra of horse heart, chicken, and yeast(C102T) ferrocytochromes c with Soret excitation. We examined the out-of-plane deformations of the heme groups by determining the relative intensities and depolarization ratios of a variety of out-of-plane and in-plane Raman active bands. Analysis of relative Raman intensities shows differences in non-planarity of the heme groups of yeast(C102T), horse heart and chicken cytochrome c. Cytochrome c has been shown to have a dominant ruffling (B 1u ) deformation by means of normal coordinate structural decomposition (NSD) analysis of the heme group in crystal structures. The presence and intensity of B 1u modes, γ 10 -γ 12 , support the indication of ruffling being the major contribution to the non-planar deformations in cytochrome c. Other types of non-planar deformations like doming (A 2U ) and waving (E g ) can be deduced from the Raman activity of γ 5 (A 2u ), γ 21 and γ 22 (E g ). The depolarization ratios of γ 5 , γ 10 , γ 11 and γ 12 are larger than 0.125, indicating the presence of other deformations such as saddling (B 2u ) and propellering (A 1u ), which is again in agreement with the crystal structures of horse heart and yeast ferrocytochrome c. An analysis of the intensities and depolarization ratios of out-of-plane modes revealed that ruffling is comparable in yeast and horse heart cytochrome c, saddling is larger and doming as well as propellering are lower in yeast cytochrome c. With respect to doming and ruffling our results contradict values obtained from the NSD analysis of the corresponding crystal structures. With respect to saddling, our data are in agreement with the crystal structure. The NSD analysis of heme structures resulting from MD simulations did not correlate very well with the spectroscopically obtained results concerning the ruffling and doming coordinate, whereas a qualitative agreement was again obtained for saddling.

  13. 4-Hydroxyestradiol induces mammary epithelial cell transformation through Nrf2-mediated heme oxygenase-1 overexpression

    OpenAIRE

    Park, Sin-Aye; Lee, Mee-Hyun; Na, Hye-Kyung; Surh, Young-Joon

    2016-01-01

    Estrogen (17?-estradiol, E2) undergoes oxidative metabolism by CYP1B1 to form 4-hydroxyestradiol (4-OHE2), a putative carcinogenic metabolite of estrogen. Our previous study showed that 4-OHE2-induced production of reactive oxygen species contributed to neoplastic transformation of human breast epithelial (MCF-10A) cells. In this study, 4-OHE2, but not E2, increased the expression of heme oxygenase-1 (HO-1), a sensor and regulator of oxidative stress, in MCF-10A cells. Silencing the HO-1 gene...

  14. Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin

    Directory of Open Access Journals (Sweden)

    Benvenisti-Zarom Luna

    2004-09-01

    Full Text Available Abstract Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed

  15. Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1.

    OpenAIRE

    Otto, B R; Kusters, J G; Luirink, J; de Graaf, F K; Oudega, B

    1996-01-01

    An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open readi...

  16. Mono- and binuclear non-heme iron chemistry from a theoretical perspective

    Czech Academy of Sciences Publication Activity Database

    Rokob, T. A.; Chalupský, Jakub; Bím, Daniel; Andrikopoulos, Prokopis C.; Srnec, Martin; Rulíšek, Lubomír

    2016-01-01

    Roč. 21, 5/6 (2016), s. 619-644 ISSN 0949-8257 R&D Projects: GA ČR(CZ) GJ15-10279Y; GA ČR(CZ) GA14-31419S; GA ČR GA15-19143S Grant - others:COST(XE) CM1305 Institutional support: RVO:61388963 ; RVO:61388955 Keywords : non-heme iron * density functional theory * multireference methods * dioxygen activation * reactivity Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.894, year: 2016

  17. Moessbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

    Energy Technology Data Exchange (ETDEWEB)

    Harami, Taikan, E-mail: harami.taikan@jaea.go.jp [Japan Atomic Energy Agency (Japan); Kitao, Shinji; Kobayashi, Yasuhiro [Kyoto University, Research Reactor Institute (Japan); Mitsui, Takaya [Japan Atomic Energy Agency (Japan)

    2008-01-15

    The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Moessbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Moessbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.

  18. Heme iron content in lamb meat is differentially altered upon boiling, grilling, or frying as assessed by four distinct analytical methods.

    Science.gov (United States)

    Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

    2013-01-01

    Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

  19. Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

    Directory of Open Access Journals (Sweden)

    Azin Pourkhalili

    2013-01-01

    Full Text Available Lamb meat is regarded as an important source of highly bioavailable iron (heme iron in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

  20. LC-MS of Metmyoglobin at pH = 2: Separation and Characterization of Apomyoglobin and Heme by ESI-MS and UV-Vis

    Science.gov (United States)

    Stynes, Helen Cleary; Layo, Araceli; Smith, Richard W.

    2004-01-01

    The protein species of apomyoglobin (apoMb) and heme are freed and segregated from the aqueous protein solution of metmyoglobin by liquid chromatography, and are distinguished by UV-Vis absorption or electrospray ionization mass spectrometry (ESI-MS). This is an ingenious and effective approach to characterize apomyoglobin and heme, while students…

  1. Structural and Functional Models of Non-Heme Iron Enzymes : A Study of the 2-His-1-Carboxylate Facial Triad Structural Motif

    NARCIS (Netherlands)

    Bruijnincx, P.C.A.

    2007-01-01

    The structural and functional modeling of a specific group of non-heme iron enzymes by the synthesis of small synthetic analogues is the topic of this thesis. The group of non-heme iron enzymes with the 2-His-1-carboxylate facial triad has recently been established as a common platform for the

  2. Up-regulation of A1M/α1-microglobulin in skin by heme and reactive oxygen species gives protection from oxidative damage.

    Science.gov (United States)

    Olsson, Magnus G; Allhorn, Maria; Larsson, Jörgen; Cederlund, Martin; Lundqvist, Katarina; Schmidtchen, Artur; Sørensen, Ole E; Mörgelin, Matthias; Akerström, Bo

    2011-01-01

    During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α(1)-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (K(d) = 0.96×10(-6) M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.

  3. Heme oxygenase is not involved in the anti-proliferative effects of statins on pancreatic cancer cells

    International Nuclear Information System (INIS)

    Vanova, K.; Boukalova, S.; Gbelcova, H.; Muchova, L.; Neuzil, J.; Gurlich, R.; Ruml, T.; Vitek, L.

    2016-01-01

    Pancreatic cancer is recognized as one of the most fatal tumors due to its aggressiveness and resistance to therapy. Statins were previously shown to inhibit the proliferation of cancer cells via various signaling pathways. In healthy tissues, statins activate the heme oxygenase pathway, nevertheless the role of heme oxygenase in pancreatic cancer is still controversial. The aim of this study was to evaluate, whether anti-proliferative effects of statins in pancreatic cancer cells are mediated via the heme oxygenase pathway. In vitro effects of various statins and hemin, a heme oxygenase inducer, on cell proliferation were evaluated in PA-TU-8902, MiaPaCa-2 and BxPC-3 human pancreatic cancer cell lines. The effect of statins on heme oxygenase activity was assessed and heme oxygenase-silenced cells were used for pancreatic cancer cell proliferation studies. Cell death rate and reactive oxygen species production were measured in PA-TU-8902 cells, followed by evaluation of the effect of cerivastatin on GFP-K-Ras trafficking and expression of markers of invasiveness, osteopontin (SPP1) and SOX2. While simvastatin and cerivastatin displayed major anti-proliferative properties in all cell lines tested, pravastatin did not affect the cell growth at all. Strong anti-proliferative effect was observed also for hemin. Co-treatment of cerivastatin and hemin increased anti-proliferative potential of these agents, via increased production of reactive oxygen species and cell death compared to individual treatment. Heme oxygenase silencing did not prevent pancreatic cancer cells from the tumor-suppressive effect of cerivastatin or hemin. Cerivastatin, but not pravastatin, protected Ras protein from trafficking to the cell membrane and significantly reduced expressions of SPP1 (p < 0.05) and SOX2 (p < 0.01). Anti-proliferative effects of statins and hemin on human pancreatic cancer cell lines do not seem to be related to the heme oxygenase pathway. While hemin triggers reactive

  4. Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS.

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    Natália Pereira de Almeida Nogueira

    Full Text Available Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.

  5. NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis*

    Science.gov (United States)

    Adamczack, Julia; Hoffmann, Martin; Papke, Ulrich; Haufschildt, Kristin; Nicke, Tristan; Bröring, Martin; Sezer, Murat; Weimar, Rebecca; Kuhlmann, Uwe; Hildebrandt, Peter; Layer, Gunhild

    2014-01-01

    Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1. PMID:25204657

  6. Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

    Directory of Open Access Journals (Sweden)

    Nitin Puri

    2017-01-01

    Full Text Available Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin-a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO is a stress response system which reduces oxidative stress. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in hepatic tissues from lean and obese mice. Obese mice exhibited hyperglycemia (p<0.05; increased levels of proinflammatory cytokines (MCP-1, IL-6, p<0.05; oxidative stress (p<0.05; and increased hepatic hepcidin levels (p<0.05. Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p<0.05. However, this effect is accompanied by a significant decline in ferritin expression. Additionally, there are reduced insulin receptor phosphorylation and attenuation of metabolic regulators pAMPK, pAKT, and pLKB1. Cobalt protoporphyrin- (CoPP- induced HO-1 upregulation in obese mice reversed these alterations (p<0.05, while attenuating hepatic hepcidin levels. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP (p<0.05. Our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin.

  7. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo [Food and Drug Administration, Silver Spring, MD (United States); Yamada, Kenneth M. [National Institutes of Health, Bethesda, MD (United States); Dhawan, Subhash, E-mail: subhash.dhawan@fda.hhs.gov [Food and Drug Administration, Silver Spring, MD (United States)

    2017-03-15

    We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retarding ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection. - Highlights: •Hemin treatment protected monocyte-derived macrophages against Zika virus (ZIKV) infection. •Innate cellular protection against ZIKV infection correlated with Nrf2-dependent HO-1 expression. •Stimulation of innate cellular responses may provide a therapeutic strategy against ZIKV infection.

  8. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

    International Nuclear Information System (INIS)

    Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo; Yamada, Kenneth M.; Dhawan, Subhash

    2017-01-01

    We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retarding ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection. - Highlights: •Hemin treatment protected monocyte-derived macrophages against Zika virus (ZIKV) infection. •Innate cellular protection against ZIKV infection correlated with Nrf2-dependent HO-1 expression. •Stimulation of innate cellular responses may provide a therapeutic strategy against ZIKV infection.

  9. Structures of the multicomponent Rieske non-heme iron toluene 2, 3-dioxygenase enzyme system

    Energy Technology Data Exchange (ETDEWEB)

    Friemann, Rosmarie [Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden); Lee, Kyoung [Department of Microbiology, Changwon National University, Changwon, Kyoungnam 641-773 (Korea, Republic of); Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242 (United States); Brown, Eric N. [Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (United States); Gibson, David T. [Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242 (United States); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden); Ramaswamy, S., E-mail: s-ramaswamy@uiowa.edu [Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (United States); Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden)

    2009-01-01

    The crystal structures of the three-component toluene 2, 3-dioxygenase system provide a model for electron transfer among bacterial Rieske non-heme iron dioxygenases. Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2, 3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe–2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe–2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.

  10. Non-coding RNAs and heme oxygenase-1 in vaccinia virus infection

    International Nuclear Information System (INIS)

    Meseda, Clement A.; Srinivasan, Kumar; Wise, Jasen; Catalano, Jennifer; Yamada, Kenneth M.; Dhawan, Subhash

    2014-01-01

    Highlights: • Heme oxygenase-1 (HO-1) induction inhibited vaccinia virus infection of macrophages. • Reduced infectivity inversely correlated with increased expression of non-coding RNAs. • The regulation of HO-1 and ncRNAs suggests a novel host defense response against vaccinia virus infection. - Abstract: Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression, and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection

  11. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    International Nuclear Information System (INIS)

    Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

    2014-01-01

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress

  12. Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

    International Nuclear Information System (INIS)

    Park, Kiyoung; Li, Ning; Kwak, Yeonju; Srnec, Martin

    2017-01-01

    Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2 . Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reaction shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.

  13. Inhibitory effects of a novel Val to Thr mutation on the distal heme of human catalase.

    Science.gov (United States)

    Mashhadi, Zahra; Boeglin, William E; Brash, Alan R

    2014-11-01

    True catalases efficiently breakdown hydrogen peroxide, whereas the catalase-related enzyme allene oxide synthase (cAOS) is completely unreactive and instead metabolizes a fatty acid hydroperoxide. In cAOS a Thr residue adjacent to the distal His restrains reaction with H2O2 (Tosha et al. (2006) J. Biol. Chem. 281:12610; De Luna et al. (2013) J. Phys. Chem. B 117: 14635) and its mutation to the consensus Val of true catalases permits the interaction. Here we investigated the effects of the reciprocal experiment in which the Val74 of human catalase is mutated to Thr, Ser, Met, Pro, or Ala. The Val74Thr substitution decreased catalatic activity by 3.5-fold and peroxidatic activity by 3-fold. Substitution with Ser had similar negative effects (5- and 3-fold decreases). Met decreased catalatic activity 2-fold and eliminated peroxidatic activity altogether, whereas the Val74Ala substitution was well tolerated. (The Val74Pro protein lacked heme). We conclude that the conserved Val74 of true catalases helps optimize catalysis. There are rare substitutions of Val74 with Ala, Met, or Pro, but not with Ser of Thr, possibly due their hydrogen bonding affecting the conformation of His75, the essential distal heme residue for activity in catalases. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  14. Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia.

    Science.gov (United States)

    Hascalovici, Jacob R; Song, Wei; Vaya, Jacob; Khatib, Soliman; Fuhrman, Bianca; Aviram, Michael; Schipper, Hyman M

    2009-01-01

    Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.

  15. Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

    Science.gov (United States)

    Smirnov, Valeriy V; Roth, Justine P

    2014-10-01

    Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

  16. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  17. Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme.

    Science.gov (United States)

    Brown, Breann L; Kardon, Julia R; Sauer, Robert T; Baker, Tania A

    2018-04-03

    5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Hormonal fluctuations during the estrous cycle modulate Heme Oxygenase-1 expression in the uterus

    Directory of Open Access Journals (Sweden)

    Maria Laura Zenclussen

    2014-03-01

    Full Text Available Deletion of the Heme Oxygenase-1 (Hmox1 locus in mice results in intrauterine lethality. The expression of the heme catabolyzing enzyme encoded by this gene, namely HO 1, is required to successfully support reproductive events. We have previously observed that HO-1 acts at several key events in reproduction ensuring pregnancy. HO-1 defines ovulation, positively influences implantation and placentation and ensures fetal growth and survival. Here, we embarked on a study aimed to determine whether hormonal changes during the estrous cycle in the mouse define HO-1 expression, thus influencing receptivity. We analyzed the serum levels of progesterone and estrogen by ELISA and HO-1 mRNA expression in uterus by real time RT-PCR at the metestrus, proestrus, estrus and diestrus phases of the estrous cycle. Further, we studied the HO-1 protein expression by Western Blot upon hormone addition to cultured uterine AN3 cells. We observed that HO-1 variations in uterine tissue correlated to changes in hormonal levels at different phases of the estrus cycle. In vitro, HO-1 protein levels in AN3 cells augmented after the addition of physiological concentrations of progesterone and estradiol, which confirmed our in vivo observations. Our data suggest an important role for hormones in HO-1 regulation in uterus that has a significant impact in receptivity and later on blastocyst implantation.

  19. Acute stress-induced antinocicept