Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane.

Science.gov (United States)

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-04-01

The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

Structural features and dynamic investigations of the membrane-bound cytochrome P450 17A1.

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Cui, Ying-Lu; Xue, Qiao; Zheng, Qing-Chuan; Zhang, Ji-Long; Kong, Chui-Peng; Fan, Jing-Rong; Zhang, Hong-Xing

2015-10-01

Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the crystallographic structures available for CYP17A1, no membrane-bound structural features of this enzyme at atomic level are available. Accumulating evidence has indicated that the interactions between bounded CYPs and membrane could contribute to the recruitment of lipophilic substrates. To this end, we have investigated the effects on structural characteristics in the presence of the membrane for CYP17A1. The MD simulation results demonstrate a spontaneous insertion process of the enzyme to the lipid. Two predominant modes of CYP17A1 in the membrane are captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. The measured heme tilt angles show good consistence with experimental data, thereby verifying the validity of the structural models. Moreover, conformational changes induced by the membrane might have impact on the accessibility of the active site to lipophilic substrates. The dynamics of internal aromatic gate formed by Trp220 and Phe224 are suggested to regulate tunnel opening motions. The knowledge of the membrane binding characteristics could guide future experimental and computational works on membrane-bound CYPs so that various investigations of CYPs in their natural, lipid environment rather than in artificially solubilized forms may be achieved. Copyright © 2015. Published by Elsevier B.V.

Biogenesis of the yeast cytochrome bc1 complex.

Science.gov (United States)

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

International Nuclear Information System (INIS)

Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

2008-01-01

Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore

Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3 enzymes

DEFF Research Database (Denmark)

Li, Mengqiu; Khan, Sanobar; Rong, Honglin

2017-01-01

The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from...... Escherichia coli, cytochrome bo3, for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055–16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P......-side of single HCOs. Proton transport activity of cytochrome bo3 was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH 6.4–8.4, while proton release at the P-side had an optimum pH of ~ 7.4, suggesting...

Production and characterization of yeast cytochrome c antibodies; immunological studies of mutants with altered cytochrome c synthesis

International Nuclear Information System (INIS)

Matner, R.R.

1980-01-01

Mutations at the structural gene, CYC1, for iso-1-cytochrome c and at the structural gene, CYC7, for iso-2-cytochrome c can reduce the levels of the respective proteins by varying degrees in Saccharomyces cerevisiae. Mutations at two other loci, cyc2 and cyc3, that are unlinked to either of the structural genes, specifically reduced the levels of both iso-cytochromes c. The cyc2 mutations can cause as low as 10 to 20% of the normal level and cyc3 mutations can cause complete deficiencies. We have explored the possiblity that the CYC2 and CYC3 loci code for maturation functions in the biosynthesis of cytochrome c. The approach used to characterize the nature of the cyc2 and cyc3 induced deficiencies of cytochrome c involved four steps. The results were used to propose possible roles for the CYC2 and CYC3 encoded functions. The CYC3 encoded function is hypothesized to be enzymatic heme attachment. CYC2 may code for a protein that binds and transports apo-cytochrome c through the outer mitochondrial membrane and/or enhances the activity of the heme attachment enzyme

Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

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Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

2017-04-01

The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

Isolation and purification of membrane-bound cytochrome c from ...

African Journals Online (AJOL)

Administrator

2007-05-02

ferrochrome and redox spectra showed the presence of heme-c. Key words: Cytochrome c, respiratory chain and Proteus mirabilis. INTRODUCTION. Proteus mirabilis is facultative anaerobic, rod-shaped, gram negative bacterium.

Cell-secreted flavins bound to membrane cytochromes dictate electron transfer reactions to surfaces with diverse charge and pH.

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Okamoto, Akihiro; Kalathil, Shafeer; Deng, Xiao; Hashimoto, Kazuhito; Nakamura, Ryuhei; Nealson, Kenneth H

2014-07-11

The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.

Cytochrome b 6 f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow.

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Dumas, Louis; Chazaux, Marie; Peltier, Gilles; Johnson, Xenie; Alric, Jean

2016-09-01

Both the structure and the protein composition of thylakoid membranes have an impact on light harvesting and electron transfer in the photosynthetic chain. Thylakoid membranes form stacks and lamellae where photosystem II and photosystem I localize, respectively. Light-harvesting complexes II can be associated to either PSII or PSI depending on the redox state of the plastoquinone pool, and their distribution is governed by state transitions. Upon state transitions, the thylakoid ultrastructure and lateral distribution of proteins along the membrane are subject to significant rearrangements. In addition, quinone diffusion is limited to membrane microdomains and the cytochrome b 6 f complex localizes either to PSII-containing grana stacks or PSI-containing stroma lamellae. Here, we discuss possible similarities or differences between green algae and C3 plants on the functional consequences of such heterogeneities in the photosynthetic electron transport chain and propose a model in which quinones, accepting electrons either from PSII (linear flow) or NDH/PGR pathways (cyclic flow), represent a crucial control point. Our aim is to give an integrated description of these processes and discuss their potential roles in the balance between linear and cyclic electron flows.

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1.

Directory of Open Access Journals (Sweden)

Alexandr N Simonov

Full Text Available Cytochrome P450c17 (P450 17A1, CYP17A1 is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.

Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b/sub 5/ and cytochrome P-450/sub cam/

Energy Technology Data Exchange (ETDEWEB)

Vickery, L; Salmon, A; Sauer, K

1975-01-01

Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b/sub 5/ and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome b/sub 5/ from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450/sub cam/ from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome b/sub 5/ are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450/sub cam/ also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

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Balodite, Elina; Strazdina, Inese; Galinina, Nina; McLean, Samantha; Rutkis, Reinis; Poole, Robert K; Kalnenieks, Uldis

2014-09-01

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase. © 2014 The Authors.

Removal of Bound Triton X-100 from Purified Bovine Heart Cytochrome bc1

OpenAIRE

Varhač, Rastislav; Robinson, Neal C.; Musatov, Andrej

2009-01-01

Cytochrome bc1 isolated from Triton X-100 solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nmol of the enzyme. Purified cytochrome bc1 is fully active; however, protein bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc1 was accomplished by incubation with Bio-Beads SM-2 in presence of sodium cholate. Sodium cholate is critical since it does not interfere with the adsorpt...

The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

Science.gov (United States)

Bickar, D; Turrens, J F; Lehninger, A L

1986-11-05

When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.

Tributyltin interacts with mitochondria and induces cytochrome c release.

Science.gov (United States)

Nishikimi, A; Kira, Y; Kasahara, E; Sato, E F; Kanno, T; Utsumi, K; Inoue, M

2001-01-01

Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis. PMID:11368793

Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

International Nuclear Information System (INIS)

Ekstroem, G.; Norsten, C.; Cronholm, T.; Ingelman-Sundberg, M.

1987-01-01

Deuterium isotope effects [/sup D/(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled of [1,1- 2 H 2 ] ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1- 13 C]- and [ 2 H 6 ] ethanol or [2,2,2- 2 H 3 ]- and [1,1- 2 H 2 ] ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The /sup D/(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM 2 oxidized the alcohol with /sup D/(V/K) of about 2.8 and 1.8, respectively. Addition of Fe/sup III/EDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect. Incubations of all cytochrome P-450 containing systems of the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1- 2 H] ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen. The data indicate that cytochrome P-450 dependent ethanol oxidation is not stereospecific and that cleavage of the C 1 -H bond appears to be a rate-determining step in the catalysis by the ethanol-inducible form of P-450. The contribution of hydroxyl radicals in ethanol oxidation by the various enzymic systems is discussed

A complex of cardiac cytochrome c1 and cytochrome c.

Science.gov (United States)

Chiang, Y L; Kaminsky, L S; King, T E

1976-01-10

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of

Structure and function of the tetraheme cytochrome associated to the reaction center of Roseobacter denitrificans.

Science.gov (United States)

Garcia, D; Richaud, P; Breton, J; Verméglio, A

1994-01-01

We have characterized the tetrahemic RC bound cytochrome isolated from the quasi-photosynthetic bacterium Roseobacter denitrificans in terms of absorption spectrum, redox property and orientation with respect to the membrane plane. The heme, designated H1, which possesses the highest redox midpoint potential (+290 mV), absorbs at 555 nm. Its plane makes an angle of 40 degrees with the membrane plane. The second high potential heme, H2 (+240 mV), peaks at 554 nm and makes a tilt of 55 degrees with the membrane. The two low potential hemes, L1 and L2, present a similar and rather high redox midpoint potential (+90 mV). They absorb at 553 nm and 550 nm. One of these hemes is oriented at 40 degrees while the other makes an angle of 90 degrees with the membrane plane. The soluble cytochrome c551 completes the cyclic electron transfer between the RC and the bc1 complex. Both the oxidation and the re-reduction of cytochrome c551 are diffusible processes. Under semi-aerobic conditions, one of the low potential hemes is photo-oxidized under illumination but only extremely slowly re-reduced. This explains the requirement of high aerobic conditions for growth of Roseobacter denitrificans cells in the light.

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

Science.gov (United States)

Papa, S; Lorusso, M; Izzo, G; Capuano, F

1981-02-15

component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

Science.gov (United States)

Iswari, S.; Palta, Jiwan P.

1989-01-01

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

Conjugation of cytochrome c with hydrogen titanate nanotubes: novel conformational state with implications for apoptosis

Energy Technology Data Exchange (ETDEWEB)

Ray, Moumita; Mazumdar, Shyamalava [Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India); Chatterjee, Sriparna; Das, Tanmay; Bhattacharyya, Somnath; Ayyub, Pushan, E-mail: somnath@tifr.res.in, E-mail: pushan@tifr.res.in, E-mail: shyamal@tifr.res.in [Department of Condensed Matter Physics and Materials Science, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India)

2011-10-14

We show that hydrogen titanate (H{sub 2}Ti{sub 3}O{sub 7}) nanotubes form strongly associated reversible nano-bio-conjugates with the vital respiratory protein, cytochrome c. Resonance Raman spectroscopy along with direct electrochemical studies indicate that in this nano-bio-conjugate, cytochrome c exists in an equilibrium of two conformational states with distinctly different formal redox potentials and coordination geometries of the heme center. The nanotube-conjugated cytochrome c also showed enhanced peroxidase activity similar to the membrane-bound protein that is believed to be an apoptosis initiator. This suggests that such a nanotube-cytochrome c conjugate may be a good candidate for cancer therapy applications.

Protein and DNA technologies for functional expression of membrane-associated cytochromes P450 in bacterial cell factories

DEFF Research Database (Denmark)

Vazquez Albacete, Dario

450 engineering guidelines and serves as platform to improve performance of microbial cells, thereby boosting recombinant production of complex plant P450-derived biochemicals. The knowledge generated, could guide future reconstruction of functional plant metabolic pathways leading to high valuable...... potential as medicines, fuels or food for humans. Plants conquered different environments thereby developing adaptation strategies based on the biosynthesis of a myriad of compounds. Unfortunately they are present in small amounts in plants and are too complex and to produce by organic chemical synthesis....... In most of biosynthetic pathways leading to these chemicals the cytochrome P450 enzyme family (P450s) is responsible for their final functionalization. However, the membrane-bound nature of P450s, makes their expression in microbial hosts a challenge. In order to meet the global demand for these natural...

Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b₅₅₈ (SdhC).

Science.gov (United States)

Baureder, Michael; Hederstedt, Lars

2011-11-01

Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

Science.gov (United States)

Renault, Hugues; De Marothy, Minttu; Jonasson, Gabriella; Lara, Patricia; Nelson, David R; Nilsson, IngMarie; André, François; von Heijne, Gunnar; Werck-Reichhart, Danièle

2017-08-01

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Radiation-induced damage of membranes

International Nuclear Information System (INIS)

Yonei, Shuji

1977-01-01

An outline of membranous structure was stated, and radiation-induced damage of membranes were surveyed. By irradiation, permeability of membranes, especially passive transportation mechanism, was damaged, and glycoprotein in the surface layers of cells and the surface layer structures were changed. The intramembranous damage was induced by decrease of electrophoresis of nuclear mambranes and a quantitative change of cytochrome P450 of microsomal membranes of the liver, and peroxidation of membranous lipid and SH substitute damage of membranous protein were mentioned as the mechanism of membranous damage. Recovery of membranous damage depends on radiation dose and temperature, and membranous damage participates largely in proliferation death. (tsunoda, M.)

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

Science.gov (United States)

Lei, Li; Egli, Martin

2016-04-01

Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

Cytochrome oxidase assembly does not require catalytically active cytochrome C.

Science.gov (United States)

Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

2003-03-14

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

A common pathway for regulation of nutritive blood flow to the brain: arterial muscle membrane potential and cytochrome P450 metabolites.

Science.gov (United States)

Harder, D R; Roman, R J; Gebremedhin, D; Birks, E K; Lange, A R

1998-12-01

Perfusion pressure to the brain must remain relatively constant to provide rapid and efficient distribution of blood to metabolically active neurones. Both of these processes are regulated by the level of activation and tone of cerebral arterioles. The active state of cerebral arterial muscle is regulated, to a large extent, by the level of membrane potential. At physiological levels of arterial pressure, cerebral arterial muscle is maintained in an active state owing to membrane depolarization, compared with zero pressure load. As arterial pressure changes, so does membrane potential. The membrane is maintained in a relatively depolarized state because of, in part, inhibition of K+ channel activity. The activity of K+ channels, especially the large conductance Ca(2+)-activated K+ channel (KCa) is dependent upon the level of 20-HETE produced by arterial muscle. As arterial pressure increases, so does cytochrome P450 (P4504A) activity. P4504A enzymes catalyse omega-hydroxylation of arachidonic acid and formation of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent inhibitor of KCa which maintains membrane depolarization and muscle cell activation. Astrocytes also metabolize AA via P450 enzymes of the 2C11 gene family to produce epoxyeicosatrienoic acids (EETs). Epoxyeicosatrienoic acids are released from astrocytes by glutamate which 'spills over' during neuronal activity. These locally released EETs shunt blood to metabolically active neurones providing substrate to support neuronal function. This short paper will discuss the findings which support the above scenario, the purpose of which is to provide a basis for future studies on the molecular mechanisms through which cerebral blood flow matches metabolism.

Electronic Connection Between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

Science.gov (United States)

Sarewicz, Marcin; Osyczka, Artur

2015-01-01

Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria. PMID:25540143

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase.

Science.gov (United States)

Woronowicz, Kamil; Sha, Daniel; Frese, Raoul N; Sturgis, James N; Nanda, Vikas; Niederman, Robert A

2011-08-01

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.

Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

International Nuclear Information System (INIS)

Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

1986-01-01

In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda A.; Evlyukhin, Andrey B.; Goodilin, Eugene A.

2015-01-01

Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow...... due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living...... mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman...

Variations in epidermal cytochrome oxidase activity after local irradiation

International Nuclear Information System (INIS)

Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

1982-01-01

Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

Science.gov (United States)

Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

2014-10-01

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. Copyright © 2014 Elsevier B.V. All rights reserved.

Biochemistry and Ecology of Novel Cytochromes Catalyzing Fe(II) Oxidation by an Acidophilic Microbial Community

Science.gov (United States)

Singer, S. W.; Jeans, C. J.; Thelen, M. P.; Verberkmoes, N. C.; Hettich, R. C.; Chan, C. S.; Banfield, J. F.

2007-12-01

An acidophilic microbial community found in the Richmond Mine at Iron Mountain, CA forms abundant biofilms in extremely acidic (pHindicated that several variants of Cyt579 were present in Leptospirillum strains. Intact protein MS analysis identified the dominant variants in each biofilm and documented multiple N-terminal cleavage sites for Cyt579. By combining biochemical, geochemical and microbiological data, we established that the sequence variation and N-terminal processing of Cyt579 are selected by ecological conditions. In addition to the soluble Cyt579, the second cytochrome appears as a much larger protein complex of ~210 kDa predominant in the biofilm membrane fraction, and has an alpha-band absorption at 572 nm. The 60 kDa cytochrome subunit, Cyt572, resides in the outer membrane of LeptoII, and readily oxidizes Fe(II) at low pH (0.95 - 3.0). Several genes encoding Cyt572 were localized within a recombination hotspot between two strains of LeptoII, causing a large range of variation in the sequences. Genomic sequencing and MS proteomic studies established that the variants were also selected by ecological conditions. A general mechanistic model for Fe(II) oxidation has been developed from these studies. Initial Fe(II) oxidation by Cyt572 occurs at the outer membrane. Cyt572 then transfers electrons to Cyt579, perhaps representing an initial step in energy flow to the biofilm community. Amino acid variations and post-translational modifications of these unique cytochromes may represent fine-tuning of function in response to local environmental conditions.

Study on the apoptosis mediated by cytochrome c and factors that affect the activation of bovine longissimus muscle during postmortem aging.

Science.gov (United States)

Zhang, Jiaying; Yu, Qunli; Han, Ling; Chen, Cheng; Li, Hang; Han, Guangxing

2017-06-01

This study investigates whether bovine longissimus muscle cell apoptosis occurs during postmortem aging and whether apoptosis is dependent on the mitochondria pathway. This study also determines the apoptosis process mediated by cytochrome c after its release from mitochondria and the factors that affect the activation processes. Results indicate that apoptotic nuclei were detected at 12 h postmortem. Cytochrome c release from the mitochondria to the cytoplasm activated the caspase-9 and caspase-3 at early postmortem aging and the activation of caspase-9 occurs before the activation of caspase-3. The pH level decreased during the first 48 h postmortem, whereas the mitochondria membrane permeability increased from 6 to 12 h. Results demonstrate that an apoptosis process of bovine muscle occurred during postmortem aging. Apoptosis was dependent on the mitochondria pathway and occurred at early postmortem aging. Increased mitochondria membrane permeability and low pH are necessary conditions for the release of cytochrome c during postmortem aging.

Lipids in the Structure of Photosystem I, Photosystem II and the Cytochrome b6f Complex

NARCIS (Netherlands)

Kern, Jan; Zouni, Athina; Guskov, Albert; Krauss, Norbert; Wada, Hajime; Murata, Norio

2009-01-01

This chapter describes the data accumulated in the last decade regarding the specific function of lipids in oxygenic photosynthesis, based on crystal structures of at least 3.0 Å resolution of the main photosynthetic membrane protein—pigment complexes, photosystem I, photosystem II and cytochrome

Duodenal Cytochrome b (DCYTB in Iron Metabolism: An Update on Function and Regulation

Directory of Open Access Journals (Sweden)

Darius J. R. Lane

2015-03-01

Full Text Available Iron and ascorbate are vital cellular constituents in mammalian systems. The bulk-requirement for iron is during erythropoiesis leading to the generation of hemoglobin-containing erythrocytes. Additionally; both iron and ascorbate are required as co-factors in numerous metabolic reactions. Iron homeostasis is controlled at the level of uptake; rather than excretion. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance non-heme iron absorption in the gut; ascorbate regulates iron homeostasis. The involvement of ascorbate in dietary iron absorption extends beyond the direct chemical reduction of non-heme iron by dietary ascorbate. Among other activities; intra-enterocyte ascorbate appears to be involved in the provision of electrons to a family of trans-membrane redox enzymes; namely those of the cytochrome b561 class. These hemoproteins oxidize a pool of ascorbate on one side of the membrane in order to reduce an electron acceptor (e.g., non-heme iron on the opposite side of the membrane. One member of this family; duodenal cytochrome b (DCYTB; may play an important role in ascorbate-dependent reduction of non-heme iron in the gut prior to uptake by ferrous-iron transporters. This review discusses the emerging relationship between cellular iron homeostasis; the emergent “IRP1-HIF2α axis”; DCYTB and ascorbate in relation to iron metabolism.

The dimerization of the yeast cytochrome bc1 complex is an early event and is independent of Rip1.

Science.gov (United States)

Conte, Annalea; Papa, Benedetta; Ferramosca, Alessandra; Zara, Vincenzo

2015-05-01

In Saccharomyces cerevisiae the mature cytochrome bc1 complex exists as an obligate homo-dimer in which each monomer consists of ten distinct protein subunits inserted into or bound to the inner mitochondrial membrane. Among them, the Rieske iron-sulfur protein (Rip1), besides its catalytic role in electron transfer, may be implicated in the bc1 complex dimerization. Indeed, Rip1 has the globular domain containing the catalytic center in one monomer while the transmembrane helix interacts with the adjacent monomer. In addition, the lack of Rip1 leads to the accumulation of an immature bc1 intermediate, only loosely associated with cytochrome c oxidase. In this study we have investigated the biogenesis of the yeast cytochrome bc1 complex using epitope tagged proteins to purify native assembly intermediates. We showed that the dimerization process is an early event during bc1 complex biogenesis and that the presence of Rip1, differently from previous proposals, is not essential for this process. We also investigated the multi-step model of bc1 assembly thereby lending further support to the existence of bona fide subcomplexes during bc1 maturation in the inner mitochondrial membrane. Finally, a new model of cytochrome bc1 complex assembly, in which distinct intermediates sequentially interact during bc1 maturation, has been proposed. Copyright © 2015 Elsevier B.V. All rights reserved.

Ubiquinol-cytochrome c reductase (Complex III) electrochemistry at multi-walled carbon nanotubes/Nafion modified glassy carbon electrodes

International Nuclear Information System (INIS)

Pelster, Lindsey N.; Minteer, Shelley D.

2012-01-01

Highlights: ► The electron transport chain is important to the understanding of metabolism in the living cell. ► Ubiquinol-cytochrome c reductase is a membrane bound complex of the electron transport chain (Complex III). ► The paper details the first bioelectrochemical characterization of ubiquinol-cytochrome c reductase at an electrode. - Abstract: Electron transport chain complexes are critical to metabolism in living cells. Ubiquinol-cytochrome c reductase (Complex III) is responsible for carrying electrons from ubiquinol to cytochrome c, but the complex has not been evaluated electrochemically. This work details the bioelectrochemistry of ubiquinol-cytochrome c reductase of the electron transport chain of tuber mitochondria. The characterization of the electrochemistry of this enzyme is investigated in carboxylated multi-walled carbon nanotube/tetrabutyl ammonium bromide-modified Nafion ® modified glassy carbon electrodes by cyclic voltammetry. Increasing concentrations of cytochrome c result in a catalytic response from the active enzyme in the nanotube sandwich. The experiments show that the enzyme followed Michaelis–Menten kinetics with a K m for the immobilized enzyme of 2.97 (±0.11) × 10 −6 M and a V max of 6.31 (±0.82) × 10 −3 μmol min −1 at the electrode, but the K m and V max values decreased compared to the free enzyme in solution, which is expected for immobilized redox proteins. This is the first evidence of ubiquinol-cytochrome c reductase bioelectrocatalysis.

Characterisation of MtoD from Sideroxydans lithotrophicus: a cytochrome c electron shuttle used in lithoautotrophic growth.

Directory of Open Access Journals (Sweden)

christopher eBeckwith

2015-04-01

Full Text Available The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidised at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (for ATP synthesis and NADH for autotrophic processes such as carbon fixation.As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolated mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical and crystallographic characterisation of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochromes c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle.

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

NARCIS (Netherlands)

Muntyan, M.S.; Cherepanov, D.A.; Malinen, A.M.; Bloch, D.A.; Sorokin, D.Y.; Severina, I.I.; Ivashina, T.V.; Lahti, R.; Muyzer, G.; Skulachev, V.P.

2015-01-01

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which

Identification of human cytochrome P450s as autoantigens.

Science.gov (United States)

Manns, M P; Johnson, E F

1991-01-01

Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Science.gov (United States)

Shak, S; Goldstein, I M

1985-09-01

Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.

Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

International Nuclear Information System (INIS)

Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

1985-01-01

The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H 2 O 2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

Science.gov (United States)

Meyer, Terry; Van Driessche, Gonzalez; Ambler, Richard; Kyndt, John; Devreese, Bart; Van Beeumen, Jozef; Cusanovich, Michael

2010-10-01

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Role of cytochrome B in the processing of the subunits of complex III in the yeast mitochondria

International Nuclear Information System (INIS)

Sen, K.G.

1986-01-01

The work described in this dissertation deals with the effect of cytochrome b on the biogenesis and assembly of the subunits of complex III in the mitochondrial membrane of the yeast Saccharomyces cerevisiae. The cytochrome b-mutants (Box mutants of S. cerevisiae form an excellent system to study such a role of cytochome B. The amounts of cytochrome c 1 in the mitochrondria, as determined both spectroscopically and immunologically, were not affected by the absence of cytochrome b. Pulse labelling of the cells with ( 35 S) methionine in the presence of CCCP showed the accumulation of the precursors to the core protein I and the iron-sulfur protein in similar amounts in the mutant Box 6-2 and the wild type cells. Synthesis of the iron sulfur protein and the cytochrome c 1 by in vitro translation of mRNA isolated from wild type and mutant Box 6-2 in a rabbit reticulocyte lysate system, also confirmed that the synthesis of the nuclear encoded subunits was not affected in the mutants. Pulse labeling of the cells in the absence of CCCP and subsequent chase with cold methionine, however, showed much less of the mature subunits of core protein I and the iron-sulfur protein in the mitochrondria of the mutant cells relative to the wild type. These results indicate that cytochrome b is necessary for the proper processing of certain subunits of complex III

Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex.

Science.gov (United States)

Hao, Ge-Fei; Wang, Fu; Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Huang, Li-Shar; Wu, Jia-Wei; Berry, Edward A; Yang, Guang-Fu

2012-07-11

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20 507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.

Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c

Science.gov (United States)

Muenzner, Julia; Toffey, Jason R.; Hong, Yuning; Pletneva, Ekaterina V.

2014-01-01

Interactions of cytochrome c (cyt c) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein’s function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt c unfolding and dramatically enhances the protein’s peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt c to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt c unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to “open” extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt c structure exposes the heme group, which enhances the protein’s peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes. PMID:23713573

Flow-alignment of bicellar lipid mixtures: orientations of probe molecules and membrane-associated biomacromolecules in lipid membranes studied with polarized light

KAUST Repository

Kogan, Maxim; Beke-Somfai, Tamá s; Nordé n, Bengt

2011-01-01

Bicelles are excellent membrane-mimicking hosts for a dynamic and structural study of solutes with NMR, but the magnetic fields required for their alignment are hard to apply to optical conditions. Here we demonstrate that bicellar mixtures can be aligned by shear forces in a Couette flow cell, to provide orientation of membrane-bound retinoic acid, pyrene and cytochrome c (cyt c) protein, conveniently studied with linear dichroism spectroscopy. © 2011 The Royal Society of Chemistry.

De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions

DEFF Research Database (Denmark)

Christensen, Ulla; Vazquez Albacete, Dario; Søgaard, Karina Marie

2017-01-01

Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading......-type E. coli strains using standard growth media. Furthermore, sequences encoding a small synthetic peptide and a small bacterial membrane anchor markedly enhance the expression of all six genes. For one of the CYPs, the length of the linker region between the predicted N-terminal transmembrane segment...

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

Science.gov (United States)

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

Science.gov (United States)

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

Science.gov (United States)

Reynafarje, B; Alexandre, A; Davies, P; Lehninger, A L

1982-01-01

The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0. PMID:6296824

Study of the interaction of cytochrome c and ferredoxine with the double membrane of chloroplast

International Nuclear Information System (INIS)

Neuburger, M.; Joyard, J.; Douce, R.

1975-01-01

The adsorption of two 59 Fe-labelled proteins on the chloroplast envelope was studied. The former molecule used was ferredoxine extracted from spinach leaves, the latter was cytochrome c, extracted from yeast (Saccharomyces cerevisiae D 261). The chloroplast envelope is thought to be involved in the transport of some proteins such as ferredoxine synthetized in the cytoplasm [fr

Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

Science.gov (United States)

Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

2013-01-01

Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence.

Science.gov (United States)

Simon, Matthieu; Metzinger-Le Meuth, Valérie; Chevance, Soizic; Delalande, Olivier; Bondon, Arnaud

2013-01-01

In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV-visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.

Mtr Extracellular Electron Transfer Pathways in Fe(III)-reducing or Fe(II)-oxidizing Bacteria: A Genomic Perspective

Energy Technology Data Exchange (ETDEWEB)

Shi, Liang; Rosso, Kevin M.; Zachara, John M.; Fredrickson, Jim K.

2012-12-01

Originally discovered in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), the Mtr (i.e., metal-reducing) pathway exists in all characterized strains of metal-reducing Shewanella. The protein components identified to date for the Mtr pathway of MR-1 include four multi-heme c-type cytochromes (c-Cyts), CymA, MtrA, MtrC and OmcA, and a porin-like, outer membrane protein MtrB. They are strategically positioned along the width of the MR-1 cell envelope to mediate electron transfer from the quinone/quinol pool in the inner-membrane to the Fe(III)-containing minerals external to the bacterial cells. A survey of microbial genomes revealed homologues of the Mtr pathway in other dissimilatory Fe(III)-reducing bacteria, including Aeromonas hydrophila, Ferrimonas balearica and Rhodoferax ferrireducens, and in the Fe(II)-oxidizing bacteria Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1. The widespread distribution of Mtr pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria emphasizes the importance of this type of extracellular electron transfer pathway in microbial redox transformation of Fe. Their distribution in these two different functional groups of bacteria also emphasizes the bi-directional nature of electron transfer reactions carried out by the Mtr pathways. The characteristics of the Mtr pathways may be shared by other pathways used by microorganisms for exchanging electrons with their extracellular environments.

The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6.

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Lydia Tome-Stangl

Full Text Available Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α-helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein "halves" for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.

Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

Science.gov (United States)

Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

2006-10-01

The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength

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Diana Campelo

2017-10-01

Full Text Available NADPH-cytochrome P450 reductase (CPR is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction, a linker (hinge, and a connecting/FAD domain (NADPH oxidation. It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state to an ensemble of open conformations (unlocked state, the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

Science.gov (United States)

Chen, Xi; Moerschell, Richard P; Pearce, David A; Ramanan, Durga D; Sherman, Fred

2005-02-01

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the rho+/rho- state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.

Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

International Nuclear Information System (INIS)

Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

2009-01-01

We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca 2+ -mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1

Carbonic anhydrase activity of integral-functional complexes of thylakoid membranes of spinach chloroplasts

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A. V. Semenihin

2015-06-01

Full Text Available Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1 within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1. Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

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

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.

Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

International Nuclear Information System (INIS)

Montaigne, David; Marechal, Xavier; Baccouch, Riadh; Modine, Thomas; Preau, Sebastien; Zannis, Konstantinos; Marchetti, Philippe; Lancel, Steve; Neviere, Remi

2010-01-01

The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solution containing 1 μM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt max of 105 ± 8 mN/s in control hearts vs. 49 ± 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 ± 0.2 in control hearts vs. 2.2 ± 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 ± 1 μM cytochrome c/min/mg in control hearts vs. 14 ± 3 μM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.

Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia coli

OpenAIRE

Kaderbhai, Mustak A.; Ugochukwu, Cynthia C.; Kelly, Steven L.; Lamb, David C.

2001-01-01

CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell ext...

Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection

International Nuclear Information System (INIS)

Carthy, Christopher M.; Yanagawa, Bobby; Luo Honglin; Granville, David J.; Yang, Decheng; Cheung, Paul; Cheung, Caroline; Esfandiarei, Mitra; Rudin, Charles M.; Thompson, Craig B.; Hunt, David W.C.; McManus, Bruce M.

2003-01-01

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release

Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

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Pengfei Ge

2013-10-01

Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

NMR comparison of prokaryotic and eukaryotic cytochromes c

International Nuclear Information System (INIS)

Chau, Meihing; Cai, Meng Li; Timkovich, R.

1990-01-01

1 H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degree C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c and mutants of yeast iso-1 cytochrome reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent

Effect of irradiation on membrane-bound rabit liver mitochondrial enzymes in embryogenesis

International Nuclear Information System (INIS)

Mirakhmedov, A.K.; Muradillaev, A.; Khan, M.Z.; Khamidov, D. Kh.

1982-01-01

Effect of irradiation on protein content of inner mitochondrial membrane and on activity of certain enzymes of respiratory chain of hepatic mitochondria has been studied. Within 24 and 48 hr after total irradiation (200 R) of pregnant rabbits, the protein content of the inner membranes of 25-30 day-old embryos and the mothers was broken with the increase in the thickness and densitometric height of the protein spots. Changes were seen in NADH-oxidase, succinate oxidase and in cytochrome-c-oxidase activities of mitochondria of 20 day-old embryos within 4 hr after irradiation and within 1 hr after irradiation in adult rabbits. The NADH-oxidase and the succinate oxidase activities of 30 day-old embryos were insensitive to the effect of irradiation. The cytochrome-c-oxidase activity increased in mitochondria of 25-30 day-old embryos upon 24 hr of irradiation. Substantial depression of the thermostability of the NADH-oxidase system was seen within 24 hr after irradiation while cytochrome-c-oxidase did not change its thermostability. The unequal disturbances of the emzyme activity and thermostability upon the total irradiation are connected with the different state of mitochondria and with the specificity of enzymes of the respiratory chain. (author)

A novel Geobacteraceae-specific outer membrane protein J (OmpJ is essential for electron transport to Fe (III and Mn (IV oxides in Geobacter sulfurreducens

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Schiffer Marianne

2005-07-01

Full Text Available Abstract Background Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III reduction in the Geobacter species that are the predominant Fe(III reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. Results When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J, was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III and insoluble Mn (IV oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal

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

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

Science.gov (United States)

Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

2001-01-01

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that

Induction of Ca2+-dependent cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength.

Science.gov (United States)

Dubinin, M V; Vedernikov, A A; Khoroshavina, E I; Samartsev, V N

2014-06-01

In liver mitochondria loaded with Ca2+ or Sr(2+), α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca2+-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca2+ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca2+ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca2+ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca2+ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca2+ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca2+ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca2+ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron

Nanoscopic dynamics of bicontinous microemulsions: effect of membrane associated protein.

Science.gov (United States)

Sharma, V K; Hayes, Douglas G; Urban, Volker S; O'Neill, Hugh M; Tyagi, M; Mamontov, E

2017-07-19

Bicontinous microemulsions (BμE) generally consist of nanodomains formed by surfactant in a mixture of water and oil at nearly equal proportions and are potential candidates for the solubilization and purification of membrane proteins. Here we present the first time report of nanoscopic dynamics of surfactant monolayers within BμEs formed by the anionic surfactant sodium dodecyl sulfate (SDS) measured on the nanosecond to picosecond time scale using quasielastic neutron scattering (QENS). BμEs investigated herein consisted of middle phases isolated from Winsor-III microemulsion systems that were formed by mixing aqueous and oil solutions under optimal conditions. QENS data indicates that surfactants undergo two distinct motions, namely (i) lateral motion along the surface of the oil nanodomains and (ii) localized internal motion. Lateral motion can be described using a continuous diffusion model, from which the lateral diffusion coefficient is obtained. Internal motion of surfactant is described using a model which assumes that a fraction of the surfactants' hydrogens undergoes localized translational diffusion that could be considered confined within a spherical volume. The effect of cytochrome c, an archetypal membrane-associated protein known to strongly partition near the surfactant head groups in BμEs (a trend supported by small-angle X-ray scattering [SAXS] analysis), on the dynamics of BμE has also been investigated. QENS results demonstrated that cytochrome c significantly hindered both the lateral and the internal motions of surfactant. The lateral motion was more strongly affected: a reduction of the lateral diffusion coefficient by 33% was measured. This change is mainly attributable to the strong association of cytochrome c with oppositely charged SDS. In contrast, analysis of SAXS data suggested that thermal fluctuations (for a longer length and slower time scale compared to QENS) were increased upon incorporation of cytochrome c. This study

Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking.

Science.gov (United States)

Ducassou, Lionel; Dhers, Laura; Jonasson, Gabriella; Pietrancosta, Nicolas; Boucher, Jean-Luc; Mansuy, Daniel; André, François

2017-09-01

Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms. We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model. The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A' and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA' and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities. This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Change of the NADPH depending superoxide producing and ferri hemoglobin reducing activities of cytochrome b558 from spleen cells and erythrocytes membranes induced by the radiation of different character

International Nuclear Information System (INIS)

Melkonyan, L.G.; Simonyan, R.M.; Simonyan, M.A.; Sekoyan, E.S.

2009-01-01

After the X radiation, UVA radiation and ultrasound radiation of new isoforms of cytochrome cyt b 5 58 from rats erythrocyte membranes - EM (cyt b 5 58III) and from spleen cell membranes (SCM) in vitro, as well as after the radiation of EM ex vivo, the suppression of both NADPH depending O 2 - producing and ferrihemoglobin (ferriHb)-reducing activities of cyt b 5 58 from EM and SCM in homogeneous (in solution) and heterogeneous phases (in EM and SCM) at various scopes takes place. These changes are associated with the destabilization of EM and SCM, conditioned by the change of the aggregation degree of these hemoproteins in EM and SCM, hemoproteins as a result of the influence of the hydrogen peroxide formed during radiolysis and photolysis of the water medium. After He-Ne laser radiation of the cyt b 5 58 from EM and SCM in vitro an increase of the NADPH depending O 2 - producing and ferriHb-reducing activities of the cyt b 5 58 from EM and SCM in homogenous and heterogeneous phases (in membranes) takes place. It is supposed that the suppression (by X-, UVA- and US-radiation) and the stimulation (by He-Ne laser radiation) of the immune system activity and the oxygen homeostasis are associated with the corresponding decrease and increase of the NADPH depending O 2 - producing and ferriHb-reducings activity of the new isoforms of cyt b 5 58 from EM and SCM in homogeneous and heterogeneous phases

Physiological and transcriptional approaches reveal connection between nitrogen and manganese cycles in Shewanella algae C6G3

Science.gov (United States)

Aigle, Axel; Bonin, Patricia; Iobbi-Nivol, Chantal; Méjean, Vincent; Michotey, Valérie

2017-03-01

To explain anaerobic nitrite/nitrate production at the expense of ammonium mediated by manganese oxide (Mn(IV)) in sediment, nitrate and manganese respirations were investigated in a strain (Shewanella algae C6G3) presenting these features. In contrast to S. oneidensis MR-1, a biotic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during anaerobic growth with Mn(IV) under condition of limiting electron acceptor, concomitantly, with a higher electron donor stoichiometry than expected. This low and reproducible transitory accumulation is the result of production and consumption since the strain is able to dissimilative reduce nitrate into ammonium. Nitrite production in Mn(IV) condition is strengthened by comparative expression of the nitrate/nitrite reductase genes (napA, nrfA, nrfA-2), and rates of the nitrate/nitrite reductase activities under Mn(IV), nitrate or fumarate conditions. Compared with S. oneidensis MR-1, S. algae contains additional genes that encode nitrate and nitrite reductases (napA-α and nrfA-2) and an Outer Membrane Cytochrome (OMC)(mtrH). Different patterns of expression of the OMC genes (omcA, mtrF, mtrH and mtrC) were observed depending on the electron acceptor and growth phase. Only gene mtrF-2 (SO1659 homolog) was specifically expressed under the Mn(IV) condition. Nitrate and Mn(IV) respirations seem connected at the physiological and transcriptional levels.

The cytochrome p450 homepage.

Science.gov (United States)

Nelson, David R

2009-10-01

The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

Economics of membrane occupancy and respiro-fermentation

Science.gov (United States)

Zhuang, Kai; Vemuri, Goutham N; Mahadevan, Radhakrishnan

2011-01-01

The simultaneous utilization of efficient respiration and inefficient fermentation even in the presence of abundant oxygen is a puzzling phenomenon commonly observed in bacteria, yeasts, and cancer cells. Despite extensive research, the biochemical basis for this phenomenon remains obscure. We hypothesize that the outcome of a competition for membrane space between glucose transporters and respiratory chain (which we refer to as economics of membrane occupancy) proteins influences respiration and fermentation. By incorporating a sole constraint based on this concept in the genome-scale metabolic model of Escherichia coli, we were able to simulate respiro-fermentation. Further analysis of the impact of this constraint revealed differential utilization of the cytochromes and faster glucose uptake under anaerobic conditions than under aerobic conditions. Based on these simulations, we propose that bacterial cells manage the composition of their cytoplasmic membrane to maintain optimal ATP production by switching between oxidative and substrate-level phosphorylation. These results suggest that the membrane occupancy constraint may be a fundamental governing constraint of cellular metabolism and physiology, and establishes a direct link between cell morphology and physiology. PMID:21694717

Structure and expression of cytochrome f in an Oenothera plastome mutant.

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Johnson, E M; Sears, B B

1990-06-01

The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.

Science.gov (United States)

Glerum, D M; Tzagoloff, A

1997-08-04

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced rhodobacter capsulatus cytochrome c(2) to the cytochrome bc(1) complex mediated by the conformation of the rieske iron-sulfur protein

International Nuclear Information System (INIS)

Devanathan, S.; Salamon, Z.; Tollin, G.; Fitch, J.C.; Meyer, T.E.; Berry, E.A.; Cusanovich, M.A.

2007-01-01

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to the oxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 M. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 binds less strongly (Kd = 0.11 M) but ∼30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c

Reduction of reversed micelle entrapped cytochrome c and cytochrome c3 by electrons generated by pulse radiolysis or by pyrene photoionization

International Nuclear Information System (INIS)

Vlsser, A.J.W.G.; Fendler, J.H.

1982-01-01

Horse heart cytochrome c and cytochrome c 3 , isolated from Desulfovibrio vulgaris, have been incorporated in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) entrapped water pools in heptane. The absorption spectra of the cytochromes have been found to be strongly dependent on the water to AOT concentration ratios. The proteins solubilized in heptane by the AOT reversed micelles have retained their ability to mediate electron transfer. They reacted very rapidly with hydrated electrons, generated pulse radiolytically or, alternatively, formed in the laser photoionization of pyrene

Plastocyanin/cytochrome c6 interchange in Scenedesmus vacuolatus.

Science.gov (United States)

Miramar, M Dolores; Inda, Luis A; Saraiva, Lígia M; Peleato, M Luisa

2003-12-01

Plastocyanin and cytochrome c6 from the green alga Scenedesmus vacuolatus were immunoquantified in cells grown under different concentrations of copper and iron. Plastocyanin expression was constitutive, its synthesis was not significantly affected by iron availability, and increases with copper availability. On the contrary, cytochrome c6 synthesis is repressed by copper, and only residual amounts of the protein were detected at 0.1 micromol/L copper. Under copper deficiency, cytochrome c6 is slightly dependent on iron. In natural environments, plastocyanin seems to be the predominant electron donor to P700.

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states.

Science.gov (United States)

Ogura, T; Yoshikawa, S; Kitagawa, T

1985-12-17

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).

Thermodynamics and kinetics of reduction and species conversion at a hydrophobic surface for mitochondrial cytochromes c and their cardiolipin adducts

International Nuclear Information System (INIS)

Ranieri, Antonio; Di Rocco, Giulia; Millo, Diego; Battistuzzi, Gianantonio; Bortolotti, Carlo A.; Lancellotti, Lidia; Borsari, Marco; Sola, Marco

2015-01-01

Highlights: • Cytochrome c and its adduct with cardiolipin can be immobilized on a hydrophobic SAM. • Adsorbed cytochrome c and its adduct undergo extensive unfolding and axial ligand substitution. • An equilibrium between a six-coordinated and a five-coordinated form is observed in both cases. • The reduced five-coordinated form is stabilized by cardiolipin binding. • Immobilized cytochrome c exchanges electrons more slowly upon cardiolipin binding. - Abstract: Cytochrome c (cytc) and its adduct with cardiolipin (CL) were immobilized on a hydrophobic SAM-coated electrode surface yielding a construct which mimics the environment experienced by the complex at the inner mitochondrial membrane where it plays a role in cell apoptosis. Under these conditions, both species undergo an equilibrium between a six-coordinated His/His-ligated and a five-coordinated His/- ligated forms stable in the oxidized and in the reduced state, respectively. The thermodynamics of the oxidation-state dependent species conversion were determined by temperature-dependent diffusionless voltammetry experiments. CL binding stabilizes the immobilized reduced His/- ligated form of cytc which was found previously to catalytically reduce dioxygen. Here, this adduct is also found to show pseudoperoxidase activity, catalysing reduction of hydrogen peroxide. These effects would impart CL with an additional role in the cytc-mediated peroxidation leading to programmed cell death. Moreover, immobilized cytc exchanges electrons more slowly upon CL binding possibly due to changes in solvent reorganization effects at the protein-SAM interface

The SMARTCyp cytochrome P450 metabolism prediction server

DEFF Research Database (Denmark)

Rydberg, Patrik; Gloriam, David Erik Immanuel; Olsen, Lars

2010-01-01

The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism.......The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism....

THE REDOX PATHWAY OF Pseudomonas aeruginosa CYTOCHROME C BIOGENESIS

Directory of Open Access Journals (Sweden)

Eva Di Silvio

2012-06-01

Full Text Available Cytochrome c contains heme covalently bound to the polypeptide chain through two thioether bonds between the heme vinyl groups and the two cysteines of the conserved heme- binding motif of the apoprotein. Surprisingly, the biochemical events leading to the synthesis of the functional holoprotein in the cell are largely unknown. In the human pathogen Pseudomonas aeruginosa, the biogenesis of Cytc is mediated by a group of membrane or membrane-anchored proteins (CcmABCDEFGHI, exposing their active site to the periplasm. The Ccm proteins involved in the necessary reduction of apoCyt disulfide bond are CcmG and CcmH. Here we present the structural and functional characterization of these two redox-active proteins. We determined the crystal structure of CcmG, both in the oxidized and the reduced state. CcmG is a membrane-anchored thioredoxinlike protein acting as a mild reductant in the redox pathway of Cytc biogenesis. The 3D structure of the soluble periplasmic domain of CcmH revealed that it adopts a peculiar three-helix bundle fold that is different from that of canonical thiol-oxidoreductases. Moreover, we present protein-protein interaction experiments aiming at elucidating the molecular mechanism of the reduction of apoCyt disulfide bond for heme attachment in vivo. On the basis of the structural and functional data on CcmG, CcmH and their interactions, we propose an assembly line for Cytc biogenesis in P. aeruginosa in which reduced CcmH specifically recognizes, binds and reduces oxidized apoCyt via the formation of a mixed disulfide complex, which is subsequently resolved by CcmG.

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.

Cytochrome P450 monooxygenases and insecticide resistance in insects.

OpenAIRE

Bergé, J B; Feyereisen, R; Amichot, M

1998-01-01

Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

Science.gov (United States)

Huber, Warren J.; Backes, Wayne L.

2009-01-01

Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

NARCIS (Netherlands)

Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

1987-01-01

A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

Hunting for low abundant redox proteins in plant plasma membranes.

Science.gov (United States)

Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

2009-04-13

Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (−)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Çelik, Haydar; Koşar, Müberra; Arinç, Emel

2013-01-01

Highlights: • We assessed inhibitory effects of 8 dietary flavonoids on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase. • The flavonol myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. • We investigated kinetics of myricetin-induced inhibition in detail. • We explored the structure–inhibitory activity relationship of compounds. • Modulation of cytochrome b5 reduction indicates a potential for myricetin to lead to some food–drug/xenobiotic interactions. - Abstract: The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. Myricetin inhibited b5 reductase noncompetitively with a K i of 0.21 μM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis–Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC 50 = 9.8 μM). The remaining flavonoids were inactive within the concentration range tested (1–50 μM). Analysis of structure–activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics

Evidence from Studies with Acifluorfen for Participation of a Flavin-Cytochrome Complex in Blue Light Photoreception for Phototropism of Oat Coleoptiles 12

Science.gov (United States)

Leong, Ta-Yan; Briggs, Winslow R.

1982-01-01

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor. PMID:16662593

Effectiveness of cytochrome C and cepharanthin for leukopenia following multidisciplinary treatment

International Nuclear Information System (INIS)

Tabata, Kumiko; Endow, Masaru; Suzuki, Hirotoshi

1986-01-01

Leukopenia is one of important problems for multidisciplinary treatment of malignant tumor. We could not be able to take a continuous cancer therapy because of leukopenia. And then we had a study of effectiveness combination treatment of cytochrome C with cepharanthin for leukopenia of cancer patient. We carried on the study of 3 classifications of treatment as follows, a) cytochrome C only, b) combined cytochrome C with cepharanthin, and c) control group without drugs. Bone marrow potentiality is individual differentiation and then the group was administrated both cytochrome C and cepharanthin following radiotherapy associated with postoperative breast cancer. The above description lead to conclusion that combination treatment of cytochrome C and cepharanthin was available for protective drugs from multidisciplinary treatment induced leukemia. (author)

The reaction of neuroglobin with potential redox protein partners cytochrome b5  and cytochrome c

DEFF Research Database (Denmark)

Fago, Angela; Mathews, A.J.; Moens, L.

2006-01-01

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b5 is relatively slow (k=6×102M-1s-1 at pH 7.0) and thus is unlikely to be of physiological...... significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2×107M-1s-1) with an apparent overall equilibrium constant of 1μM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis...

Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

DEFF Research Database (Denmark)

Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

2011-01-01

The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR und...... to serve as an effective electron transferring "nano-machine"....

A positive feedback-based gene circuit to increase the production of a membrane protein

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Gennis Robert B

2010-05-01

Full Text Available Abstract Background Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

Science.gov (United States)

Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

1976-03-01

Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

Science.gov (United States)

Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

2012-01-01

Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

A novel alkaloid, evodiamine causes nuclear localization of cytochrome-c and induces apoptosis independent of p53 in human lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Mohan, Vijay [School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat (India); Agarwal, Rajesh [Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, CO (United States); Singh, Rana P., E-mail: ranaps@hotmail.com [School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat (India); Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi (India)

2016-09-02

Lung cancer is the most frequently diagnosed malignancy that contributes to high proportion of deaths globally among patients who die due to cancer. Chemotherapy remains the common mode of treatment for lung cancer patients though with limited success. We assessed the biological effects and associated molecular changes of evodiamine, a plant alkaloid, on human lung cancer A549 and H1299 cells along with other epithelial cancer and normal lung SAEC cells. Our data showed that 20–40 μM evodiamine treatment for 24–48 h strongly (up to 73%, P < 0.001) reduced the growth and survival of these cancer cells. However, it also moderately inhibited growth and survival of SAEC cells. A strong inhibition (P < 0.001) was observed on clonogenicity of A549 cells. Further, evodiamine increased (4-fold) mitochondrial membrane depolarization with 6-fold increase in apoptosis and a slight increase in Bax/Bcl-2 ratio. It increased the cytochrome-c release from mitochondria into the cytosol as well as nucleus. Cytosolic cytochrome-c activated cascade of caspase-9 and caspase-3 intrinsic pathway, however, DR5 and caspase-8 extrinsic pathway was also activated which could be due to nuclear cytochrome-c. Pan-caspase inhibitor (z-VAD.fmk) partially reversed evodiamine induced apoptosis. An increase in p53 as well as its serine 15 phosphorylation was also observed. Pifithrin-α, a p53 inhibitor, slightly inhibited growth of A549 cells and under p53 inhibitory condition evodiamine-induced apoptosis could not be reversed. Together these findings suggest that evodiamine is a strong inducer of apoptosis in lung epithelial cancer cells independent of their p53 status and that could involve both intrinsic as well as extrinsic pathway of apoptosis. Thus evodiamine could be a potential anticancer agent against lung cancer. - Highlights: • Evodiamine, a novel plant alkaloid, relatively selectively inhibited growth and survival of human lung cancer cells. • Increased cancer cell

New insight into the mechanism of mitochondrial cytochrome c function

DEFF Research Database (Denmark)

Chertkova, Rita V; Brazhe, Nadezda A; Bryantseva, Tatiana V

2017-01-01

We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76...... with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c....

Influence of polyhalogenated aromatic hydrocarbons on the induction, activity, and stabilization of cytochrome P450

International Nuclear Information System (INIS)

Voorman, R.

1987-01-01

In the course of experiments evaluating the metabolism of polybrominated biphenyls by cytochrome P450 isozymes induced by 3,4,5,3',4',5'-hexabromobiphenyl (HBB), it was discovered that the inducer remained closely associated with cytochrome P450d. Subsequent purification of cytochromes from HBB treated rates revealed a 0.5:1 association of HBB to cytochrome P450d but virtually none with cytochrome P450c or cytochrome b5. Immunochemical quantitation of cytochrome P450d in the same microsomes yielded a ratio of P450d:HBB that approached unity. Measurement of cytochrome P450d estradiol 2-hydroxylase indicated non-competitive or mixed type inhibition caused by HBB at a concentration of 10-1000 nM. Inhibition was specific to cytochrome P450d since estradiol 2-hydroxylase catalyzed by cytochrome P450h was unaffected by HBB. The ability of HCB and isosafrole to stabilize cytochrome P450d, and thus indirectly influence regulation of the enzyme, was evaluated by treating rats with a dose of TCDD sufficient to produce maximum induction of cytochromes P450c and P450d via the Ah receptor, yet insufficient to bind to the enzyme. Subsequent treatment of these animals with HCB or isosafrole and a radiolabeled amino acid, revealed a significant increase in cytochrome P450d specific content relative to cytochrome P450c and significant retention of the radiolabel in P450d relative to rats treated only with TCDD

HiPIP oxido-reductase activity in membranes from aerobically grown cells of the facultative phototroph Rhodoferax fermentans

DEFF Research Database (Denmark)

Hochkoeppler, Alejandro; Kofod, Pauli; Zannoni, Davide

1995-01-01

The role of the periplasmically located, water-soluble, HiPIP (high-potential iron-sulfur protein) in the respiratory chain of the facultative phototroph Rhodoferax fermentans has been examined. The oxidized HiPIP is reduced by succinate-dependent respiration via the bc 1 complex, this reaction...... being inhibited by myxothiazol and/or stigmatellin. The reduced HiPIP can be oxidized by the membrane-bound cytochrome oxidase, this reaction being inhibited by 0.1 mM cyanide. We conclude that aerobically grown Rf. fermentans contains a redox chain in which HiPIP mediates electron transfer between...... the bc 1 complex and the cb-type cytochrome oxidase....

Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

Science.gov (United States)

Van Pelt, F N; Meierink, Y J; Blaauboer, B J; Weterings, P J

1990-12-01

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

Science.gov (United States)

Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

2017-08-22

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

Elucidating cytochrome C release from mitochondria: insights from an in silico three-dimensional model.

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Tam, Zhi Yang; Cai, Yi Hui; Gunawan, Rudiyanto

2010-11-17

Mitochondrial regulation of apoptosis depends on the programmed release of proapoptotic proteins such as cytochrome c (Cyt c) through the outer mitochondrial membrane (OMM). Although a few key processes involved in this release have been identified, including the liberation of inner membrane-bound Cyt c and formation of diffusible pores on the OMM, other details like the transport of Cyt c within complex mitochondrial compartments, e.g., the cristae and crista junctions, are not yet fully understood (to our knowledge). In particular, a remodeling of the inner mitochondrial membrane accompanying apoptosis seen in a few studies, in which crista junctions widen, has been hypothesized to be a necessary step in the Cyt c release. Using a three-dimensional spatial modeling of mitochondrial crista and the crista junction, model simulations and analysis illustrated how the interplay among solubilization of Cyt c, fast diffusion of Cyt c, and OMM permeabilization gives rise to the observed experimental release profile. Importantly, the widening of the crista junction was found to have a negligible effect on the transport of free Cyt c from cristae. Finally, model simulations showed that increasing the fraction of free/loosely-bound Cyt c can sensitize the cell to apoptotic stimuli in a threshold manner, which may explain increased sensitivity to cell death associated with aging. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mitochondrial cytochrome c biogenesis: no longer an enigma.

Science.gov (United States)

Babbitt, Shalon E; Sutherland, Molly C; San Francisco, Brian; Mendez, Deanna L; Kranz, Robert G

2015-08-01

Cytochromes c (cyt c) and c1 are heme proteins that are essential for aerobic respiration. Release of cyt c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for the import of apocytochrome c (apocyt c). Thus, HCCS affects cellular levels of cyt c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles of heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

Energy Technology Data Exchange (ETDEWEB)

Volkov, Alexander N., E-mail: ovolkov@vub.ac.be; Nuland, Nico A. J. van [Vrije Universiteit Brussel, Jean Jeener NMR Centre, Structural Biology Brussels (Belgium)

2013-07-15

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc-CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.

In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids

DEFF Research Database (Denmark)

Breinholt, Vibeke; Offord, E.A.; Brouwer, C.

2002-01-01

Human and mouse liver microsomes And membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin...... were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodietyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin. respectively, Microsomal flavonoid metabolism as potently inhibited by the CYP1A2...... inhibitors. fluvoxamine and alpha-naphthoflavone. Recombinant CYP1A2 as capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation. but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant...

Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing.

Science.gov (United States)

Hough, Michael A; Andrew, Colin R

2015-01-01

Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins. © 2015 Elsevier Ltd. All rights reserved.

A cytosolic cytochrome b 5-like protein in yeast cell accelerating the electron transfer from NADPH to cytochrome c catalyzed by Old Yellow Enzyme

International Nuclear Information System (INIS)

Nakagawa, Manabu; Yamano, Toshio; Kuroda, Kiyo; Nonaka, Yasuki; Tojo, Hiromasa; Fujii, Shigeru

2005-01-01

A 410-nm absorbing species which enhanced the reduction rate of cytochrome c by Old Yellow Enzyme (OYE) with NADPH was found in Saccharomyces cerevisiae. It was solubilized together with OYE by the treatment of yeast cells with 10% ethyl acetate. The purified species showed visible absorption spectra in both oxidized and reduced forms, which were the same as those of the yeast microsomal cytochrome b 5 . At least 14 amino acid residues of the N-terminal region coincided with those of yeast microsomal b 5 , but the protein had a lower molecular weight determined to be 12,600 by SDS-PAGE and 9775 by mass spectrometry. The cytochrome b 5 -like protein enhanced the reduction rate of cytochrome c by OYE, and a plot of the reduction rates against its concentration showed a sigmoidal curve with an inflexion point at 6 x 10 -8 M of the protein

MOLECULAR DYNAMICS STUDY OF CYTOCHROME C – LIPID COMPLEXES

Directory of Open Access Journals (Sweden)

V. Trusova

2017-10-01

Full Text Available The interactions between a mitochondrial hemoprotein cytochrome c (cyt c and the model lipid membranes composed of zwitterionic lipid phosphatidylcholine (PC and anionic lipids phosphatidylglycerol (PG, phosphatidylserine (PS or cardiolipin (CL were studied using the method of molecular dynamics. It was found that cyt c structure remains virtually unchanged in the protein complexes with PC/PG or PC/PS bilayers. In turn, protein binding to PC/CL bilayer is followed by the rise in cyt c radius of gyration and root-mean-square fluctuations. The magnitude of these changes was demonstrated to increase with the anionic lipid content. The revealed effect was interpreted in terms of the partial unfolding of polypeptide chain in the region Ala15-Leu32, widening of the heme crevice and enhancement of the conformational fluctuations in the region Pro76-Asp93 upon increasing the CL molar fraction from 5 to 25%. The results obtained seem to be of utmost importance in the context of amyloidogenic propensity of cyt c.

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

Science.gov (United States)

Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

The novel cytochrome c6 of chloroplasts: a case of evolutionary bricolage?

Science.gov (United States)

Howe, Christopher J; Schlarb-Ridley, Beatrix G; Wastl, Juergen; Purton, Saul; Bendall, Derek S

2006-01-01

Cytochrome c6 has long been known as a redox carrier of the thylakoid lumen of cyanobacteria and some eukaryotic algae that can substitute for plastocyanin in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However, a homologue of the protein has now been identified in several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This form of the protein, designated cytochrome c6A, differs from the 'conventional' cytochrome c6 in possessing a conserved insertion of 12 amino acids that includes two absolutely conserved cysteine residues. There are conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron transfer. The evidence for and against this is reviewed and the likely evolutionary history of cytochrome c6A is discussed. It is suggested that it has been converted from a primary role in electron transfer to one in regulation within the chloroplast, and is an example of evolutionary 'bricolage'.

Induction of apoptosis in cancer cells by NiZn ferrite nanoparticles through mitochondrial cytochrome C release

Directory of Open Access Journals (Sweden)

Al-Qubaisi MS

2013-10-01

Full Text Available Mothanna Sadiq Al-Qubaisi,1 Abdullah Rasedee,1,2 Moayad Husein Flaifel,3 Sahrim Hj Ahmad,3 Samer Hussein-Al-Ali,1 Mohd Zobir Hussein,4 Zulkarnain Zainal,4 Fatah H Alhassan,4 Yun H Taufiq-Yap,4 Eltayeb EM Eid,5 Ismail Adam Arbab,1 Bandar A Al-Asbahi,3 Thomas J Webster,6,7 Mohamed Ezzat El Zowalaty1,8,9 1Institute of Bioscience, 2Faculty of Veterinary Medicine, Universiti Putra Malaysia, 3Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 4Faculty of Science, Universiti Putra Malaysia, Selangor, Malaysia; 5College of Pharmacy, Qassim University, Buraidah, Saudi Arabia; 6Department of Chemical Engineering and Program in Bioengineering, Northeastern University, Boston, MA, USA; 7Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia; 8Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; 9Faculty of Public Health and Tropical Medicine, Jazan University, Jazan, Saudi Arabia Abstract: The long-term objective of the present study was to determine the ability of NiZn ferrite nanoparticles to kill cancer cells. NiZn ferrite nanoparticle suspensions were found to have an average hydrodynamic diameter, polydispersity index, and zeta potential of 254.2 ± 29.8 nm, 0.524 ± 0.013, and -60 ± 14 mV, respectively. We showed that NiZn ferrite nanoparticles had selective toxicity towards MCF-7, HepG2, and HT29 cells, with a lesser effect on normal MCF 10A cells. The quantity of Bcl-2, Bax, p53, and cytochrome C in the cell lines mentioned above was determined by colorimetric methods in order to clarify the mechanism of action of NiZn ferrite nanoparticles in the killing of cancer cells. Our results indicate that NiZn ferrite nanoparticles promote apoptosis in cancer cells via caspase-3 and caspase-9, downregulation of Bcl-2, and upregulation of Bax and p53, with cytochrome C translocation. There was a concomitant collapse of the mitochondrial membrane potential in these cancer cells when treated

The amino acid sequence of cytochrome c from Cucurbita maxima L. (pumpkin)

Science.gov (United States)

Thompson, E. W.; Richardson, M.; Boulter, D.

1971-01-01

The amino acid sequence of pumpkin cytochrome c was determined on 2μmol of protein. Some evidence was found for the occurrence of two forms of cytochrome c, whose sequences differed in three positions. Pumpkin cytochrome c consists of 111 residues and is homologous with mitochondrial cytochromes c from other plants. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7. PMID:5131733

Aspirin Induces Apoptosis through Release of Cytochrome c from Mitochondria

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Katja C. Zimmermann

2000-01-01

Full Text Available Nonsteroidal anti-inflammatory drugs (NSAID reduce the risk for cancer, due to their anti proliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

The Role of Cytochromes P450 in Infection

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Elisavet Stavropoulou

2018-01-01

Full Text Available Cytochromes are expressed in many different tissues of the human body. They are found mostly in intestinal and hepatic tissues. Cytochromes P450 (CYPs are enzymes that oxidize substances using iron and are able to metabolize a large variety of xenobiotic substances. CYP enzymes are linked to a wide array of reactions including and O-dealkylation, S-oxidation, epoxidation, and hydroxylation. The activity of the typical P450 cytochrome is influenced by a variety of factors, such as genus, environment, disease state, herbicide, alcohol, and herbal medications. However, diet seems to play a major role. The mechanisms of action of dietary chemicals, macro- and micronutrients on specific CYP isoenzymes have been extensively studied. Dietary modulation has effects upon the metabolism of xenobiotics. Cytochromes harbor intra- or interindividual and intra- or interethnic genetic polymorphisms. Bacteria were shown to express CYP-like genes. The tremendous metabolic activity of the microbiota is associated to its abundant pool of CYP enzymes, which catalyze phase I and II reactions in drug metabolism. Disease states, intestinal disturbances, aging, environmental toxic effects, chemical exposures or nutrition modulate the microbial metabolism of a drug before absorption. A plethora of effects exhibited by most of CYP enzymes can resemble those of proinflammatory cytokines and IFNs. Moreover, they are involved in the initiation and persistence of pathologic pain by directly activating sensory neurons and inflammatory cytokines.

A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

Science.gov (United States)

Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

2009-12-01

Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

Science.gov (United States)

Huber, Warren J; Backes, Wayne L

2007-10-30

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

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.

Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

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Míriam Cristina Sakuragui Matuo

2010-09-01

Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

Isolation of Highly Purified Fractions of Plasma Membrane and Tonoplast from the Same Homogenate of Soybean Hypocotyls by Free-Flow Electrophoresis 1

Science.gov (United States)

Sandelius, Anna Stina; Penel, Claude; Auderset, Guy; Brightman, Andrew; Millard, Merle; Morré, D. James

1986-01-01

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K+-stimulated, vanadate-inhibited Mg2+ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 9 PMID:16664771

Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

Energy Technology Data Exchange (ETDEWEB)

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, I-00146 Roma (Italy); Ciaccio, Chiara [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari (Italy); Sinibaldi, Federica; Santucci, Roberto [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Coletta, Massimo [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari (Italy)

2011-01-07

Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.

Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

Science.gov (United States)

Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

2007-08-03

Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

International Nuclear Information System (INIS)

Kresheck, G.C.; Erman, J.E.

1988-01-01

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (t/sub m/) were 43.9 +- 1.4 and 63.3 +- 1.6 0 C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +- 1.3 0 C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Energy Technology Data Exchange (ETDEWEB)

Kolodkin-Gal, I; Elsholz, AKW; Muth, C; Girguis, PR; Kolter, R; Losick, R

2013-04-29

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Science.gov (United States)

Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

2013-01-01

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

OpenAIRE

Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

2008-01-01

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

International Nuclear Information System (INIS)

McCue, Jeffrey M.; Driscoll, William J.; Mueller, Gregory P.

2008-01-01

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo

NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

Science.gov (United States)

Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

2013-01-01

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

Energy Technology Data Exchange (ETDEWEB)

Kang, Jung Hoon [Cheongju Univ., Cheongju (Korea, Republic of)

2013-11-15

Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD.

Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

International Nuclear Information System (INIS)

Kang, Jung Hoon

2013-01-01

Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD

Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes

DEFF Research Database (Denmark)

Juel, C; Thomsen, J J; Rentsch, R L

2007-01-01

on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects...... performance by approximately 54%. Membrane transport systems and carbonic anhydrases involved in pH regulation remained unchanged. Of the Na(+), K(+)-pump isoforms only the density of the alpha2 subunit was decreased (by 22%) after treatment. The marker enzymes cytochrom c and hexokinase remained unchanged......Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment...

{sup 13}C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

Energy Technology Data Exchange (ETDEWEB)

McCullough, Christopher R.; Pullela, Phani Kumar [Marquette University, Chemical Proteomics Facility at Marquette, Department of Chemistry (United States); Im, Sang-Choul; Waskell, Lucy [University of Michigan and VA Medical Center, Department of Anesthesiology (United States); Sem, Daniel S. [Marquette University, Chemical Proteomics Facility at Marquette, Department of Chemistry (United States)], E-mail: Daniel.sem@marquette.edu

2009-03-15

The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a {sup 13}CH{sub 3}-reporter attached. This {sup 13}C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site.

Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

International Nuclear Information System (INIS)

Ross, S.B.

1987-01-01

3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

Redox enzymes in the plant plasma membrane and their possible roles

DEFF Research Database (Denmark)

Berczi, A.; Møller, I.M.

2000-01-01

Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low-molecular-mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K-1), all of which are likely to participate in redox...... protein which has been partially purified from plant PM so far is a high-potential and ascorbate-reducible b-type cytochrome. In co-operation with vitamin K-1 and an NAD(P)H-quinone oxidoreductase, it may participate in trans-PM electron transport....

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

DEFF Research Database (Denmark)

Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Janina Zygadlo

2016-01-01

. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble...... glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed...... compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons....

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability.

Science.gov (United States)

Goldes, Matthew E; Jeakins-Cooley, Margaret E; McClelland, Levi J; Mou, Tung-Chung; Bowler, Bruce E

2016-05-01

The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0kcal/mol at 25°C and pH7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70-85), which contains the Met80 heme ligand. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

A decade of crystallization drops: crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri.

Science.gov (United States)

Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

2014-04-01

The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-D-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. © 2014 The Protein Society.

Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.

Science.gov (United States)

Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco

2011-10-21

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

In-silico assessment of protein-protein electron transfer. a case study: cytochrome c peroxidase--cytochrome c.

Directory of Open Access Journals (Sweden)

Frank H Wallrapp

Full Text Available The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, ab-initio quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47 × 10(6 s(-1 for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.

The effect of X-irradiation on vitamin E deficient rat liver mitochondrial ATPase and cytochrome c oxidase

International Nuclear Information System (INIS)

Korkut, S.

1978-01-01

Male albino rats were fed for 3 weeks on standard diets or on diets either deficient in or supplemented by vitamin E, whole-body X-irradiated and then immediately decapitated. Liver mitochondrial ATPase activity was stimulated and cytochrome c oxidase inhibited in the irradiated vitamin E deficient group. These activities were not influenced by irradiation in the rats fed on vitamin E supplemented and standard diets. The live mitochondrial vitamin E level was decreased in rats fed on the deficient diet. No differences in liver mitochondrial vitamin E levels were observed after X-irradiation of rats fed on any of the diets. The results suggest that the liver mitochondrial inner-membrane structure may be altered by a diet deficient in vitamin E. (U.K.)

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

Science.gov (United States)

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-06-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

Energy Technology Data Exchange (ETDEWEB)

Ross, S.B.

1987-01-01

/sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

In-vivo identification of direct electron transfer from Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein complexes

Energy Technology Data Exchange (ETDEWEB)

Okamoto, Akihiro [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Nakamura, Ryuhei, E-mail: nakamura@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Hashimoto, Kazuhito, E-mail: hashimoto@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); ERATO/JST, HASHIMOTO Light Energy Conversion Project (Japan)

2011-06-30

Graphical abstract: . Display Omitted Highlights: > Monolayer biofilm of Shewanella cells was prepared on an ITO electrode. > Extracellular electron transfer (EET) process was examined with series of mutants. > Direct ET was confirmed with outer-membrane-bound OmcA-MtrCAB complex. > The EET process was not prominently influenced by capsular polysaccharide. - Abstract: The direct electron-transfer (DET) property of Shewanella bacteria has not been resolved in detail due to the complexity of in vivo electrochemistry in whole-cell systems. Here, we report the in vivo assignment of the redox signal indicative of the DET property in biofilms of Shewanella oneidensis MR-1 by cyclic voltammetry (CV) with a series of mutants and a chemical marking technique. The CV measurements of monolayer biofilms formed by deletion mutants of c-type cytochromes ({Delta}mtrA, {Delta}mtrB, {Delta}mtrC/{Delta}omcA, and {Delta}cymA), and pilin ({Delta}pilD), capsular polysaccharide ({Delta}SO3177) and menaquinone ({Delta}menD) biosynthetic proteins demonstrated that the electrochemical redox signal with a midpoint potential at 50 mV (vs. SHE) was due to an outer-membrane-bound OmcA-MtrCAB protein complex of decaheme cytochromes, and did not involve either inner-membrane-bound CymA protein or secreted menaquinone. Using the specific binding affinity of nitric monoxide for the heme groups of c-type cytochromes, we further confirmed this conclusion. The heterogeneous standard rate constant for the DET process was estimated to be 300 {+-} 10 s{sup -1}, which was two orders of magnitude higher than that previously reported for the electron shuttling process via riboflavin. Experiments using a mutant unable to produce capsular polysaccharide ({Delta}SO3177) revealed that the DET property of the OmcA-MtrCAB complex was not influenced by insulating and hydrophilic extracellular polysaccharide. Accordingly, under physiological conditions, S. oneidensis MR-1 utilizes a high density of outer-membrane

Monoclonal antibodies to drosophila cytochrome P-450's

International Nuclear Information System (INIS)

Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

1987-01-01

Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

Effect of Pulsed Electric Field on Membrane Lipids and Oxidative Injury of Salmonella typhimurium.

Science.gov (United States)

Yun, Ou; Zeng, Xin-An; Brennan, Charles S; Han, Zhong

2016-08-22

Salmonella typhimurium cells were subjected to pulsed electric field (PEF) treatment at 25 kV/cm for 0-4 ms to investigate the effect of PEF on the cytoplasmic membrane lipids and oxidative injury of cells. Results indicated that PEF treatment induced a decrease of membrane fluidity of Salmonella typhimurium (S. typhimuriumi), possibly due to the alterations of fatty acid biosynthesis-associated gene expressions (down-regulation of cfa and fabA gene expressions and the up-regulation of fabD gene expression), which, in turn, modified the composition of membrane lipid (decrease in the content ratio of unsaturated fatty acids to saturated fatty acids). In addition, oxidative injury induced by PEF treatment was associated with an increase in the content of malondialdehyde. The up-regulation of cytochrome bo oxidase gene expressions (cyoA, cyoB, and cyoC) indicated that membrane damage was induced by PEF treatment, which was related to the repairing mechanism of alleviating the oxidative injury caused by PEF treatment. Based on these results, we achieved better understanding of microbial injury induced by PEF, suggesting that micro-organisms tend to decrease membrane fluidity in response to PEF treatment and, thus, a greater membrane fluidity might improve the efficiency of PEF treatment to inactivate micro-organisms.

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Kärenlampi, S O; Marin, E; Hänninen, O O

1981-02-15

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

Artificial membranes with selective nanochannels for protein transport

KAUST Repository

Sutisna, Burhannudin

2016-09-05

A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

Artificial membranes with selective nanochannels for protein transport

KAUST Repository

Sutisna, Burhannudin; Polymeropoulos, Georgios; Mygiakis, E.; Musteata, Valentina-Elena; Peinemann, Klaus-Viktor; Smilgies, D. M.; Hadjichristidis, Nikolaos; Nunes, Suzana Pereira

2016-01-01

A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

Fast prediction of cytochrome P450 mediated drug metabolism

DEFF Research Database (Denmark)

Rydberg, Patrik Åke Anders; Poongavanam, Vasanthanathan; Oostenbrink, Chris

2009-01-01

Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory...... calculations, for predicting activation energies for aliphatic and aromatic oxidations by cytochromes P450 is developed and compared with several other methods. Although the applicability of the method is currently limited to a subset of P450 reactions, these reactions describe more than 90...

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

OpenAIRE

Kärenlampi, S O; Marin, E; Hänninen, O O

1981-01-01

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture...

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

Energy Technology Data Exchange (ETDEWEB)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

2013-02-15

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

International Nuclear Information System (INIS)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A.

2013-01-01

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

International Nuclear Information System (INIS)

Stiborová, Marie; Moserová, Michaela; Černá, Věra; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J.; Wolf, C. Roland; Schmeiser, Heinz H.; Phillips, David H.; Frei, Eva; Arlt, Volker M.

2014-01-01

In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b 5 , and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b 5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b 5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b 5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR

Polarography of cytochrome c in ammoniacal buffers containing cobalt ions. The effect of the protein conformation.

Science.gov (United States)

Brabec, V

1985-12-01

Catalytic currents yielded by cytochrome c in ammoniacal buffers containing cobalt ions at a dropping mercury electrode (Brdicka's catalytic currents) were investigated by means of direct current, differential pulse, normal pulse (NP) and phase-selective alternating current polarography. It was found that Brdicka's catalytic current of cytochrome c, (the more negative part of Brdicka's double wave, wave B) is influenced by the presence of cytochrome c denaturants in the background solution. The wave B rose with the increasing concentrations of urea and sodium perchlorate, and increased in parallel with absorbance changes at 409 and 695 nm measured for identical cytochrome c solutions. The latter absorbance changes reflect unfolding of cytochrome c molecules in the bulk of solution by these denaturants. The results of NP polarography (a technique working with large potential excursion during the drop lifetime) indicate that in Brdicka's solution cytochrome c could extensively be unfolded due to its adsorption at the mercury electrode, polarized to potentials around that of zero charge.

Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

International Nuclear Information System (INIS)

Kim, Seong K.; Stark, Benjamin C.; Webster, Dale A.

2005-01-01

The operon (cyo) encoding the Na + -pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H + transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na + -pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na + against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na + through the D-channel to the exterior and transports four H + through the K-channel for the reduction of each O 2

Heterogeneous binding of sigma radioligands in the rat brain and liver; Possible relationship to subforms of cytochrome P-450

Energy Technology Data Exchange (ETDEWEB)

Ross, S B [Research Laboratories, Astra Research Centre AB, Soedertaejle (Sweden)

1991-01-01

The binding of four sigma receptor ligands, {sup 3}H-(+)-N-allyl-N-normetazocine ({sup 3}H-(+)-SKF 10,047), {sup 3}H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ({sup 3}H-(+)-3-PPP), {sup 3}H-haloperidol and {sup 3}H-N,N'-di(o-totyl)guanidine ({sup 3}H-DTG), and the cytochrome P450IID6 ligand and dopamine uptake inhibitor {sup 3}H-1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine ({sup 3}H-GBR 12935) to membranal preparations of rat liver or whole rat brain was examined regarding kinetical properties and inhibition by various compounds with affinity for sigma binding sites or cytochrome P-450. In rat brain the density of binding sites was increased in order (+)-SKF 10,047<(+)-3-PPPcytochrome P-450 inhibitor proadifen (SKF 525A), like haloperidol, was a potent inhibitor of the binding of {sup 3}H-(+)-SKF 10,047, {sup 3}H-(+)-3-PPP and {sup 3}H-haloperidol to the liver and brain preparations, less active in inhibiting the binding of {sup 3}H-DTG and least effective on the binding of {sup 3}H-GBR 12935. Another cytochrome P-450 inhibitor, L-lobeline, was particularly potent in inhibiting the binding of {sup 3}H-DTG but was also quite potent inhibitor of the binding of the other sigma ligands. It was less potent in inhibiting the binding of {sup 3}H-GBR 12935. The binding of the latter ligand was potently inhibited by the analogous compound GBR 12909 but of the other compounds examined only L-lobeline, proadifen, haloperidol, DTG and (+)-3-PPP had IC50 values below 10 {mu}M. (Abstract Truncated)

Charge Transfer at the Qo-Site of the Cytochrome bc1 Complex Leads to Superoxide Production

DEFF Research Database (Denmark)

Bøgh Salo, Adrian; Husen, Peter; Solov'yov, Ilia A

2017-01-01

The cytochrome bc1 complex is the third protein complex in the electron transport chain of mitochondria or photosynthetic bacteria, and it serves to create an electrochemical gradient across a cellular membrane, which is used to drive ATP synthesis. The purpose of this study is to investigate...... interactions involving an occasionally trapped oxygen molecule (O2) at the so-called Qo site of the bc1 complex, which is one of the central active sites of the protein complex, where redox reactions are expected to occur. The investigation focuses on revealing the possibility of the oxygen molecule...... to influence the normal operation of the bc1 complex and acquire an extra electron, thus becoming superoxide, a biologically toxic free radical. The process is modeled by applying quantum chemical calculations to previously performed classical molecular dynamics simulations. Investigations reveal several...

Prevention of LDL-suppression of HMG-CoA reductase (HMGR) activity by progesterone (PG): evidence for cytochrome P-450 involvement

International Nuclear Information System (INIS)

Sexton, R.C.; Gupta, A.; Panini, S.R.; Rudney, H.

1987-01-01

Incubation of rat intestinal epithelial cells (IEC-6) with PG has been reported by us to prevent the suppression of HMGR activity by LDL. In the present study, addition of LDL and PG to IEC-6 cells resulted in a 2 fold increase in cellular free cholesterol (CH) in 24 h, while HMGR activity remained elevated. PG did not affect the internalization and degradation of [ 125 I] LDL nor the accumulation of free [ 3 H] CH in cells incubated with [ 3 H-cholesteryl linoleate]-LDL. Also, PG did not affect the intracellular transport of LDL-derived [ 3 H] CH to the plasma membrane nor the efflux of the [ 3 H] CH into medium containing human high density lipoprotein. Addition of LDL to cells, in which the cellular CH was radiolabeled from [ 3 H] acetate, resulted in an increased formation of radiolabeled oxysterols, detected by HPLC, and a corresponding decrease in HMGR activity. PG attenuated both the LDL-induced formation of oxysterols and suppression of HMGR activity. PG inhibited cytochrome P-450 dependent oxidation of benzphetamine, aminopyrine and aniline by liver microsomes from phenobarbitol treated rats. These results suggest PG may prevent LDL suppression of HMGR activity in IEC-6 cells by inhibiting cytochrome P-450 dependent formation of regulatory oxysterols

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

Science.gov (United States)

Vanlerberghe, G C; McIntosh, L

1992-12-01

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

Unique organizational and functional features of the cytochrome c maturation system in Shewanella oneidensis.

Directory of Open Access Journals (Sweden)

Miao Jin

Full Text Available Shewanella are renowned for their ability to respire on a wide range of electron acceptors, which has been partially accredited to the presence of a large number of the c-type cytochromes. In the model species S. oneidensis MR-1, at least 41 genes encode c-type cytochromes that are predicted to be intact, thereby likely functional. Previously, in-frame deletion mutants for 36 of these genes were obtained and characterized. In this study, first we completed the construction of an entire set of c-type cytochrome mutants utilizing a newly developed att-based mutagenesis approach, which is more effective and efficient than the approach used previously by circumventing the conventional cloning. Second, we investigated the cytochrome c maturation (Ccm system in S. oneidensis. There are two loci predicted to encode components of the Ccm system, SO0259-SO0269 and SO0476-SO0478. The former is proven essential for cytochrome c maturation whereas the latter is dispensable. Unlike the single operon organization observed in other γ-proteobacteria, genes at the SO0259-SO0269 locus are uniquely organized into four operons, ccmABCDE, scyA, SO0265, and ccmFGH-SO0269. Functional analysis revealed that the SO0265 gene rather than the scyA and SO0269 genes are relevant to cytochrome c maturation.

Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling.

Science.gov (United States)

Sturgis, James N; Niederman, Robert A

2008-01-01

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc (1) complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc (1) complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.

Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

Science.gov (United States)

Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

2012-02-01

Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

Cytochrome c interaction with hematite ({alpha}-Fe{sub 2}O{sub 3}) surfaces

Energy Technology Data Exchange (ETDEWEB)

Eggleston, Carrick M. [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States)]. E-mail: carrick@uwyo.edu; Khare, Nidhi [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States); Lovelace, David M. [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States)

2006-02-15

The interaction of metalloproteins such as cytochromes with oxides is of interest for a number of reasons, including molecular catalysis of environmentally important mineral-solution electron transfer reactions (e.g., dehalogenations) and photovoltaic applications. Iron reduction by bacteria, thought to be cytochrome mediated, is of interest for geochemical and environmental remediation reasons. As a baseline for understanding cytochrome interaction with ferric oxide surfaces, we report on the interaction of mitochondrial cytochrome c (Mcc), a well-studied protein, with hematite ({alpha}-Fe{sub 2}O{sub 3}) surfaces. Mcc sorbs strongly to hematite from aqueous solution in a narrow pH range corresponding to opposite charge on Mcc and hematite (between pH 8.5 and 10, Mcc is positively charged and hematite surfaces are negatively charged). Cyclic voltammetry of Mcc using hematite electrodes gives redox potentials characteristic of Mcc in a native conformational state, with no evidence for unfolding on the hematite surface. Atomic force microscopy imaging is consistent with a loosely attached adsorbate that is easily deformed by the AFM tip. In phosphate-containing solution, Mcc adhers to the surface more strongly. These results establish hematite as a viable material for electrochemical and spectroscopic characterization of cytochrome-mineral interaction.

JS-K, a nitric oxide prodrug, induces cytochrome c release and caspase activation in HL-60 myeloid leukemia cells.

Science.gov (United States)

Udupi, Vidya; Yu, Margaret; Malaviya, Swati; Saavedra, Joseph E; Shami, Paul J

2006-10-01

Nitric oxide (NO) induces differentiation and apoptosis in acute myelogenous leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K induces apoptosis in HL-60 cells by a caspase-dependent mechanism. The purpose of this study was to determine the pathway through which JS-K induces apoptosis. We show that JS-K alters mitochondrial membrane potential (DeltaPsim) and induces cytochrome c release from mitochondria into the cytoplasm. Treatment with JS-K resulted in activation of Caspase (Casp) 9, Casp 3 and Casp 8. JS-K constitutes a promising lead for a new class of anti-leukemic agents.

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.

In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

Science.gov (United States)

Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

2010-03-30

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome C3

Energy Technology Data Exchange (ETDEWEB)

Wall, Judy D

2013-04-11

The central objective of our proposed research was twofold: 1) to investigate the structure-function relationship of Desulfovibrio desulfuricans (now Desulfovibrio alaskensis G20) cytochrome c3 with uranium and 2) to elucidate the mechanism for uranium reduction in vitro and in vivo. Physiological analysis of a mutant of D. desulfuricans with a mutation of the gene encoding the type 1 tetraheme cytochrome c3 had demonstrated that uranium reduction was negatively impacted while sulfate reduction was not if lactate were the electron donor. This was thought to be due to the presence of a branched pathway of electron flow from lactate leading to sulfate reduction. Our experimental plan was to elucidate the structural and mechanistic details of uranium reduction involving cytochrome c3.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

Science.gov (United States)

Sacco, James C; Trepanier, Lauren A

2010-01-01

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (Phydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Science.gov (United States)

Gray, K A; Dutton, P L; Daldal, F

1994-01-25

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

Science.gov (United States)

Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

2004-12-01

In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Science.gov (United States)

Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

2014-09-02

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

Energy Technology Data Exchange (ETDEWEB)

Wang, Zheming; Shi, Zhi; Shi, Liang; White, Gaye F.; Richardson, David J.; Clarke, Thomas A.; Fredrickson, Jim K.; Zachara, John M.

2015-08-25

Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, some evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin that are proposed to mediate electron transfer (Marsili et al., 2008). In this work, we used methyl viologen (MV•+)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of secreted flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200 µM) by MELs ([MV•+] ~ 42 µM and MtrABC ≤ 1 nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MV•+ and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 seconds. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (≤ 1 µM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. However, at higher FMN concentrations (> 1 µM), the reaction rates for both steps decreased and varied inversely with FMN concentration, indicating that FMN inhibited the MEL to Fe(III)-oxide electron transfer

Cytochrome c6B of Synechococcus sp. WH 8102 – Crystal structure and basic properties of novel c6-like family representative

International Nuclear Information System (INIS)

Zatwarnicki, Pawel; Barciszewski, Jakub; Krzywda, Szymon; Jaskolski, Mariusz; Kolesinski, Piotr; Szczepaniak, Andrzej

2014-01-01

Highlights: • Crystal structure of cytochrome c 6B from Synechococcus sp. WH 8102 was solved. • Basic biophysical properties of cytochrome c 6B were determined. • Cytochrome c 6B exhibits similar architecture to cytochrome c 6 . • Organization of heme binding pocket of cytochrome c 6B differs from that of c 6 . • Midpoint potential of cytochrome c 6B is significantly lower than of cytochrome c 6 . - Abstract: Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c 6 participates in electron transfer from cytochrome b 6 f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c 6A from Arabidopsis thaliana was identified as a protein responsible for disulfide bond formation in response to intracellular redox state changes and c 550 is well known element of photosystem II. However, function of cytochromes marked as c 6B , c 6C and c M as well as the physiological process in which they take a part still remain unidentified. Here we present the first structural and biophysical analysis of cytochrome from the c 6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Purified protein was crystallized and its structure was refined at 1.4 Å resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c 6 , are slightly red-shifted α band of UV–Vis spectrum as well as relatively low midpoint potential (113.2 ± 2.2 mV). Although, physiological function of cytochrome c 6B has yet to be determined its properties probably exclude the participation of this protein in electron trafficking between b 6 f complex and photosystem I

Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

International Nuclear Information System (INIS)

Meier, U.T.; Meyer, U.A.

1987-01-01

The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single [ 125 I]-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

Science.gov (United States)

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

North African genetic variation of cytochrome and sulfotransferase ...

African Journals Online (AJOL)

in these genes have shown relevant ethnic differences among Sub-Saharan .... This cytochrome catalyzes a big amount of oxidative reactions of substances like ... with samples of European and African origin (because of the scarce data ...

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Directory of Open Access Journals (Sweden)

Shilpi Khare

2015-07-01

Full Text Available Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1 in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50 of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450

International Nuclear Information System (INIS)

Marinello, A.J.; Bansal, S.K.; Paul, B.; Koser, P.L.; Love, J.; Struck, R.F.; Gurtoo, H.L.

1984-01-01

The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [chloroethyl-3H]cyclophosphamide [( chloroethyl-3H]CP) and [4-14C]cyclophosphamide [( 4-14C]CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP, were also investigated. The metabolism of [chloroethyl-3H]CP and [4-14C]CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar. The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between [4-14C]CP and [chloroethyl-3H]CP metabolism. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of [chloroethyl-3H]CP to nucleic acids and almost exclusive binding of [4-14C]CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of NADPH. The results confirmed covalent association between [14C]acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of [14C]acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations

Aquifex aeolicus membrane hydrogenase for hydrogen biooxidation: Role of lipids and physiological partners in enzyme stability and activity

Energy Technology Data Exchange (ETDEWEB)

Infossi, Pascale; Lojou, Elisabeth; Giudici-Orticoni, Marie-Therese [Unite de Bioenergetique et Ingenierie des Proteines, UPR 9036, Institut de Microbiologie de la Mediterranee - CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Chauvin, Jean-Paul [Institut de Biologie du developpement de Marseille Luminy, UMR 6216, Parc Scientifique de Luminy, 163 Avenue de Luminy, BP 907, 13009 Marseille (France); Herbette, Gaetan [Spectropole FI 1739, Aix-Marseille Universite case 511, Faculte de St Jerome Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20 (France); Brugna, Myriam [Unite de Bioenergetique et Ingenierie des Proteines, UPR 9036, Institut de Microbiologie de la Mediterranee - CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Universite de Provence, 3 Place Victor Hugo, 13331 Marseille Cedex 03 (France)

2010-10-15

Hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus is a good candidate for biotechnological devices thanks to its ability to oxidize hydrogen at high temperature, even in the presence of oxygen and CO. In order to enhance the enzyme stability and the catalytic efficiency, we investigated the hydrogen oxidation process with hydrogenase I embedded in a physiological-like environment. Hydrogenase I partners in the metabolic chain, namely membrane quinone and cytochrome b, were purified and fully characterized. The complex hydrogenase I-cytochrome b was inserted into liposomes. Surface Plasmon Resonance revealed that quinone took part in the stabilization of the complex. By use of molecular modelization and electrochemistry analysis, enzyme stability has been demonstrated to be stronger and enzymatic efficiency to be five times higher when hydrogenase is embedded into the liposomes. This result raises the possibility of using hydrogenases as biocatalysts in fuel cells. (author)

Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

DEFF Research Database (Denmark)

Gnanasekaran, Thiyagarajan; Vavitsas, Konstantinos; Andersen-Ranberg, Johan

2015-01-01

in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45-55 μg/g plant dry weight of isopimaric acid four days after the infiltration with the modified...... in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co...

Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

Science.gov (United States)

Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

1988-11-01

In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

Genetic defects of cytochrome c oxidase assembly

Czech Academy of Sciences Publication Activity Database

Pecina, Petr; Houšťková, H.; Hansíková, H.; Zeman, J.; Houštěk, Josef

2004-01-01

Roč. 53, Suppl. 1 (2004), s. S213-S223 ISSN 0862-8408 R&D Projects: GA ČR GA303/03/0749 Institutional research plan: CEZ:AV0Z5011922 Keywords : cytochrome c oxidase * mitochondrial disorders Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.140, year: 2004

Membrane Transporters for Bilirubin and Its Conjugates: A Systematic Review

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Jovana Čvorović

2017-12-01

Full Text Available Background: Bilirubin is a highly-hydrophobic tetrapyrrole which binds to plasma albumin. It is conjugated in the liver to glucuronic acid, and the water-soluble glucuronides are excreted in urine and bile. The membrane transporters of bilirubin diglucuronide are well-known. Still undefined are however the transporters performing the uptake of bilirubin from the blood into the liver, a process known to be fast and not rate-limited. The biological importance of this process may be appraised by considering that in normal adults 200–300 mg of bilirubin are produced daily, as a result of the physiologic turnover of hemoglobin and cellular cytochromes. Nevertheless, research in this field has yielded controversial and contradicting results. We have undertaken a systematic review of the literature, believing in its utility to improve the existing knowledge and promote further advancements.Methods: We have sourced the PubMed database until 30 June 2017 by applying 5 sequential searches. Screening and eligibility criteria were applied to retain research articles reporting results obtained by using bilirubin molecules in membrane transport assays in vitro or by assessing serum bilirubin levels in in vivo experiments.Results: We have identified 311 articles, retaining 44, reporting data on experimental models having 6 incremental increases of complexity (isolated proteins, membrane vesicles, cells, organ fragments, in vivo rodents, and human studies, demonstrating the function of 19 membrane transporters, encoded by either SLCO or ABC genes. Three other bilirubin transporters have no gene, though one, i.e., bilitranslocase, is annotated in the Transporter Classification Database.Conclusions: This is the first review that has systematically examined the membrane transporters for bilirubin and its conjugates. Paradoxically, the remarkable advancements in the field of membrane transport of bilirubin have pointed to the elusive mechanism(s enabling

Metallic nickel nano- and fine particles induce JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway

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Castranova Vincent

2009-04-01

Full Text Available Abstract Background Carcinogenicity of nickel compounds has been well documented. However, the carcinogenic effect of metallic nickel is still unclear. The present study investigates metallic nickel nano- and fine particle-induced apoptosis and the signal pathways involved in this process in JB6 cells. The data obtained from this study will be of benefit for elucidating the pathological and carcinogenic potential of metallic nickel particles. Results Using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, we found that metallic nickel nanoparticles exhibited higher cytotoxicity than fine particles. Both metallic nickel nano- and fine particles induced JB6 cell apoptosis. Metallic nickel nanoparticles produced higher apoptotic induction than fine particles. Western-blot analysis showed an activation of proapoptotic factors including Fas (CD95, Fas-associated protein with death domain (FADD, caspase-8, death receptor 3 (DR3 and BID in apoptotic cells induced by metallic nickel particles. Immunoprecipitation (IP western blot analysis demonstrated the formation of the Fas-related death-inducing signaling complex (DISC in the apoptotic process. Furthermore, lamin A and beta-actin were cleaved. Moreover, we found that apoptosis-inducing factor (AIF was up-regulated and released from mitochondria to cytoplasm. Interestingly, although an up-regulation of cytochrome c was detected in the mitochondria of metallic nickel particle-treated cells, no cytochrome c release from mitochondria to cytoplasm was found. In addition, activation of antiapoptotic factors including phospho-Akt (protein kinase B and Bcl-2 was detected. Further studies demonstrated that metallic nickel particles caused no significant changes in the mitochondrial membrane permeability after 24 h treatment. Conclusion In this study, metallic nickel nanoparticles caused higher cytotoxicity and apoptotic induction than fine particles in JB6 cells. Apoptotic cell death

Interface Adsorption Taking the Most Advantageous Conformation for Electron Transfer Between Graphene and Cytochrome c.

Science.gov (United States)

Hu, Benfeng; Ge, Zhenpeng; Li, Xiaoyi

2015-07-01

Most designed functions in biomedical nanotechnology are directly influenced by interactions of biological molecules with nano surfaces. Here, we explored and detected the most favorable adsorption conformation of cytochrome c on graphene by measuring the adsorption energy, the number of contact atoms, and the minimal distance between protein and surface. From the root mean square deviation of the protein backbone, the radius of gyration, and the proportion of secondary structure, it is revealed that cytochrome c does not deform significantly and the secondary structures are preserved to a large extent. The residues, Lys, Phe and Thr contribute significantly to the adsorption of cytochrome c to graphene. The long hydrophobic and flexible alkyl tail of Lys, the π-π stacking interaction between Phe and graphene, and the presence of abundant Thr constitute the driving force for the stable adsorption of cytochrome c on graphene. Cytochrome c is adsorbed to graphene with the group heme lying almost perpendicular to the graphene, and the distance between Fe atom and the graphene is 10.15 A, which is shorter than that between electron donor and receptor in many other biosystems. All the results suggest that the most favorable adsorption takes the most advantageous conformation for electron transfer, which promotes significantly the electron transfer between graphene and cytochrome c. The findings might provide new and important information for designs of biomedical devices or products with graphene-based nanomaterials.

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b

DEFF Research Database (Denmark)

Grimmelikhuijzen, C J; Marres, C A; Slater, Conor

1975-01-01

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reo...

Data for molecular dynamics simulations of B-type cytochrome c oxidase with the Amber force field

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Longhua Yang

2016-09-01

Full Text Available Cytochrome c oxidase (CcO is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. This article presents parameters for the cofactors of ba3-type CcO that are compatible with the all-atom Amber ff12SB and ff14SB force fields. Specifically, parameters were developed for the CuA pair, heme b, and the dinuclear center that consists of heme a3 and CuB bridged by a hydroperoxo group. The data includes geometries in XYZ coordinate format for cluster models that were employed to compute proton transfer energies and derive bond parameters and point charges for the force field using density functional theory. Also included are the final parameter files that can be employed with the Amber leap program to generate input files for molecular dynamics simulations with the Amber software package. Based on the high resolution (1.8 Å X-ray crystal structure of the ba3-type CcO from Thermus thermophilus (Protein Data Bank ID number PDB: 3S8F, we built a model that is embedded in a POPC lipid bilayer membrane and solvated with TIP3P water molecules and counterions. We provide PDB data files of the initial model and the equilibrated model that can be used for further studies.

Effects of Muscle-Specific Oxidative Stress on Cytochrome c Release and Oxidation-Reduction Potential Properties.

Science.gov (United States)

Ke, Yiling; Mitacek, Rachel M; Abraham, Anupam; Mafi, Gretchen G; VanOverbeke, Deborah L; DeSilva, Udaya; Ramanathan, Ranjith

2017-09-06

Mitochondria play a significant role in beef color. However, the role of oxidative stress in cytochrome c release and mitochondrial degradation is not clear. The objective was to determine the effects of display time on cytochrome c content and oxidation-reduction potential (ORP) of beef longissimus lumborum (LL) and psoas major (PM) muscles. PM discolored by day 3 compared with LL. On day 0, mitochondrial content and mitochondrial oxygen consumption were greater in PM than LL. However, mitochondrial content and oxygen consumption were lower (P stress can affect cytochrome c release and ORP changes.

Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

Energy Technology Data Exchange (ETDEWEB)

Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu [Department of Environmental Sciences and LSU Superfund Research Center, Louisiana State University A& M College, Baton Rouge, LA 70803 (United States); Lomnicki, Slawo M., E-mail: slomni1@lsu.edu [Department of Environmental Sciences and LSU Superfund Research Center, Louisiana State University A& M College, Baton Rouge, LA 70803 (United States); Backes, Wayne L., E-mail: wbacke@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

2015-12-01

Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2–CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • Particulate matter (PM) competitively inhibited CYP1A2 activity. • EPFRs were much more potent CYP1A2 inhibitors than other types of PM. • PM interacts differently with different forms of P450. • PM

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

OpenAIRE

Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

2012-01-01

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. O...

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

NARCIS (Netherlands)

Van Wielink, J E; Reijnders, W N; Van Spanning, R J; Oltmann, L F; Stouthamer, A.H.

1986-01-01

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

Science.gov (United States)

Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

1999-01-01

Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

Early Decrease in Respiration and Uncoupling Event Independent of Cytochrome c Release in PC12 Cells Undergoing Apoptosis

Science.gov (United States)

Berghella, Libera; Ferraro, Elisabetta

2012-01-01

Cytochrome c is a key molecule in mitochondria-mediated apoptosis. It also plays a pivotal role in cell respiration. The switch between these two functions occurs at the moment of its release from mitochondria. This process is therefore extremely relevant for the fate of the cell. Since cytochrome c mediates respiration, we studied the changes in respiratory chain activity during the early stages of apoptosis in order to contribute to unravel the mechanisms of cytochrome c release. We found that, during staurosporine (STS)- induced apoptosis in PC12 cells, respiration is affected before the release of cytochrome c, as shown by a decrease in the endogenous uncoupled respiration and an uncoupling event, both occurring independently of cytochrome c release. The decline in the uncoupled respiration occurs also upon Bcl-2 overexpression (which inhibits cytochrome c release), while the uncoupling event is inhibited by Bcl-2. We also observed that the first stage of nuclear condensation during STS-induced apoptosis does not depend on the release of cytochrome c into the cytosol and is a reversibile event. These findings may contribute to understand the mechanisms affecting mitochondria during the early stages of apoptosis and priming them for the release of apoptogenic factors. PMID:22666257

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study.

Science.gov (United States)

Magalhães, Pedro R; Oliveira, A Sofia F; Campos, Sara R R; Soares, Cláudio M; Baptista, António M

2017-02-27

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286 I , Y288 I , and K362 I . All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

Modeling of interaction between cytochrome c and the WD domains of Apaf-1: bifurcated salt bridges underlying apoptosome assembly.

Science.gov (United States)

Shalaeva, Daria N; Dibrova, Daria V; Galperin, Michael Y; Mulkidjanian, Armen Y

2015-05-27

Binding of cytochrome c, released from the damaged mitochondria, to the apoptotic protease activating factor 1 (Apaf-1) is a key event in the apoptotic signaling cascade. The binding triggers a major domain rearrangement in Apaf-1, which leads to oligomerization of Apaf-1/cytochrome c complexes into an apoptosome. Despite the availability of crystal structures of cytochrome c and Apaf-1 and cryo-electron microscopy models of the entire apoptosome, the binding mode of cytochrome c to Apaf-1, as well as the nature of the amino acid residues of Apaf-1 involved remain obscure. We investigated the interaction between cytochrome c and Apaf-1 by combining several modeling approaches. We have applied protein-protein docking and energy minimization, evaluated the resulting models of the Apaf-1/cytochrome c complex, and carried out a further analysis by means of molecular dynamics simulations. We ended up with a single model structure where all the lysine residues of cytochrome c that are known as functionally-relevant were involved in forming salt bridges with acidic residues of Apaf-1. This model has revealed three distinctive bifurcated salt bridges, each involving a single lysine residue of cytochrome c and two neighboring acidic resides of Apaf-1. Salt bridge-forming amino acids of Apaf-1 showed a clear evolutionary pattern within Metazoa, with pairs of acidic residues of Apaf-1, involved in bifurcated salt bridges, reaching their highest numbers in the sequences of vertebrates, in which the cytochrome c-mediated mechanism of apoptosome formation seems to be typical. The reported model of an Apaf-1/cytochrome c complex provides insights in the nature of protein-protein interactions which are hard to observe in crystallographic or electron microscopy studies. Bifurcated salt bridges can be expected to be stronger than simple salt bridges, and their formation might promote the conformational change of Apaf-1, leading to the formation of an apoptosome. Combination of

Cytochrome P450s and molecular epidemiology

Science.gov (United States)

Gonzalez, Frank J.; Gelboin, Harry V.

1993-03-01

Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

Oxygen and xenobiotic reductase activities of cytochrome P450.

NARCIS (Netherlands)

Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

1995-01-01

The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

[Immunomodulators with an 8-azasteroid structure as inducers of liver cytochrome P-450].

Science.gov (United States)

Kuz'mitskiĭ, B B; Dad'kov, I G; Mashkovich, A E; Stoma, O V; Slepneva, L M

1990-01-01

Two structural analogues of D-homo-8-azasteroids, both an immunostimulant and an immunodepressant, are inductors of the liver cytochrome P-450 in animals. This capability was shown by means of both a decrease of the hexenal sleep duration in the pharmacological test and an increase of the quantity of cytochrome P-450 and the rate of N-demethylation of aminopyrine in the biochemical assays.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

OpenAIRE

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confo...

The anti-mycobacterial activity of the cytochrome bcc inhibitor Q203 can be enhanced by small-molecule inhibition of cytochrome bd.

NARCIS (Netherlands)

Lu, P.; Asseri, A.H.O.; Kremer, Martijn; Maaskant, Janneke; Ummels, Roy; Lill, H.; Bald, D.

2018-01-01

Mycobacterial energy metabolism currently attracts strong attention as new target space for development of anti-tuberculosis drugs. The imidazopyridine Q203 targets the cytochrome bcc complex of the respiratory chain, a key component in energy metabolism. Q203 blocks growth of Mycobacterium

The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance.

Directory of Open Access Journals (Sweden)

Julia Leclerc

Full Text Available Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

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Yuny Erwanto

2013-03-01

Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

Role of Asp544 in subunit I for Na+ pumping by Vitreoscilla cytochrome bo

International Nuclear Information System (INIS)

Chung, Yeon T.; Stark, Benjamin C.; Webster, Dale A.

2006-01-01

The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H + pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na + pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na + as well as decreased stimulation in respiratory activity in the presence of Na + . Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

Science.gov (United States)

Laforge, Mireille; Limou, Sophie; Harper, Francis; Casartelli, Nicoletta; Rodrigues, Vasco; Silvestre, Ricardo; Haloui, Houda; Zagury, Jean-Francois; Senik, Anna; Estaquier, Jerome

2013-01-01

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells. PMID:23658518

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

International Nuclear Information System (INIS)

Hiraoka, Yoshinori; Granja, Ana Teresa; Fialho, Arsenio M.; Schlarb-Ridley, Beatrix G.; Das Gupta, Tapas K.; Chakrabarty, Ananda M.; Yamada, Tohru

2005-01-01

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c 551 from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c 551 , the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G 1 to S phase, as well as at the G 2 /M phase at higher concentrations. Unlike P. aeruginosa cytochrome c 551 which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G 2 /M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 deg. C, suggesting energy requirement in the entry process

Cytochrome oxidase as an indicator of ice storage and frozen storage

DEFF Research Database (Denmark)

Godiksen, Helene; Jessen, Flemming

2001-01-01

in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

Science.gov (United States)

González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

2015-08-11

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

Rieske iron-sulfur protein of the cytochrome bc(1) complex: a potential target for fungicide discovery.

Science.gov (United States)

Yang, Wen-Chao; Li, Hui; Wang, Fu; Zhu, Xiao-Lei; Yang, Guang-Fu

2012-07-23

The cytochrome bc(1) complex (complex III, cyt bc(1)) is an essential component of cellular respiration. Cyt bc(1) has three core subunits that are required for its catalytic activity: cytochrome b, cytochrome c(1), and the Rieske iron-sulfur protein (ISP). Although most fungicides inhibit this enzyme by binding to the cytochrome b subunit, resistance to these fungicides has developed rapidly due to their widespread application. Resistance is mainly associated with mutations in cytochrome b, the only subunit encoded by mitochondrial DNA. Recently, the flexibility and motion of the ISP and its essential role in electron transfer have received intense attention; this leads us to propose a new classification of cyt bc(1) inhibitors (three types of Q(o) inhibitors) that mobilize, restrict, or fix the rotation of the ISP. Importantly, the strengths of the ISP-inhibitor interactions correlate with inhibitor activity and the development of resistance to Q(o) inhibitors, thereby offering clues for designing novel cyt bc(1) inhibitors with high potency and a low risk of resistance. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrochemical determination of hydrogen peroxide using Rhodobacter capsulatus cytochrome c peroxidase at a gold electrode

NARCIS (Netherlands)

De Wael, K.; Buschop, H.; Heering, H.A.; De Smet, L.; Van Beeumen, J.; Devreese, B.; Adriaens, A.

2007-01-01

We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cytochrome c peroxidase (RcCCP) at a gold electrode modified with 4,4?-bipyridyl. RcCCP shows no additional oxidation or reduction peaks compared to the electrochemistry of only HHC, which indicates that it

Covalent modification of cytochrome c by reactive metabolites of furan.

Science.gov (United States)

Phillips, Martin B; Sullivan, Mathilde M; Villalta, Peter W; Peterson, Lisa A

2014-01-21

Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan.

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.

Science.gov (United States)

Yun, Jiae; Malvankar, Nikhil S; Ueki, Toshiyuki; Lovley, Derek R

2016-02-01

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

Kinetics and Mechanistic Studies on the Reaction between Cytochrome c and Tea Catechins

Directory of Open Access Journals (Sweden)

Lihua Wang

2014-08-01

Full Text Available Green tea is characterized by the presence of an abundance of polyphenolic compounds, also known as catechins, including epicatechin (EC, epigallocatechin (EGC, epicatechin gallate (EGC and epigallocatechin gallate (EGCG. In addition to being a popular beverage, tea consumption has been suggested as a mean of chemoprevention. However, its mode of action is unclear. It was discovered that tea catechins can react with cytochrome c. When oxidized cytochrome c was mixed with catechins commonly found in green tea under non-steady-state conditions, a reduction of cytochrome c was observed. The reaction rate of the catechins was dependent on the pH and the nature of the catechin. The pseudo-first order rate constant obtained increased in the order of EC < ECG < EGC < EGCG, which is consistent with previously reported superoxide reduction activities and Cu2+ reduction activities of tea catechins.

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase

NARCIS (Netherlands)

van Kuilenburg, A. B.; Gorren, A. C.; Dekker, H. L.; Nieboer, P.; van Gelder, B. F.; Muijsers, A. O.

1992-01-01

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of

Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

Science.gov (United States)

Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

2011-01-01

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

On the role of cytochrome c8 in photosynthetic electron transfer of the purple non-sulfur bacterium Rhodoferax fermentans

DEFF Research Database (Denmark)

Hochkoeppler, Alejandro; Ciurli, Stefano; Kofod, Pauli

1997-01-01

We report on the isolation, purification and functional characterization of a soluble c-type cytochrome from light-grown cells of the purple phototroph Rhodoferax fermentans. This cytochrome is basic (pI = 8), has a molecular mass of 12 kDa, and is characterized by a midpoint reduction potential...... center, in a fast (sub-ms) and a slow (ms) phase. Competition experiments in the presence of both cytochrome c8 and high potential iron-sulfur protein (HiPIP), isolated from the same microorganism, show that cytochrome c8 oxidation is decreased upon addition of HiPIP. These observations suggest...

Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing

International Nuclear Information System (INIS)

Serra, A.S.; Jorge, S.R.; Silveira, C.M.; Moura, J.J.G.; Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H.; Almeida, M.G.

2011-01-01

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd 1 (cyt-cd 1 ) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c 552 (cyt-c 552 ), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 μM cyt-cd 1 /100 μM cyt-c 552 and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c 552 and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254 ± 2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c 552 and cd 1 is 9.9 x 10 3 M -1 s -1 . The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 μM; detection and quantification limits of 7 and 24 μM, respectively; sensitivity of 2.49 ± 0.08 A mol -1 cm 2 μM -1 . Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Cytochrome c biosensor for determination of trace levels of cyanide and arsenic compounds

International Nuclear Information System (INIS)

Fuku, Xolile; Iftikar, Faiza; Hess, Euodia; Iwuoha, Emmanuel; Baker, Priscilla

2012-01-01

Highlights: ► Cytochrome c biosensor for detection of KCN, As 2 O 3 and Fe 2 K (CN) was constructed. ► Detection limits in the range of 4.3–9.1 μM for the analytes were obtained using CV, SWV and EIS. ► The detection limits for the biosensor were significantly lower than current EPA and WHO guidelines. - Abstract: An electrochemical method based on a cytochrome c biosensor was developed, for the detection of selected arsenic and cyanide compounds. Boron doped diamond (BDD) electrode was used as a transducer, onto which cytochrome c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH = 7) was found to be in the range of (1.1–4.5) × 10 −8 A μM −1 and the detection limits ranged from 4.3 to 9.1 μM. The biosensor is therefore able to measure significantly lower than current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines, for these types of analytes. The protein binding was monitored as a decrease in biosensor peak currents by SWV and as an increase in biosensor charge transfer resistance by electrochemical impedance spectroscopy (EIS). EIS provided evidence that the electrocatalytic advantage of BDD electrode was not lost upon immobilisation of cytochrome c. The interfacial kinetics of the biosensor was modelled as equivalent electrical circuit based on electrochemical impedance spectroscopy data. UV–vis spectroscopy was used to confirm the binding of the protein in solution by monitoring the intensity of the soret bands and the Q bands. FTIR was used to characterise the protein in the immobilised state and to confirm that the protein was not denatured upon binding to the pre-treated bare BDD electrode. SNFTIR of cyt c immobilised at platinum electrode, was used to study the effect of oxidation state on the surface bond

Biomimetic Membranes for Multi-Redox Center Proteins

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Renate L. C. Naumann

2016-03-01

Full Text Available His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs. An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM. MRPs, such as the cytochrome c oxidase (CcO from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS and surface-enhanced resonance Raman spectroscopy (SERRS, were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs. The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart

DEFF Research Database (Denmark)

Brazhe, Nadezda; Treiman, Marek; Faricelli, Barbara

2013-01-01

We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial...... surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling...

Electrostatic models of electron-driven proton transfer across a lipid membrane

Energy Technology Data Exchange (ETDEWEB)

Smirnov, Anatoly Yu; Nori, Franco [Advanced Science Institute, RIKEN, Wako-shi, Saitama, 351-0198 (Japan); Mourokh, Lev G [Department of Physics, Queens College, The City University of New York, Flushing, NY 11367 (United States)

2011-06-15

We present two models for electron-driven uphill proton transport across lipid membranes, with the electron energy converted to the proton gradient via the electrostatic interaction. In the first model, associated with the cytochrome c oxidase complex in the inner mitochondria membranes, the electrostatic coupling to the site occupied by an electron lowers the energy level of the proton-binding site, making proton transfer possible. In the second model, roughly describing the redox loop in a nitrate respiration of E. coli bacteria, an electron displaces a proton from the negative side of the membrane to a shuttle, which subsequently diffuses across the membrane and unloads the proton to its positive side. We show that both models can be described by the same approach, which can be significantly simplified if the system is separated into several clusters, with strong Coulomb interaction inside each cluster and weak transfer couplings between them. We derive and solve the equations of motion for the electron and proton creation/annihilation operators, taking into account the appropriate Coulomb terms, tunnel couplings, and the interaction with the environment. For the second model, these equations of motion are solved jointly with a Langevin-type equation for the shuttle position. We obtain expressions for the electron and proton currents and determine their dependence on the electron and proton voltage build-ups, on-site charging energies, reorganization energies, temperature, and other system parameters. We show that the quantum yield in our models can be up to 100% and the power-conversion efficiency can reach 35%.

Electrostatic models of electron-driven proton transfer across a lipid membrane

International Nuclear Information System (INIS)

Smirnov, Anatoly Yu; Nori, Franco; Mourokh, Lev G

2011-01-01

We present two models for electron-driven uphill proton transport across lipid membranes, with the electron energy converted to the proton gradient via the electrostatic interaction. In the first model, associated with the cytochrome c oxidase complex in the inner mitochondria membranes, the electrostatic coupling to the site occupied by an electron lowers the energy level of the proton-binding site, making proton transfer possible. In the second model, roughly describing the redox loop in a nitrate respiration of E. coli bacteria, an electron displaces a proton from the negative side of the membrane to a shuttle, which subsequently diffuses across the membrane and unloads the proton to its positive side. We show that both models can be described by the same approach, which can be significantly simplified if the system is separated into several clusters, with strong Coulomb interaction inside each cluster and weak transfer couplings between them. We derive and solve the equations of motion for the electron and proton creation/annihilation operators, taking into account the appropriate Coulomb terms, tunnel couplings, and the interaction with the environment. For the second model, these equations of motion are solved jointly with a Langevin-type equation for the shuttle position. We obtain expressions for the electron and proton currents and determine their dependence on the electron and proton voltage build-ups, on-site charging energies, reorganization energies, temperature, and other system parameters. We show that the quantum yield in our models can be up to 100% and the power-conversion efficiency can reach 35%.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

2000-04-01

Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+ T cells infected with HIV.

Directory of Open Access Journals (Sweden)

Mireille Laforge

Full Text Available Productive HIV infection of CD4(+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP. Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.

Science.gov (United States)

Rybniker, Jan; Vocat, Anthony; Sala, Claudia; Busso, Philippe; Pojer, Florence; Benjak, Andrej; Cole, Stewart T

2015-07-09

Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H(+)K(+)-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study

International Nuclear Information System (INIS)

Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Tavagnacco, Claudio; Borsari, Marco

2011-01-01

Highlights: → Denaturation involves intermediate and partially unfolded forms. → An unfolded species displaying the haem with Fe coordinated by two His is observed. → Under unfolding conditions the nature of the SAM influences conformation of protein. → Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E o ' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E o ' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.

Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study

Energy Technology Data Exchange (ETDEWEB)

Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy); Tavagnacco, Claudio [Department of Chemistry, University of Trieste, via Giorgieri 1, 34127 Trieste (Italy); Borsari, Marco, E-mail: marco.borsari@unimore.it [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy)

2011-08-01

Highlights: > Denaturation involves intermediate and partially unfolded forms. > An unfolded species displaying the haem with Fe coordinated by two His is observed. > Under unfolding conditions the nature of the SAM influences conformation of protein. > Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E{sup o}' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E{sup o}' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.

Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations

Science.gov (United States)

von Moltke, Lisa L; Greenblatt, David J; Granda, Brian W; Duan, Su Xiang; Grassi, Jeffrey M; Venkatakrishnan, Karthik; Harmatz, Jerold S; Shader, Richard I

1999-01-01

Aims To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. Methods Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Results The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50) of 0.61 μm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by ≈40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. Conclusions The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition. PMID:10383565

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

Energy Technology Data Exchange (ETDEWEB)

Jan, Yi-Hua [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Baker, Angela A. [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Mishin, Vladimir [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Health Science, New York Medical College, Valhalla, NY (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

2015-10-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

International Nuclear Information System (INIS)

Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2015-01-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

Metagenomic insights into anaerobic metabolism along an Arctic peat soil profile.

Directory of Open Access Journals (Sweden)

David A Lipson

Full Text Available A metagenomic analysis was performed on a soil profile from a wet tundra site in northern Alaska. The goal was to link existing biogeochemical knowledge of the system with the organisms and genes responsible for the relevant metabolic pathways. We specifically investigated how the importance of iron (Fe oxides and humic substances (HS as terminal electron acceptors in this ecosystem is expressed genetically, and how respiratory and fermentative processes varied with soil depth into the active layer and into the upper permafrost. Overall, the metagenomes reflected a microbial community enriched in a diverse range of anaerobic pathways, with a preponderance of known Fe reducing species at all depths in the profile. The abundance of sequences associated with anaerobic metabolic processes generally increased with depth, while aerobic cytochrome c oxidases decreased. Methanogenesis genes and methanogen genomes followed the pattern of CH4 fluxes: they increased steeply with depth into the active layer, but declined somewhat over the transition zone between the lower active layer and the upper permafrost. The latter was relatively enriched in fermentative and anaerobic respiratory pathways. A survey of decaheme cytochromes (MtrA, MtrC and their homologs revealed that this is a promising approach to identifying potential reducers of Fe(III or HS, and indicated a possible role for Acidobacteria as Fe reducers in these soils. Methanogens appear to coexist in the same layers, though in lower abundance, with Fe reducing bacteria and other potential competitors, including acetogens. These observations provide a rich set of hypotheses for further targeted study.

Atomic force microscopy studies of native photosynthetic membranes.

Science.gov (United States)

Sturgis, James N; Tucker, Jaimey D; Olsen, John D; Hunter, C Neil; Niederman, Robert A

2009-05-05

In addition to providing the earliest surface images of a native photosynthetic membrane at submolecular resolution, examination of the intracytoplasmic membrane (ICM) of purple bacteria by atomic force microscopy (AFM) has revealed a wide diversity of species-dependent arrangements of closely packed light-harvesting (LH) antennae, capable of fulfilling the basic requirements for efficient collection, transmission, and trapping of radiant energy. A highly organized architecture was observed with fused preparations of the pseudocrystalline ICM of Blastochloris viridis, consiting of hexagonally packed monomeric reaction center light-harvesting 1 (RC-LH1) core complexes. Among strains which also form a peripheral LH2 antenna, images of ICM patches from Rhodobacter sphaeroides exhibited well-ordered, interconnected networks of dimeric RC-LH1 core complexes intercalated by rows of LH2, coexisting with LH2-only domains. Other peripheral antenna-containing species, notably Rhodospirillum photometricum and Rhodopseudomonas palustris, showed a less regular organization, with mixed regions of LH2 and RC-LH1 cores, intermingled with large, paracrystalline domains. The ATP synthase and cytochrome bc(1) complex were not observed in any of these topographs and are thought to be localized in the adjacent cytoplasmic membrane or in inaccessible ICM regions separated from the flat regions imaged by AFM. The AFM images have served as a basis for atomic-resolution modeling of the ICM vesicle surface, as well as forces driving segregation of photosynthetic complexes into distinct domains. Docking of atomic-resolution molecular structures into AFM topographs of Rsp. photometricum membranes generated precise in situ structural models of the core complex surrounded by LH2 rings and a region of tightly packed LH2 complexes. A similar approach has generated a model of the highly curved LH2-only membranes of Rba. sphaeroides which predicts that sufficient space exists between LH2 complexes

Supercapacitors based on c-type cytochromes using conductive nanostructured networks of living bacteria.

Science.gov (United States)

Malvankar, Nikhil S; Mester, Tünde; Tuominen, Mark T; Lovley, Derek R

2012-02-01

Supercapacitors have attracted interest in energy storage because they have the potential to complement or replace batteries. Here, we report that c-type cytochromes, naturally immersed in a living, electrically conductive microbial biofilm, greatly enhance the device capacitance by over two orders of magnitude. We employ genetic engineering, protein unfolding and Nernstian modeling for in vivo demonstration of charge storage capacity of c-type cytochromes and perform electrochemical impedance spectroscopy, cyclic voltammetry and charge-discharge cycling to confirm the pseudocapacitive, redox nature of biofilm capacitance. The biofilms also show low self-discharge and good charge/discharge reversibility. The superior electrochemical performance of the biofilm is related to its high abundance of cytochromes, providing large electron storage capacity, its nanostructured network with metallic-like conductivity, and its porous architecture with hydrous nature, offering prospects for future low cost and environmentally sustainable energy storage devices. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

Czech Academy of Sciences Publication Activity Database

Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

2013-01-01

Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain.

Science.gov (United States)

Rep, M; Grivell, L A

1996-06-17

The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.

Computational identification of putative cytochrome P450 genes in ...

African Journals Online (AJOL)

Chattha

Economically, legumes represent the second most important family of crop plants after Poacea (grass family), accounting for ... further characterization of P450 genes with both known and unknown functions. MATERIALS AND METHODS ..... Cytochrome P450. In: Somerville CR, Meyerowitz EM (eds) .The Arabidopsis book,.

Multivariate Modeling of Cytochrome P450 Enzymes for 4 ...

African Journals Online (AJOL)

Conclusion: Apart from insights into important molecular properties for CYP inhibition, the findings may also guide further investigations of novel drug candidates that are unlikely to inhibit multiple CYP sub-types. Keywords: Antimalarial, Chloroquine, Cytochrome P450, Genetic algorithm-based multiple linear regression, ...

Molecular Characterization and Functional Analysis of Three Pathogenesis-Related Cytochrome P450 Genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea

Directory of Open Access Journals (Sweden)

Xiao-Lu Xu

2015-03-01

Full Text Available Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c

International Nuclear Information System (INIS)

Ilan, Y.; Shafferman, A.; Feinberg, B.A.; Lau, Y.K.

1979-01-01

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH 3 CHOH, CO 2 , O 2 , and e - /sub aq/, and the oxidation of the reduced cytochrome c derivatives by Fe(CN) 6 3- were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partitioned from each other in some cases. In regard to cycochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region

Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release.

LENUS (Irish Health Repository)

Huber, Heinrich J

2011-03-01

Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m). However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell\\'s glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.

Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

Science.gov (United States)

Chen, Wen; Djama, Zeinab Robleh; Coffey, Michael D; Martin, Frank N; Bilodeau, Guillaume J; Radmer, Lorien; Denton, Geoff; Lévesque, C André

2013-01-01

Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

Self-assembled tethered bimolecular lipid membranes.

Science.gov (United States)

Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

2009-01-01

via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

Science.gov (United States)

Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

1994-01-01

Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

The effect of amixin and agmatine on cytochrome c release from isolated mitochondria

Directory of Open Access Journals (Sweden)

K. R. Uspenska

2017-02-01

Full Text Available Mitochondrial nicotinic acetylcholine receptors (nAChRs control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

DEFF Research Database (Denmark)

Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas

2016-01-01

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

Construction and engineering of a thermostable self-sufficient cytochrome P450

Energy Technology Data Exchange (ETDEWEB)

Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

2009-06-19

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Construction and engineering of a thermostable self-sufficient cytochrome P450

International Nuclear Information System (INIS)

Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

2009-01-01

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Hydrogen/deuterium exchange of multiply-protonated cytochrome c ions

International Nuclear Information System (INIS)

Wood, T.D.; Guan, Ziqiang; O'Connor, P.B.

1995-01-01

Low resolution measurements show gaseous multiply-protonated cytochrome c ions undergo hydrogen/deuterium (H/D) exchange with pseudo first-order kinetics at three distinct exchange levels, suggesting the co-existence of gaseous protein conformations. Although exchange levels first increase with increasing charge values, they decrease at the highest charge values, consistent with solution-phase behavior of cytochrome c, where the native structure unfolds with decreasing pH until folding into a compact A-state at lowest pH. High resolution measurements indicate the presence of at least six H/D exchange levels. Infrared (IR) laser heating and fast collisions via quadrupolar excitation (QE) increase H/D exchange levels (unfolding) while charge-stripping ions to lower charge values can increase or decrease H/D exchange levels (unfolding or folding). Wolynes has suggested studying proteins in vacuo could play an important role in delineating the contributions various forces play in the protein folding process, provided appropriate comparisons can be made between gas-phase and solution-phase structures

Natively oxidized amino acid residues in the spinach cytochrome b 6 f complex.

Science.gov (United States)

Taylor, Ryan M; Sallans, Larry; Frankel, Laurie K; Bricker, Terry M

2018-01-29

The cytochrome b 6 f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b 6 f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc 1 complex. The types of ROS produced (O 2 •-, 1 O 2 , and, possibly, H 2 O 2 ) and the site(s) of ROS production within the b 6 f complex have been the subject of some debate. Proposed sources of ROS have included the heme b p , PQ p •- (possible sources for O 2 •- ), the Rieske iron-sulfur cluster (possible source of O 2 •- and/or 1 O 2 ), Chl a (possible source of 1 O 2 ), and heme c n (possible source of O 2 •- and/or H 2 O 2 ). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b 6 f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b 6 f complex.

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

NARCIS (Netherlands)

Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The

An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

Science.gov (United States)

Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

2017-05-01

Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

2010-10-08

Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

International Nuclear Information System (INIS)

Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

2010-01-01

Research highlights: → Cytochrome c oxidase loses catalytic activity during the aging process. → Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. → Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H 2 O 2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

Rational redesign of the biodegradative enzyme cytochrome P450 cam:

International Nuclear Information System (INIS)

Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

1991-03-01

Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs

Abundance of the multiheme c-type cytochrome OmcB increases in outer biofilm layers of electrode-grown Geobacter sulfurreducens.

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Camille S Stephen

Full Text Available When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB and cytoplasmic acetate kinase (AckA were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode. Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10-20 µm distant from the electrode surface compared to inner layers (0-10 µm. In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.

The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

DEFF Research Database (Denmark)

Bønding, Signe Helbo; Henty, K.; Dingley, A.J.

2008-01-01

is associated with a small unfavourable enthalpy change (1.9 kcal mol-1) and a moderately large, favourable entropy change (14.8 cal mol-1 deg-1). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions....... one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved....... surface plasmon resonance studies provide a value of 45 μM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation...

Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold

DEFF Research Database (Denmark)

Henriques de Jesus, Maria Perestrello Ramos; Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck

2017-01-01

to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s....... Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase...... in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold...

Mesoporous silica nanotubes hybrid membranes for functional nanofiltration

International Nuclear Information System (INIS)

El-Safty, Sherif A; Shahat, Ahmed; Mekawy, Moataz; Nguyen, Hoa; Warkocki, Wojciech; Ohnuma, Masato

2010-01-01

The development of nanofiltration systems would greatly assist in the production of well-defined particles and biomolecules with unique properties. We report a direct, simple synthesis of hexagonal silica nanotubes (NTs), which vertically aligned inside anodic alumina membranes (AAM) by means of a direct templating method of microemulsion phases with cationic surfactants. The direct approach was used as soft templates for predicting ordered assemblies of surfactant/silica composites through strong interactions within AAM pockets. Thus, densely packed NTs were successfully formed in the entirety of the AAM channels. These silica NTs were coated with layers of organic moieties to create a powerful technique for the ultrafine filtration. The resulting modified-silica NTs were chemically robust and showed affinity toward the transport of small molecular particles. The rigid silica NTs inside AAM channels had a pore diameter of ≤ 4 nm and were used as ultrafine filtration systems for noble metal nanoparticles (NM NPs) and semiconductor nanocrystals (SC NCs) fabricated with a wide range of sizes (1.0-50 nm) and spherical/pyramidal morphologies. Moreover, the silica NTs hybrid membranes were also found to be suitable for separation of biomolecules such as cytochrome c (CytC). Importantly, this nanofilter design retains high nanofiltration efficiency of NM NPs, SC NCs and biomolecules after a number of reuse cycles. Such retention is crucial in industrial applications.

Resonance Raman Optical Activity and Surface Enhanced Resonance Raman Optical Activity analysis of Cytochrome C

DEFF Research Database (Denmark)

Johannessen, Christian; Abdali, Salim; White, Peter C.

2007-01-01

High quality Resonance Raman (RR) and resonance Raman Optical Activity (ROA) spectra of cytochrome c were obtained in order to perform full assignment of spectral features of the resonance ROA spectrum. The resonance ROA spectrum of cytochrome c revealed a distinct spectral signature pattern due...... to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein back-bone. Combining the intrinsic resonance enhancement of cytochrome c with surface plasmon enhancement by colloidal silver particles, the Surface Enhanced Resonance Raman Scattering (SERRS) and Chiral...... Enhanced Raman Spectroscopy (ChERS) spectra of the protein were successfully obtained at very low concentration (as low as 1 µM). The assignment of spectral features was based on the information obtained from the RR and resonance ROA spectra. Excellent agreement between RR and SERRS spectra is reported...

Significance of cytochrome P450 system responses and levels of bile fluorescent aromatic compounds in marine wildlife following oil spills

International Nuclear Information System (INIS)

Lee, R.F.; Anderson, J.W.

2005-01-01

The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 in marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a linkage to

Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

Science.gov (United States)

Chen, Xi'en; Zhang, Yalin

2015-03-10

NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

Science.gov (United States)

Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

2017-01-06

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ m hyperpolarization, a known cause of ROS and trigger of apoptosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Modulation of Erythrocyte Plasma Membrane Redox System Activity by Curcumin

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Prabhakar Singh

2016-01-01

Full Text Available Plasma membrane redox system (PMRS is an electron transport chain system ubiquitously present throughout all cell types. It transfers electron from intracellular substrates to extracellular acceptors for regulation of redox status. Curcumin, isolated from Curcuma longa, has modulatory effects on cellular physiology due to its membrane interaction ability and antioxidant potential. The present study investigates the effect of curcumin on PMRS activity of erythrocytes isolated from Wistar rats in vitro and in vivo and validated through an in silico docking simulation study using Molegro Virtual Docker (MVD. Effects of curcumin were also evaluated on level of glutathione (GSH and the oxidant potential of plasma measured in terms of plasma ferric equivalent oxidative potentials (PFEOP. Results show that curcumin significantly (p<0.01 downregulated the PMRS activity in a dose-dependent manner. Molecular docking results suggest that curcumin interacts with amino acids at the active site cavity of cytochrome b5 reductase, a key constituent of PMRS. Curcumin also increased the GSH level in erythrocytes and plasma while simultaneously decreasing the oxidant potential (PFEOP of plasma. Altered PMRS activity and redox status are associated with the pathophysiology of several health complications including aging and diabetes; hence, the above finding may explain part of the role of curcumin in health beneficial effects.

Intestinal cytochromes P450 regulating the intestinal microbiota and its probiotic profile

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Eugenia Elefterios Venizelos Bezirtzoglou

2012-09-01

Full Text Available Cytochromes P450 (CYPs enzymes metabolize a large variety of xenobiotic substances. In this vein, a plethora of studies were conducted to investigate their role, as cytochromes are located in both liver and intestinal tissues. The P450 profile of the human intestine has not been fully characterized. Human intestine serves primarily as an absorptive organ for nutrients, although it has also the ability to metabolize drugs. CYPs are responsible for the majority of phase I drug metabolism reactions. CYP3A represents the major intestinal CYP (80% followed by CYP2C9. CYP1A is expressed at high level in the duodenum, together with less abundant levels of CYP2C8-10 and CYP2D6. Cytochromes present a genetic polymorphism intra- or interindividual and intra- or interethnic. Changes in the pharmacokinetic profile of the drug are associated with increased toxicity due to reduced metabolism, altered efficacy of the drug, increased production of toxic metabolites, and adverse drug interaction. The high metabolic capacity of the intestinal flora is due to its enormous pool of enzymes, which catalyzes reactions in phase I and phase II drug metabolism. Compromised intestinal barrier conditions, when rupture of the intestinal integrity occurs, could increase passive paracellular absorption. It is clear that high microbial intestinal charge following intestinal disturbances, ageing, environment, or food-associated ailments leads to the microbial metabolism of a drug before absorption. The effect of certain bacteria having a benefic action on the intestinal ecosystem has been largely discussed during the past few years by many authors. The aim of the probiotic approach is to repair the deficiencies in the gut flora and establish a protective effect. There is a tentative multifactorial association of the CYP (P450 cytochrome role in the different diseases states, environmental toxic effects or chemical exposures and nutritional status.

Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120*

Science.gov (United States)

Baniulis, Danas; Yamashita, Eiki; Whitelegge, Julian P.; Zatsman, Anna I.; Hendrich, Michael P.; Hasan, S. Saif; Ryan, Christopher M.; Cramer, William A.

2009-01-01

The crystal structure of the cyanobacterial cytochrome b6f complex has previously been solved to 3.0-Å resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b6f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b6f complex. Purified b6f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b6f complex, determined to a resolution of 3.0Å (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme bp that is rotated 180° about the α- and γ-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme cn is similar to that previously found in the b6f complex from other sources. PMID:19189962

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

Energy Technology Data Exchange (ETDEWEB)

Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

2016-10-07

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Identification of fraud (with pig stuffs) in chicken-processed meat through information of mitochondrial cytochrome b.

Science.gov (United States)

Yacoub, Haitham A; Sadek, Mahmoud A

2017-11-01

This study was conducted to find out the fraud in chicken-processed meat ingredients to protect consumers from commercial adulteration and authentication through a reliable way: direct amplification of conserved segment of cytochrome b gene of mitochondrial DNA, in addition, using species-specific primer assay for a certain cytochrome b. The results reported that chicken-processed meats were identified as a chicken meat based on amplification of conserved cytochrome b gene of mtDNA, while different fragments sizes were produced after the application of species-specific primer as follows: 227, 157, 274, 331, 389 and 439 bp for raw meat of chicken, goat, cattle, sheep, pig and horse, respectively. The results revealed that all chicken meat products are produced with 227 bp in size. While, an adulteration with pork stuffs was observed in some of the chicken meat products using a species-specific primer of cytochrome b gene, namely, chicken luncheon and chicken burger. This study represents a reliable technique that could be used to provide a promising solution for identifying the commercial adulteration and substitutions in processed meat in retail markets.

Relative importance of driving force and electrostatic interactions in the reduction of multihaem cytochromes by small molecules.

Science.gov (United States)

Quintas, Pedro O; Cepeda, Andreia P; Borges, Nuno; Catarino, Teresa; Turner, David L

2013-06-01

Multihaem cytochromes are essential to the energetics of organisms capable of bioremediation and energy production. The haems in several of these cytochromes have been discriminated thermodynamically and their individual rates of reduction by small electron donors were characterized. The kinetic characterization of individual haems used the Marcus theory of electron transfer and assumed that the rates of reduction of each haem by sodium dithionite depend only on the driving force, while electrostatic interactions were neglected. To determine the relative importance of these factors in controlling the rates, we studied the effect of ionic strength on the redox potential and the rate of reduction by dithionite of native Methylophilus methylotrophus cytochrome c″ and three mutants at different pH values. We found that the main factor determining the rate is the driving force and that Marcus theory describes this satisfactorily. This validates the method of the simultaneous fitting of kinetic and thermodynamic data in multihaem cytochromes and opens the way for further investigation into the mechanisms of these proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

Cloning, expression and purification of cytochrome c{sub 6} from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure

Energy Technology Data Exchange (ETDEWEB)

Akazaki, Hideharu [Bio-organic Chemistry Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa-shi, Kanagawa 252-8510 (Japan); Kawai, Fumihiro [Protein Design Laboratory, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Chida, Hirotaka; Matsumoto, Yuichirou; Hirayama, Mao; Hoshikawa, Ken [Bio-organic Chemistry Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa-shi, Kanagawa 252-8510 (Japan); Unzai, Satoru [Protein Design Laboratory, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Hakamata, Wataru; Nishio, Toshiyuki; Park, Sam-Yong; Oku, Tadatake, E-mail: oku@brs.nihon-u.ac.jp [Bio-organic Chemistry Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa-shi, Kanagawa 252-8510 (Japan)

2008-08-01

The crystal structure of cytochrome c{sub 6} from the brown alga H. fusiformis has been determined at 1.6 Å resolution. The amino-acid sequence and tertiary structure of H. fusiformis cytochrome c{sub 6} were very similar to those of red algal cytochrome c{sub 6} rather than those of green algal cytochrome c{sub 6}. The primary sequence of cytochrome c{sub 6} from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 Å resolution. The crystal belonged to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 84.58, c = 232.91 Å and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c{sub 6} were most similar to those of red algal cytochrome c{sub 6}. These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c/sub 2/ gene

Energy Technology Data Exchange (ETDEWEB)

Donohue, T.J.; McEwan, A.G.; Kaplan, S.

1986-11-01

The Rhodobacter sphaeroides cytochrome c/sub 2/ functions as a mobile electron carrier in both aerobic and photosynthetic electron transport chains. Synthetic deoxyoligonucleotide probes, based on the known amino acid sequence of this protein (M/sub r/ 14,000), were used to identify and clone the cytochrome c/sub 2/ structural gene (cycA). DNA sequence analysis of the cycA gene indicated the presence of a typical procaryotic 21-residue signal sequence, suggesting that this periplasmic protein is synthesized in vivo as a precursor. Synthesis of an immunoreactive cytochrome c/sub 2/ precursor protein (M/sub r/ 15,500) was observed in vitro when plasmids containing the cycA gene were used as templates in an R. sphaeroides coupled transcription-translation system. Approximately 500 base pairs of DNA upstream of the cycA gene was sufficient to allow expression of this gene product in vitro. Northern blot analysis with an internal cycA-specific probe identified at least two possibly monocistronic transcripts present in both different cellular levels and relative stoichiometries in steady-state cells grown under different physiological conditions. The ratio of the small (740-mucleotide) and large (920-nucleotide) cycA-specific mRNA species was dependent on cultural conditions but was not affected by light intensity under photosynthetic conditions. These results suggest that the increase in the cellular level of the cytochrome c/sub 2/ protein found in photosynthetic cells was due, in part, to increased transcription of the single-copy cyc operon.

Oxygen consumption and cytochrome exidase activity of axolotl limbs muscle tissue in restoration of regenerative ability suprressed by X-irradiation

International Nuclear Information System (INIS)

Teplits, N.A.

1975-01-01

The rate of oxygen use and activity of cytochrome oxidase in a homogenate of mitochondria and nuclei of muscle tissue of axolotl limbs after suppression of their regenerative capability by x irradiation and their restoration was studied experimentally. With suppression of the regenative capability the use of oxygen was depressed. Cytochrome oxidase activity in the homogenate and mitochondria decreased compared to that of the intact limb, in the nuclei of muscle tissue it was the same or greater. With restoration of the regenerative capability of the limbs the respiration rate of the homogenate and the mitochondria increased, accompanied by increased cytochrome oxidase activity. In the nuclei the cytochrome oxidase activity did not change in the blastema stage and sharply decreased in the limb formation state. (E.T.)

Oxygen consumption and cytochrome exidase activity of axolotl limbs muscle tissue in restoration of regenerative ability suppressed by X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Teplits, N A [AN SSSR, Moscow. Inst. Biologii Razvitiya

1975-01-01

The rate of oxygen use and activity of cytochrome oxidase in a homogenate of mitochondria and nuclei of muscle tissue of axolotl limbs after suppression of their regenerative capability by x irradiation and their restoration was studied experimentally. With suppression of the regenative capability the use of oxygen was depressed. Cytochrome oxidase activity in the homogenate and mitochondria decreased compared to that of the intact limb, in the nuclei of muscle tissue it was the same or greater. With restoration of the regenerative capability of the limbs the respiration rate of the homogenate and the mitochondria increased, accompanied by increased cytochrome oxidase activity. In the nuclei the cytochrome oxidase activity did not change in the blastema stage and sharply decreased in the limb formation state.

The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1

Directory of Open Access Journals (Sweden)

Melva Louisa

2009-12-01

Full Text Available Aim: Some carotenoids such as canthaxantin, astaxanthin and beta apo-8’-carotenal were reported to have modulatoryeffect on the cytochrome P450. The present study was conducted to investigate the effects of lycopene, a nonprovitamin A carotenoid, on microsomal cytochrome P450, CYP1A2 and CYP2E1.Methods: Total cytochrome P450 levels, CYP1A2 and CYP2E1-catalyzed reactions (acetanilide 4-hydroxylation and p-nitrophenol hydroxylation were studied in the liver microsomes of male Sprague Dawley rats. Microsomes were prepared using differential centrifugation combined with calcium aggregation method. Lycopene was orally administered in the dosages of 0, 25, 50 or 100 mg/kgBW/day for 14 days in a repeated fashion. Data were analyzed using ANOVA test.Results: Total cytochrome P450 level and acetanilide 4-hydroxylase activity were unaffected by any of the treatments. The CYP2E1 probe enzyme (p-nitrophenol hydroxylase was significantly reduced by repeated administration of 100mg/ kgBW/day lycopene (7.88 + 2.04 vs 12.26 + 2.77 n mol/min/mg prot.Conclusion: The present results suggest that lycopene does not affect the total cytochrome P450 or CYP1A2 activity but it inhibits the activity of CYP2E1 (p-nitrophenol hydroxylase in the rat. (Med J Indones 2009; 18: 233-8Keywords: lycopene, cytochrome P450, CYP1A2, CYP2E1

A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

Science.gov (United States)

Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

1989-02-28

1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

Cooperative use of cytochrome cd{sub 1} nitrite reductase and its redox partner cytochrome c{sub 552} to improve the selectivity of nitrite biosensing

Energy Technology Data Exchange (ETDEWEB)

Serra, A.S.; Jorge, S.R.; Silveira, C.M.; Moura, J.J.G. [REQUIMTE - Dept. de Quimica, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H. [CIDETEC - Centro de Tecnologias Electroquimicas, Parque Tecnologico de San Sebastian, Po Miramon, 196, 20009 Donostia - San Sebastian (Spain); Almeida, M.G., E-mail: mga@dq.fct.unl.pt [REQUIMTE - Dept. de Quimica, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Escola Superior de Saude Egas Moniz, Monte de Caparica, 2829-511 Caparica (Portugal)

2011-05-05

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd{sub 1} (cyt-cd{sub 1}) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c{sub 552} (cyt-c{sub 552}), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 {mu}M cyt-cd{sub 1}/100 {mu}M cyt-c{sub 552} and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c{sub 552} and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254 {+-} 2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c{sub 552} and cd{sub 1} is 9.9 x 10{sup 3} M{sup -1} s{sup -1}. The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 {mu}M; detection and quantification limits of 7 and 24 {mu}M, respectively; sensitivity of 2.49 {+-} 0.08 A mol{sup -1} cm{sup 2} {mu}M{sup -1}. Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy

Science.gov (United States)

Forman, C. J.; Wang, N.; Yang, Z. Y.; Mowat, C. G.; Jarvis, S.; Durkan, C.; Barker, P. D.

2013-05-01

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

Science.gov (United States)

Wong-Riley, M T; Trusk, T C; Kaboord, W; Huang, Z

1994-09-01

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly

Cytochrome and Alternative Pathway Respiration in Tobacco (Effects of Salicylic Acid).

Science.gov (United States)

Rhoads, D. M.; McIntosh, L.

1993-11-01

In suspension cultures of NT1 tobacco (Nicotiana tabacum L. cv Bright Yellow) cells the cytochrome pathway capacity increased between d 3 and d 4 following subculturing and reached the highest level observed on d 7. The capacity decreased significantly by d 10 and was at the same level on d 14. Both alternative pathway capacity and the amount of the 35-kD alternative oxidase protein increased significantly between d 5 and d 6, reached the highest point observed on d 7, remained constant until d 10, and decreased by d 14. The highest capacities of the alternative and cytochrome pathways and the highest amount of the 35-kD protein were attained on the day that cell cultures reached a stationary phase of growth. Addition of salicylic acid to cell cultures on d 4 caused a significant increase in alternative pathway capacity and a dramatic accumulation of the 35-kD protein by 12 h. The alternative pathway capacity and the protein level reached the highest level observed by 16 h after salicylic acid addition, and the cytochrome pathway capacity was at about the same level at each time point. The accumulation of the 35-kD alternative oxidase protein was significantly decreased by addition of actinomycin D 1 h before salicylic acid and was blocked by addition of cycloheximide. These results indicate that de novo transcription and translation were necessary for salicylic acid to cause the maximum accumulation of the 35-kD protein.

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase.

Science.gov (United States)

Lehninger, A L; Reynafarje, B; Costa, L

1985-01-01

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

Science.gov (United States)

Furge, Laura Lowe; Fletke, Kyle J.

2007-01-01

Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding

NARCIS (Netherlands)

Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.

2009-01-01

The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy

International Nuclear Information System (INIS)

Forman, C J; Barker, P D; Wang, N; Durkan, C; Yang, Z Y; Mowat, C G; Jarvis, S

2013-01-01

Amyloid fibres displaying cytochrome b 562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias ( 562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid. (paper)

Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

Science.gov (United States)

Battistuzzi, G; Borsari, M; Sola, M; Francia, F

1997-12-23

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450.

Science.gov (United States)

Ewen, Kerstin Maria; Hannemann, Frank; Khatri, Yogan; Perlova, Olena; Kappl, Reinhard; Krug, Daniel; Hüttermann, Jürgen; Müller, Rolf; Bernhardt, Rita

2009-10-16

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.

The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress.

NARCIS (Netherlands)

Lu, P.; Heineke, M.H.; Koul, A.; Andries, K.; Cook, G.M.; Lill, H.; van Spanning, R.J.M.; Bald, D.

2015-01-01

Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc 1 complex and an aa 3 -type cytochrome

Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5.

Science.gov (United States)

Kollipara, Sireesha; Tatireddy, Shivakishore; Pathirathne, Thusitha; Rathnayake, Lasantha K; Northrup, Scott H

2016-08-25

Brownian dynamics (BD) simulations provide here a theoretical atomic-level treatment of the reduction of human ferric cytochrome b5 (cyt b5) by NADH-cytochrome b5 reductaste (cyt b5r) and several of its mutants. BD is used to calculate the second-order rate constant of electron transfer (ET) between the proteins for direct correlation with experiments. Interestingly, the inclusion of electrostatic forces dramatically increases the reaction rate of the native proteins despite the overall negative charge of both proteins. The role played by electrostatic charge distribution in stabilizing the ET complexes and the role of mutations of several amino acid residues in stabilizing or destabilizing the complexes are analyzed. The complex with the shortest ET reaction distance (d = 6.58 Å) from rigid body BD is further subjected to 1 ns of molecular dynamics (MD) in a periodic box of TIP3P water to produce a more stable complex allowed by flexibility and with a shorter average reaction distance d = 6.02 Å. We predict a docking model in which the following ion-ion interactions are dominant (cyt b5r/cyt b5): Lys162-Heme O1D/Lys163-Asp64/Arg91-Heme O1A/Lys125-Asp70.

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

Science.gov (United States)

Zahn, J A; Duncan, C; DiSpirito, A A

1994-01-01

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline. Images PMID:7928947

Prognostic Value of Cytochrome C and Cytokines in Acute Viral Encephalopathy

Directory of Open Access Journals (Sweden)

J Gordon Millichap

2006-06-01

Full Text Available Serum cytochrome c and cytokines were evaluated as prognostic predictors in 29 children (ages 9 mos to 9 yrs 11 mos with viral acute encephalopathies and multiple organ failure at Fukushima Medical University School of Medicine, Japan.

Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria*S⃞

Science.gov (United States)

Yamada, Akiko; Yamamoto, Takenori; Yamazaki, Naoshi; Yamashita, Kikuji; Kataoka, Masatoshi; Nagata, Toshihiko; Terada, Hiroshi; Shinohara, Yasuo

2009-01-01

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed. PMID:19218587

Identification of a c-Type Cytochrome Specific for Manganese Dioxide (MnO2) Reduction in Anaeromyxobacter dehalogenans Strain 2CP-C

Science.gov (United States)

Pfiffner, S. M.; Nissen, S.; Liu, X.; Chourey, K.; Vishnivetskaya, T. A.; Hettich, R.; Loeffler, F.

2014-12-01

Anaeromyxobacter dehalogenans is a metabolically versatile Deltaproteobacterium and conserves energy from the reduction of various electron acceptors, including insoluble MnO2 and ferric oxides/oxyhydroxides (FeOOH). The goal of this study was to identify c-type cytochromes involved in electron transfer to MnO2. The characterization of deletion mutants has revealed a number of c-type cytochromes involved in electron transfer to solid metal oxides in Shewanella spp. and Geobacter spp; however, a genetic system for Anaeromyxobacter is not available. The A. dehalogenans str. 2CP-C genome encodes 68 putative c-type cytochromes, which all lack functional assignments. To identify c-type cytochromes involved in electron transfer to solid MnO2, protein expression profiles of A. dehalogenans str. 2CP-C cells grown with acetate as electron donor and MnO2, ferric citrate, FeOOH, nitrate or fumarate as electron acceptors were compared. Whole cell proteomes were analyzed after trypsin proteolysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Distinct c-type cytochrome expression patterns were observed with cells grown with different electron acceptors. A. dehalogenans str. 2CP-C grown with MnO2 expressed 25 out of the 68 c-type cytochromes encoded on the genome. The c-type cytochrome Adeh_1278 was only expressed in strain 2CP-C grown with MnO2. Reverse transcription PCR confirmed that the Adeh_1278 gene was transcribed in MnO2-grown cells but not in cells grown with other terminal electron acceptors. The expression of the Adeh_1278 gene correlated with Mn(IV) reduction activity. Adeh_1278 has three heme binding motifs and is predicted to be located in the periplasm. The identification of Adeh_1278 as a protein uniquely expressed when MnO2 serves as electron acceptor suggests its utility as a biomarker for MnO2 reduction. This example demonstrates the value of the LC-MS/MS approach for identifying specific proteins of interest and making functional assignments

Interplay between cytochrome c and gibberellins during Arabidopsis vegetative development

Czech Academy of Sciences Publication Activity Database

Racca, S.; Welchen, E.; Gras, D. E.; Tarkowská, Danuše; Turečková, Veronika; Maurino, V. G.; Gonzalez, D. H.

2018-01-01

Roč. 94, č. 1 (2018), s. 105-121 ISSN 0960-7412 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * cytochrome c * DELLA protein * gibberellin * mitochondrion Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 5.901, year: 2016

Prediction of cytochrome P450 mediated metabolism

DEFF Research Database (Denmark)

Olsen, Lars; Oostenbrink, Chris; Jørgensen, Flemming Steen

2015-01-01

Cytochrome P450 enzymes (CYPs) form one of the most important enzyme families involved in the metabolism of xenobiotics. CYPs comprise many isoforms, which catalyze a wide variety of reactions, and potentially, a large number of different metabolites can be formed. However, it is often hard...... to rationalize what metabolites these enzymes generate. In recent years, many different in silico approaches have been developed to predict binding or regioselective product formation for the different CYP isoforms. These comprise ligand-based methods that are trained on experimental CYP data and structure...

High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment.

Directory of Open Access Journals (Sweden)

Theresa Tiefenbrunn

Full Text Available The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H(+ and e(- transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba(3-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O(2-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu(B atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe(a3 and Cu(B atoms that is best modeled as peroxide. The structure of ba(3-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba(3-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the

The Cytochrome b 6 f Complex: Biophysical Aspects of Its Functioning in Chloroplasts.

Science.gov (United States)

Tikhonov, Alexander N

2018-01-01

This chapter presents an overview of structural properties of the cytochrome (Cyt) b 6 f complex and its functioning in chloroplasts. The Cyt b 6 f complex stands at the crossroad of photosynthetic electron transport pathways, providing connectivity between Photosystem (PSI) and Photosysten II (PSII) and pumping protons across the membrane into the thylakoid lumen. After a brief review of the chloroplast electron transport chain, the consideration is focused on the structural organization of the Cyt b 6 f complex and its interaction with plastoquinol (PQH 2 , reduced form of plastoquinone), a mediator of electron transfer from PSII to the Cyt b 6 f complex. The processes of PQH 2 oxidation by the Cyt b 6 f complex have been considered within the framework of the Mitchell's Q-cycle. The overall rate of the intersystem electron transport is determined by PQH 2 turnover at the quinone-binding site Q o of the Cyt b 6 f complex. The rate of PQH 2 oxidation is controlled by the intrathylakoid pH in , which value determines the protonation/deprotonation events in the Q o -center. Two other regulatory mechanisms associated with the Cyt b 6 f complex are briefly overviewed: (i) redistribution of electron fluxes between alternative (linear and cyclic) pathways, and (ii) "state transitions" related to redistribution of solar energy between PSI and PSII.

Cytochrome P450-mediated metabolism of tumour promoters modifies the inhibition of intercellular communication: a modified assay for tumour promotion

DEFF Research Database (Denmark)

Vang, Ole; Wallin, H.; Doehmer, J.

1993-01-01

The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay...... B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular communication of some tumour...... promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular...

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay

International Nuclear Information System (INIS)

Gade, Sudeep Kumar; Bhattacharya, Subarna; Manoj, Kelath Murali

2012-01-01

Highlights: ► At low concentrations, cytochrome c/vitamin C do not catalyze peroxidations. ► But low levels of cytochrome c/vitamin C enhance diverse heme peroxidase activities. ► Enhancement positively correlates to the concentration of peroxide in reaction. ► Reducible additives serve as non-specific agents for redox relay in the system. ► Insight into electron transfer processes in routine and oxidative-stress states. -- Abstract: We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding – (1) the promiscuous role of cytochrome b 5 in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.

RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

Science.gov (United States)

Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

2012-01-01

NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

An extensive deletion causing overproduction of yeast iso-2-cytochrome c

International Nuclear Information System (INIS)

McKnight, G.L.; Cardillo, T.S.; Sherman, F.

1981-01-01

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event

A theoretical study on the metabolic activation of paracetamol by cytochrome P-450 : indications for a uniform oxidation mechanism

NARCIS (Netherlands)

Koymans, L.; Lenthe, J.H.; Van de Straat, R; Donné-Op den Kelder, G M; Vermeulen, N P

1989-01-01

The cytochrome P-450 mediated activation of paracetamol (PAR) to the reactive electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI) has been studied by use of SV 6-31G ab initio energy calculations and spin distributions. A simplified model for cytochrome P-450 has been used by

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

Science.gov (United States)

Hiesel, Rudolf; Brennicke, Axel

1983-01-01

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

Nitrate as a probe of cytochrome c surface: crystallographic identification of crucial "hot spots" for protein-protein recognition.

Science.gov (United States)

De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

2014-06-01

The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred. Copyright © 2014 Elsevier Inc. All rights reserved.

Certain tryptophan photoproducts are inhibitors of cytochrome P450-dependent mutagenicity

International Nuclear Information System (INIS)

Rannug, U.; Agurell, E.; Cederberg, H.; Rannug, A.

1992-01-01

Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomyces cerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Salmonella tphimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100+S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-4501A1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecapine and dehydrorutaecarpine. 20 refs., 3 figs., 4 tabs

Comparison of basal and induced cytochromes P450 in 6 species of waterfowl

Science.gov (United States)

Melancon, M.J.; Rattner, B.A.; Hoffman, D.J.; Beeman, D.; Day, D.; Custer, T.

1999-01-01

Cytochrome P450-associated monooxygenase activities were measured in control and prototype inducer-treated mallard duck, black duck, wood duck, lesser scaup, Canada goose and mute swan. Ages of the birds ranged from pipping embryos (that were treated approximately 3 days before pipping) to adults. Three or more of the following hepatic microsomal monooxygenases were assayed in each species: Benzyloxyresorufin-O-dealkylase (BROD), Ethoxyresorufin-O-dealkylase (EROD), methoxyresorufin-O-dealkylase (MROD), and pentoxyresorufin-O-dealkylase (PROD). Baseline activities differed between species, but because of differences in ages, sources of the eggs or birds, and diets, these cannot be viewed as absolute differences. The cytochrome P450 inducers utilized were beta-naphthoflavone (BNF), 3-methylcholanthrene (3MC) and phenobarbital (PB). In general, there was little response to PB; only lesser scaup were induced to greater than three times control level and most species were well under this. Responses to BNF and 3MC occurred in each species studied, but differed in which of the monooxygenases was most induced (absolute values and ratios to control values) and in relative induction between species. BROD frequently had an induction ratio EROD. Overall, lesser scaup were the most responsive, canada geese the least responsive, and the other species intermediate in responsiveness to the cytochrome P450 inducers studied.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Energy Technology Data Exchange (ETDEWEB)

Chinchilla, Diana, E-mail: Diana_Chinchilla@yahoo.com; Kilheeney, Heather, E-mail: raindropszoo@yahoo.com; Vitello, Lidia B., E-mail: lvitello@niu.edu; Erman, James E., E-mail: jerman@niu.edu

2014-01-03

Highlights: •Cytochrome c peroxidase (CcP) binds acrylonitrile in a pH-independent fashion. •The spectrum of the CcP/acrylonitrile complex is that of a 6c–ls ferric heme. •The acrylonitrile/CcP complex has a K{sub D} value of 1.1 ± 0.2 M. •CcP compound I oxidizes acrylonitrile with a maximum turnover rate of 0.61 min{sup −1}. -- Abstract: Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M{sup −1} s{sup −1} and 0.34 ± 0.15 s{sup −1}, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min{sup −1} at pH 6.0.

CYTOCHROME P450-DEPENDENT METABOLISM OF TRICHLOROETHYLENE IN THE RAT KIDNEY

Science.gov (United States)

The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite con...

INTERACTION OF AROMATIC CYTOKININS WITH HUMAN LIVER MICROSOMAL CYTOCHROMES P450

Czech Academy of Sciences Publication Activity Database

Anzenbacherová, E.; Janalík, J.; Popa, Igor; Strnad, Miroslav; Anzenbacher, P.

2005-01-01

Roč. 149, č. 2 (2005), s. 349-351 ISSN 1213-8118 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * Cyclin dependent kinase inhibitor * Cytochrome P450 Subject RIV: CE - Biochemistry http://publib.upol.cz/~obd/fulltext/Biomed/2005/2/349.pdf

The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

Science.gov (United States)

Schuster, I

1985-06-01

Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

The mechanism of electron gating in proton pumping cytochrome c oxidase: the effect of pH and temperature on internal electron transfer.

Science.gov (United States)

Brzezinski, P; Malmström, B G

1987-10-29

Electron-transfer reactions following flash photolysis of the mixed-valence cytochrome oxidase-CO complex have been measured at 445, 598 and 830 nm between pH 5.2 and 9.0 in the temperature range of 0-25 degrees C. There is a rapid electron transfer from the cytochrome a3-CuB pair to CuA (time constant: 14200 s-1), which is followed by a slower electron transfer to cytochrome a. Both the rate and the amplitude of the rapid phase are independent of pH, and the rate in the direction from CuA to cytochrome a3-CuB is practically independent of temperature. The second phase depends strongly on pH due to the titration of an acid-base group with pKa = 7.6. The equilibrium at pH 7.4 corresponds to reduction potentials of 225 and 345 mV for cytochrome a and a3, respectively, from which it is concluded that the enzyme is in a different conformation compared to the fully oxidized form. The results have been used to suggest a series of reaction steps in a cycle of the oxidase as a proton pump. Application of the electron-transfer theory to the temperature-dependence data suggests a mechanism for electron gating in the pump. Reduction of both cytochrome a and CuA leads to a conformational change, which changes the structure of cytochrome a3-CuB in such a way that the reorganizational barrier for electron transfer is removed and the driving force is increased.

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

Jacobsen Catalyst as a Cytochrome P450 Biomimetic Model for the Metabolism of Monensin A

Directory of Open Access Journals (Sweden)

Bruno Alves Rocha

2014-01-01

Full Text Available Monensin A is a commercially important natural product isolated from Streptomyces cinnamonensins that is primarily employed to treat coccidiosis. Monensin A selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. In this study, we evaluated the Jacobsen catalyst as a cytochrome P450 biomimetic model to investigate the oxidation of monensin A. Mass spectrometry analysis of the products from these model systems revealed the formation of two products: 3-O-demethyl monensin A and 12-hydroxy monensin A, which are the same ones found in in vivo models. Monensin A and products obtained in biomimetic model were tested in a mitochondrial toxicity model assessment and an antimicrobial bioassay against Staphylococcus aureus, S. aureus methicillin-resistant, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. Our results demonstrated the toxicological effects of monensin A in isolated rat liver mitochondria but not its products, showing that the metabolism of monensin A is a detoxification metabolism. In addition, the antimicrobial bioassay showed that monensin A and its products possessed activity against Gram-positive microorganisms but not for Gram-negative microorganisms. The results revealed the potential of application of this biomimetic chemical model in the synthesis of drug metabolites, providing metabolites for biological tests and other purposes.

Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

DEFF Research Database (Denmark)

Thuesen, Marianne Hallberg; Nørgaard, Allan; Hansen, Anne Merete

2003-01-01

The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein...

Preferential hydroxylation over epoxidation catalysis by a horseradish peroxidase mutant: a cytochrome P450 mimic.

Science.gov (United States)

de Visser, Sam P

2007-10-25

Density functional theory calculations are presented on the catalytic properties of a horseradish peroxidase mutant whereby the axial nitrogen atom is replaced by phosphorus. This mutant has never been studied experimentally and only one theoretical report on this system is known (de Visser, S. P. J. Phys. Chem. B 2006, 110, 20759-20761). Thus, a one-atom substitution in horseradish peroxidase changes the properties of the catalytic center of the enzyme to more cytochrome P450-type qualities. In particular, the phosphorus-substituted horseradish peroxidase mutant reacts with substrates via a unique reactivity pattern, whereby alkanes are regioselectively hydroxylated even in the presence of a double bond. Reaction barriers of propene epoxidation and hydroxylation are almost identical to ones observed for a cytochrome P450 catalyst and significantly higher than those obtained for a horseradish peroxidase catalyst. It is shown that the regioselectivity difference is entropy and thermally driven and that the electron-transfer processes that occur during the reaction mechanism follow cytochrome P450-type patterns in the hydroxylation reaction.

Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

DEFF Research Database (Denmark)

Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

1997-01-01

In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms...

The Use of Cytochrome b Gene as a Specific Marker of the Rat Meat (Rattus norvegicus on Meat and Meat Products

Directory of Open Access Journals (Sweden)

C. Sumantri

2012-04-01

Full Text Available Falsification of the origin of livestock meat and its processed with rat meat is a problem that must be overcome to ensure food safety. One way that is often used to detect forgeries by using cytochrome b gene as a marker. The purpose of this study was to create a specific primer derived from cytochrome b sequences in rat (Rattus norvegicus as the DNA marker to detect any contamination of rat meat on fresh livestock meat and its processed meat products. Meatballs were made from beef meat with the addition of rat 1%-25%, and the meatballs were obtained from traditional markets. DNA extraction was conducted from seven species (goat, chicken, cattle, sheep, pig, horse, and rat by using phenol-chloroform. The highest success rate in detecting the presence of rat meat in a mixture of beef meatballs at concentration of 15% was 100%. The specific fragment of cytochrome b gene in R. norvegicus has no similarity with the cytochrome b gene from six other species, so it can be used as molecular markers to detect the presence of rat meat contamination in the processed of meat products. Amplified fragment length for goats, chickens, cattle, sheep, pigs, horses, and rats 157, 227, 274, 331, 398, 439 and 603 bp respectively. The amplification of cytochrome b gene in seven species of animals with different fragment length indicated the specificity of cytochrome b gene sequences among species.

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay

Energy Technology Data Exchange (ETDEWEB)

Gade, Sudeep Kumar; Bhattacharya, Subarna [Heme and Flavo Proteins Laboratory, 204, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu 632014 (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [Heme and Flavo Proteins Laboratory, 204, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu 632014 (India)

2012-03-09

Highlights: Black-Right-Pointing-Pointer At low concentrations, cytochrome c/vitamin C do not catalyze peroxidations. Black-Right-Pointing-Pointer But low levels of cytochrome c/vitamin C enhance diverse heme peroxidase activities. Black-Right-Pointing-Pointer Enhancement positively correlates to the concentration of peroxide in reaction. Black-Right-Pointing-Pointer Reducible additives serve as non-specific agents for redox relay in the system. Black-Right-Pointing-Pointer Insight into electron transfer processes in routine and oxidative-stress states. -- Abstract: We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b{sub 5} in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.

Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

International Nuclear Information System (INIS)

Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

2009-01-01

A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity.

Science.gov (United States)

Lei, Haotian; Bowler, Bruce E

2018-06-01

Structural studies of yeast iso-1-cytochrome c (L.J. McClelland, T.-C. Mou, M.E. Jeakins-Cooley, S.R. Sprang, B.E. Bowler, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 6648-6653) show that modest movement of Ω-loop D (residues 70-85, average RMSD versus the native structure: 0.81 Å) permits loss of Met80-heme ligation creating an available coordination site to catalyze the peroxidase activity mediated by cytochrome c early in apoptosis. However, Ala81 and Gly83 move significantly (RMSDs of 2.18 and 1.26 Å, respectively). Ala81 and Gly83 evolve to Ile and Val, respectively, in human cytochrome c and peroxidase activity decreases 25-fold relative to the yeast protein at pH 7. To test the hypothesis that these residues evolved to restrict the peroxidase activity of cytochrome c, A81I and G83V variants of yeast iso-1-cytochrome c were prepared. For both variants, the apparent pK a of the alkaline transition increases by 0.2 to 0.3 relative to the wild type (WT) protein and the rate of opening the heme crevice is slowed. The cooperativity of acid unfolding is decreased for the G83V variant. At pH 7 and 8, the catalytic rate constant, k cat , for the peroxidase activity of both variants decreases relative to WT, consistent with the effects on alkaline isomerization. Below pH 7, the loss in the cooperativity of acid unfolding causes k cat for peroxidase activity to increase for the G83V variant relative to WT. Neither variant decreases k cat to the level of the human protein, indicating that other residues also contribute to the low peroxidase activity of human cytochrome c. Copyright © 2018 Elsevier Inc. All rights reserved.

Interaction of rocuronium with human liver cytochromes P450

OpenAIRE

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-01-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

Science.gov (United States)

Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R; Battaile, Kevin P; Lovell, Scott; Benson, David R; Zhu, Hao

2010-09-24

NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain.

Patterning of electrically conductive poly(aniline-co-aniline sulfonic acid) and its application in the immobilization of cytochrome c

International Nuclear Information System (INIS)

Oh, Se Young; Oh, Il Soo; Choi, Jeong-Woo

2004-01-01

We have synthesized poly(aniline-co-aniline sulfonic acid) and then investigated the feasibility of application as a specific and electrically conductive binding template for biomolecules. Poly(aniline-co-aniline sulfonic acid)s were prepared by oxidation polymerization of aniline and aniline sulfonic acid under various ratios. A fine pattern of the conducting copolyaniline was obtained by using a deep UV lithographic technique. Cytochrome c was immobilized onto the photochemically patterned conducting copolyaniline with a self-assembly method. Physical and electrochemical properties of the self-assembled cytochrome c monolayer were studied from atomic force microscopy and cyclic voltammetry. The self-assembled cytochrome c monolayer immobilized onto the copolyaniline with a high electrical conductivity showed a high electrochemical activity

N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

International Nuclear Information System (INIS)

Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo; Yun, Yeon-Sook; Hong, Sung Hee

2009-01-01

N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.

Membranes, methods of making membranes, and methods of separating gases using membranes

Science.gov (United States)

Ho, W. S. Winston

2012-10-02

Membranes, methods of making membranes, and methods of separating gases using membranes are provided. The membranes can include at least one hydrophilic polymer, at least one cross-linking agent, at least one base, and at least one amino compound. The methods of separating gases using membranes can include contacting a gas stream containing at least one of CO.sub.2, H.sub.2S, and HCl with one side of a nonporous and at least one of CO.sub.2, H.sub.2S, and HCl selectively permeable membrane such that at least one of CO.sub.2, H.sub.2S, and HCl is selectively transported through the membrane.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

Science.gov (United States)

Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

2015-10-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

Science.gov (United States)

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

Science.gov (United States)

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-01-01

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

Role of gemfibrozil as an inhibitor of CYP2C8 and membrane transporters.

Science.gov (United States)

Tornio, Aleksi; Neuvonen, Pertti J; Niemi, Mikko; Backman, Janne T

2017-01-01

Cytochrome P450 (CYP) 2C8 is a drug metabolizing enzyme of major importance. The lipid-lowering drug gemfibrozil has been identified as a strong inhibitor of CYP2C8 in vivo. This effect is due to mechanism-based inhibition of CYP2C8 by gemfibrozil 1-O-β-glucuronide. In vivo, gemfibrozil is a fairly selective CYP2C8 inhibitor, which lacks significant inhibitory effect on other CYP enzymes. Gemfibrozil can, however, have a smaller but clinically meaningful inhibitory effect on membrane transporters, such as organic anion transporting polypeptide 1B1 and organic anion transporter 3. Areas covered: This review describes the inhibitory effects of gemfibrozil on CYP enzymes and membrane transporters. The clinical drug interactions caused by gemfibrozil and the different mechanisms contributing to the interactions are reviewed in detail. Expert opinion: Gemfibrozil is a useful probe inhibitor of CYP2C8 in vivo, but its effect on membrane transporters has to be taken into account in study design and interpretation. Moreover, gemfibrozil could be used to boost the pharmacokinetics of CYP2C8 substrate drugs. Identification of gemfibrozil 1-O-β-glucuronide as a potent mechanism-based inhibitor of CYP2C8 has led to recognition of glucuronide metabolites as perpetrators of drug-drug interactions. Recently, also acyl glucuronide metabolites of clopidogrel and deleobuvir have been shown to strongly inhibit CYP2C8.

Cyanide inhibition and pyruvate-induced recovery of cytochrome c oxidase

Czech Academy of Sciences Publication Activity Database

Nůsková, Hana; Vrbacký, Marek; Drahota, Zdeněk; Houštěk, Josef

2010-01-01

Roč. 42, č. 5 (2010), s. 395-403 ISSN 0145-479X R&D Projects: GA ČR(CZ) GA303/07/0781; GA MŠk(CZ) 1M0520; GA MŠk OC08017 Institutional research plan: CEZ:AV0Z50110509 Keywords : cytochrom c oxidase * cyanide * oxygen affinity Subject RIV: CE - Biochemistry Impact factor: 3.637, year: 2010

Substrate binding in the active site of cytochrome P450cam

NARCIS (Netherlands)

Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

2005-01-01

We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

[ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism].

Science.gov (United States)

Lemeshko, V V; Kaliman, P A; Belostotskaia, L I; Uchitel', A A

1982-04-01

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.

Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

Science.gov (United States)

Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

2005-01-01

Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

Directory of Open Access Journals (Sweden)

Jorge L Porfido

Full Text Available Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious.We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs.This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.

Application of dynamic membranes in anaerobic membranes in anaerobic membrane bioreactor systems

NARCIS (Netherlands)

Erşahin, M.E.

2015-01-01

Anaerobic membrane bioreactors (AnMBRs) physically ensure biomass retention by the application of a membrane filtration process. With growing application experiences from aerobic membrane bioreactors (MBRs), the combination of membrane and anaerobic processes has received much attention and become

A study on the interaction of horse heart cytochrome c with some conventional and ionic liquid surfactants probed by ultraviolet-visible and fluorescence spectroscopic techniques

Science.gov (United States)

Mondal, Satyajit; Das, Bijan

2018-06-01

The interactions of a protein cytochrome c with some selected conventional and ionic liquid surfactants have been investigated at pH 7.4 using ultraviolet-visible and fluorescence spectroscopic techniques. We used four conventional surfactants - cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), sodium N-dodecanoylsarcosinate (SDDS), and N-decanoyl-N-methylglucamine (Mega 10), and a surface active ionic liquid 1-hexadecyl-3-methylimidazolium chloride (C16MeImCl). All the investigated surfactants were found to induce an unfolding of the protein cytochrome c. In presence of CTAB, SDDS and C16MeImCl, the heme iron atom was found to loose methionine from its axial position. Differential binding of the surfactant monomers and their micelles to the protein molecules was inferred. The ionic surfactants were found to be more effective than the nonionic one in unfolding the investigated protein. However, the extent of binding of CTAB/C16MeImCl to cytochrome c reaches a plateau past the critical micellization concentration (cmc) of the surfactant. For each of the cytochrome c-DTAB, cytochrome c-SDDS and cytochrome c-Mega 10 system, although there exists an inflection in the surfactant-binding, saturation point could not be detected. It has been demonstrated from the ultraviolet-visible spectral studies that the oxidation state of iron in cytochrome c does not change when the protein binds with the investigated surfactants.

Insight into mitochondrial structure and function from electron tomography.

Science.gov (United States)

Frey, T G; Renken, C W; Perkins, G A

2002-09-10

In recent years, electron tomography has provided detailed three-dimensional models of mitochondria that have redefined our concept of mitochondrial structure. The models reveal an inner membrane consisting of two components, the inner boundary membrane (IBM) closely apposed to the outer membrane and the cristae membrane that projects into the matrix compartment. These two components are connected by tubular structures of relatively uniform size called crista junctions. The distribution of crista junction sizes and shapes is predicted by a thermodynamic model based upon the energy of membrane bending, but proteins likely also play a role in determining the conformation of the inner membrane. Results of structural studies of mitochondria during apoptosis demonstrate that cytochrome c is released without detectable disruption of the outer membrane or extensive swelling of the mitochondrial matrix, suggesting the formation of an outer membrane pore large enough to allow passage of holo-cytochrome c. The possible compartmentation of inner membrane function between the IBM and the cristae membrane is also discussed.

Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4)

DEFF Research Database (Denmark)

Andersen, Niels Højmark; Jensen, Thomas Jon; Nørgaard, Allan

2002-01-01

F stutzeri cytochrome c. is a di-haem protein, composed of two globular domains each with His-Met coordinated haem. and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and ...

Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

NARCIS (Netherlands)

Bienen, E. J.; Maturi, R. K.; Pollakis, G.; Clarkson, A. B.

1993-01-01

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens.

Science.gov (United States)

Seidel, Julian; Hoffmann, Maren; Ellis, Katie E; Seidel, Antonia; Spatzal, Thomas; Gerhardt, Stefan; Elliott, Sean J; Einsle, Oliver

2012-04-03

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

A study on the interaction of horse heart cytochrome c with some conventional and ionic liquid surfactants probed by ultraviolet-visible and fluorescence spectroscopic techniques.

Science.gov (United States)

Mondal, Satyajit; Das, Bijan

2018-06-05

The interactions of a protein cytochrome c with some selected conventional and ionic liquid surfactants have been investigated at pH7.4 using ultraviolet-visible and fluorescence spectroscopic techniques. We used four conventional surfactants - cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), sodium N-dodecanoylsarcosinate (SDDS), and N-decanoyl-N-methylglucamine (Mega 10), and a surface active ionic liquid 1-hexadecyl-3-methylimidazolium chloride (C 16 MeImCl). All the investigated surfactants were found to induce an unfolding of the protein cytochrome c. In presence of CTAB, SDDS and C 16 MeImCl, the heme iron atom was found to loose methionine from its axial position. Differential binding of the surfactant monomers and their micelles to the protein molecules was inferred. The ionic surfactants were found to be more effective than the nonionic one in unfolding the investigated protein. However, the extent of binding of CTAB/C 16 MeImCl to cytochrome c reaches a plateau past the critical micellization concentration (cmc) of the surfactant. For each of the cytochrome c-DTAB, cytochrome c-SDDS and cytochrome c-Mega 10 system, although there exists an inflection in the surfactant-binding, saturation point could not be detected. It has been demonstrated from the ultraviolet-visible spectral studies that the oxidation state of iron in cytochrome c does not change when the protein binds with the investigated surfactants. Copyright © 2018 Elsevier B.V. All rights reserved.

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem

2017-10-12

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and/or impedance sensors) mounted on the porous surface. In another example, a membrane distillation (MD) process includes the membrane. Processing circuitry can be configured to monitor outputs of the plurality of sensors. The monitored outputs can be used to determine membrane degradation, membrane fouling, or to provide an indication of membrane replacement or cleaning. The sensors can also provide temperatures or temperature differentials across the porous surface, which can be used to improve modeling or control the MD process.

Flower colour and cytochromes P450.

Science.gov (United States)

Tanaka, Yoshikazu; Brugliera, Filippa

2013-02-19

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

Chemical labeling studies on isolated and vesicular bovine heart mitochondrial cytochrome c oxidase

International Nuclear Information System (INIS)

Venzke, K.S.; Reynolds, K.A.; Prochaska, L.J.

1987-01-01

Bovine heart cytochrome c oxidase dispersed in Triton X-100, Tween 80, or dodecyl maltoside was reacted with the water-soluble reagents [ 35 S]-diazonium benzene sulfonate (DABS) (10-100 μM) or [ 125 I]-iodo-DABS (34-55 nM) to map the surface topography of the enzyme in different protein aggregation states. Both reagents gave similar labeling profiles of the enzyme under all conditions. Subunits II, III, and VII were extensively labeled by DABS, while subunits I and VI were unreactive with DABS in each detergent. Subunit V exhibited an increase in DABS labeling when the enzyme was reacted in Tween 80 as compared to the enzyme in Triton X-100 or dodecyl maltoside. Also, components b and c showed an increase in DABS reactivity when the enzyme was modified in dodecyl maltoside. In general, the labeling profile of the enzyme in dodecyl maltoside resembled that of the enzyme in Triton X-100, emphasizing that the mechanism of dispersal of the enzyme by both detergents is similar. Cytochrome c oxidase incorporated into phosphatidylglycerol:phosphatidylcholine(1:20)(w:w) phospholipid vesicles (COV) by cholate dialysis was reacted with DABS and subunits II and III were significantly labeled. Approximately 65-70% of the enzyme in COV was oriented with the cytochrome c binding domain facing the extravesicular medium, as determined by comparison of the DABS labeling in subunit IV in detergent-lysed and intact COV

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.; Choi, Seung Hak

2012-01-01

. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.

2012-06-24

Membrane reactors are generally applied in high temperature reactions (>400 °C). In the field of fine chemical synthesis, however, much milder conditions are generally applicable and polymeric membranes were applied without their damage. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane is to be used in. In this chapter a review of up to date literature about polymers and configuration catalyst/ membranes used in some recent polymeric membrane reactors is given. The new emerging concept of polymeric microcapsules as catalytic microreactors has been proposed. © 2012 Bentham Science Publishers. All rights reserved.

The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (Triticum aestivum L.) under waterlogging.

Science.gov (United States)

Qi, Yuan-Hong; Mao, Fang-Fang; Zhou, Zhu-Qing; Liu, Dong-Cheng; Min-Yu; Deng, Xiang-Yi; Li, Ji-Wei; Mei, Fang-Zhu

2018-05-02

It has been shown in mammalian systems that the mitochondria can play a key role in the regulation of apoptosis by releasing intermembrane proteins (such as cytochrome c) into the cytosol. Cytochrome c released from the mitochondria to the cytoplasm activates proteolytic enzyme cascades, leading to specific nuclear DNA degradation and cell death. This pathway is considered to be one of the important regulatory mechanisms of apoptosis. Previous studies have shown that endosperm cell development in wheat undergoes specialized programmed cell death (PCD) and that waterlogging stress accelerates the PCD process; however, little is known regarding the associated molecular mechanism. In this study, changes in mitochondrial structure, the release of cytochrome c, and gene expression were studied in the endosperm cells of the wheat (Triticum aestivum L.) cultivar "huamai 8" during PCD under different waterlogging durations. The results showed that waterlogging aggravated the degradation of mitochondrial structure, increased the mitochondrial permeability transition (MPT), and decreased mitochondrial transmembrane potential (ΔΨm), resulting in the advancement of the endosperm PCD process. In situ localization and western blotting of cytochrome c indicated that with the development of the endosperm cell, cytochrome c was gradually released from the mitochondria to the cytoplasm, and waterlogging stress led to an advancement and increase in the release of cytochrome c. In addition, waterlogging stress resulted in the increased expression of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT), suggesting that the mitochondrial permeability transition pore (MPTP) may be involved in endosperm PCD under waterlogging stress. The MPTP inhibitor cyclosporine A effectively suppressed cell death and cytochrome c release during wheat endosperm PCD. Our results indicate that the mitochondria play important roles in the PCD of endosperm cells and that

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

DEFF Research Database (Denmark)

Markussen, Mette; Heiberg, Ann-Charlotte; Fredholm, Merete

2008-01-01

over-express the Cyp2a1 gene. TGhe altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared...... expression profiles, from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal...... resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant...

4-Alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines

International Nuclear Information System (INIS)

Lee, J.S.; Jacobsen, N.E.; Ortiz de Montellano, P.R.

1988-01-01

Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4,-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine matabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4- 2 H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4- 2 H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms

Differential action on cancer and normal tissue by adrenochrome monoaminoguanidine methanesulfonate and cytochrome C combined with radiotherapy

International Nuclear Information System (INIS)

Nakatsugawa, S.; Sugahara, T.

1994-01-01

The possibility that radioprotective effects on potent natural killer (NK) cells by adrenochrome monoaminoguanidine methanesulfonate (AMM) + cytochrome C during radiotherapy (RT) for lung cancer might result in the radiosensitization of human lung cancer cells in vivo is examined. Human lung cancer xenografts in the right hind legs of KSN mice (10 weeks old) were locally irradiated with 20 Gy of X ray. AMM (10 mg/kg/day) and/or cytochrome C (CCC) (5 mg/kg/day) were given intraperitoneally immediately before or after RT, followed by daily administration for 4 days. Natural killer activities of host splenocytes were also tested with the standard 51 Cr releasing assay with YAC-1 cells as target cells. In a clinical study, 65 patients with lung cancer were treated with more than 50 Gy of RT with or without combination with AMM + CCC, OK-432 or AMM + CCC + OK-432. Before and after RT, lymphocyte subsets in the peripheral blood were examined with dichromatic analysis using an Ortho Spectrum IIIFCM system and fluorescent MABs. In this study, the change in the absolute number of each subset was investigated. AMM + cytochrome C augumented NK activity in KSN nude mice, protected potent NK cells in patients with lung cancer against RT and sensitized the human lung cancer xenografts to RT. AMM + cytochrome C may have potential as a differential modulator of radiosensitivity of normal tissues and of tumors. 8 refs., 2 figs., 1 tab

Benzo[alpyrene induction of cytochrome P450 1A1/1A2 in the lymph nodes of rats.

Science.gov (United States)

Borodin, Yu I; Safina, A F; Maiborodin, I V; Grishanova, A Yu

2003-12-01

Studies of mesenteric lymph nodes of rats by indirect immunoperoxidase method using monoclonal antibodies to cytochrome P450 1A/1A2 after oral dose of benzo[a]pyrene showed the presence of these cytochrome forms in monocytes, macrophages, reticular and litoral cells, cell detritus, and liquid contents of the paracortical zone and medullary substance sinuses. Oxidation of various exo- and endogenous toxins in the lymph nodes was revealed.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

Energy Technology Data Exchange (ETDEWEB)

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

2011-08-05

Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

International Nuclear Information System (INIS)

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

2011-01-01

Highlights: → Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. → First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. → Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. → Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. → Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b 5 and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

Science.gov (United States)

Witkamp, R F; Nijmeijer, S M; van Miert, A S

1996-01-01

Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

Science.gov (United States)

Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

2012-02-15

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

Genome-Wide Analysis, Classification, Evolution, and Expression Analysis of the Cytochrome P450 93 Family in Land Plants

OpenAIRE

Du, Hai; Ran, Feng; Dong, Hong-Li; Wen, Jing; Li, Jia-Na; Liang, Zhe

2016-01-01

Cytochrome P450 93 family (CYP93) belonging to the cytochrome P450 superfamily plays important roles in diverse plant processes. However, no previous studies have investigated the evolution and expression of the members of this family. In this study, we performed comprehensive genome-wide analysis to identify CYP93 genes in 60 green plants. In all, 214 CYP93 proteins were identified; they were specifically found in flowering plants and could be classified into ten subfamilies?CYP93A?K, with t...

Cloning, expression, crystallization and preliminary X-ray characterization of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus

International Nuclear Information System (INIS)

Ichiki, Shin-ichi; Nakamura, Shota; Ohkubo, Tadayasu; Kobayashi, Yuji; Hasegawa, Jun; Uchiyama, Susumu; Nishihara, Hirofumi; Mizuta, Keiko; Sambongi, Yoshihiro

2005-01-01

Cytochrome c 552 of a moderate thermophile, H. thermoluteolus, was overexpressed in E. coli and crystallized for X-ray diffraction study. The amino-acid sequence of cytochrome c 552 (PH c 552 ) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c 552 ), and from a mesophile, Pseudomonas aeruginosa (PA c 551 ). The PH c 552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c 552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222 1 , with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 Å. The crystals diffract X-rays to around 2.1 Å resolution

Modification of plasma membrane electron transport in cultured rose cells by UV-C radiation and fungal elicitor

International Nuclear Information System (INIS)

Murphy, T.M.; Auh, C.K.; Schorr, R.; Grobe, C.

1991-01-01

Previous experiments have shown that treatments of suspension-cultured cells of Rosa damascena Mill. with UV radiation or with fungal elicitors stimulates the synthesis of H 2 O 2 by the cells. To test the hypothesis that this synthesis involves reduction of O 2 at the plasma membrane and to identify the mechanism of the reduction, we have determined the effects of UV and elicitor on redox reactions associated with the plasma membrane. Elicitor prepared from cell walls of Phytophthora sp. (14 μg solids/ml) inhibited the reduction of ferricyanide by intact cells by 98%; UV-C (primarily 254 nm, up to 19,500 J/m 2 ) inhibited this reduction by 40%. Neither treatment inhibited the reduction of Fe(III)-EDTA by intact cells. Intact cells oxidized NADH in the absence of external oxidizing agent, and the rate of oxidation was increased by UV and elicitor. Cells that were poisoned with arsenite and CCCP catalyzed the reduction of Fe(III)-EDTA in the presence of external NADH, and this ability was slightly stimulated by UV and elicitor. UV irradiation (6,480 J/m 2 ) of cells resulted in a 27% inhibition of the specific activity of NADH-ferricyanide oxidoreductase in plasma membrane isolated from those cells. Elicitor treatment of cells for at least 90 min resulted in a 50% inhibition of the enzyme's specific activity in isolated plasma membrane; this inhibition was reversed by addition of Triton-X100 in the assay mixture. The results suggest that UV and elicitor alter the flow of electrons in the plasma membrane, reversibly inhibiting NADH-cytochrome b reductase, the putative key enzyme in the pathway of ferricyanide reduction, and stimulating (or at least not inhibiting) the pathway of Fe(III)-EDTA reduction

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

Energy Technology Data Exchange (ETDEWEB)

Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

2014-06-01

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

International Nuclear Information System (INIS)

Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

2014-01-01

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

International Nuclear Information System (INIS)

Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W.

2007-01-01

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB

Membrane fouling mechanism of biofilm-membrane bioreactor (BF-MBR): Pore blocking model and membrane cleaning.

Science.gov (United States)

Zheng, Yi; Zhang, Wenxiang; Tang, Bing; Ding, Jie; Zheng, Yi; Zhang, Zhien

2018-02-01

Biofilm membrane bioreactor (BF-MBR) is considered as an important wastewater treatment technology that incorporates advantages of both biofilm and MBR process, as well as can alleviate membrane fouling, with respect to the conventional activated sludge MBR. But, to be efficient, it necessitates the establishment of proper methods for the assessment of membrane fouling. Four Hermia membrane blocking models were adopted to quantify and evaluate the membrane fouling of BF-MBR. The experiments were conducted with various operational conditions, including membrane types, agitation speeds and transmembrane pressure (TMP). Good agreement between cake formation model and experimental data was found, confirming the validity of the Hermia models for assessing the membrane fouling of BF-MBR and that cake layer deposits on membrane. Moreover, the influences of membrane types, agitation speeds and transmembrane pressure on the Hermia pore blocking coefficient of cake layer were investigated. In addition, the permeability recovery after membrane cleaning at various operational conditions was studied. This work confirms that, unlike conventional activated sludge MBR, BF-MBR possesses a low degree of membrane fouling and a higher membrane permeability recovery after cleaning. Copyright © 2017 Elsevier Ltd. All rights reserved.

Membrane dynamics

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

Science.gov (United States)

Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

2015-08-01

Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

Science.gov (United States)

Donnelly, Mark

2006-08-01

The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor

DEFF Research Database (Denmark)

Jensen, Kenneth; Johnston, Jonathan B.; Montellano, Paul R. Ortiz de

2012-01-01

The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multif...

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem; Karam, Ayman M.

2017-01-01

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

Science.gov (United States)

Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

2017-05-01

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anchoring a plant cytochrome P450 via PsaM to the thylakoids in Synechococcus sp. PCC 7002: evidence for light-driven biosynthesis.

Directory of Open Access Journals (Sweden)

Lærke Münter Lassen

Full Text Available Plants produce an immense variety of specialized metabolites, many of which are of high value as their bioactive properties make them useful as for instance pharmaceuticals. The compounds are often produced at low levels in the plant, and due to their complex structures, chemical synthesis may not be feasible. Here, we take advantage of the reducing equivalents generated in photosynthesis in developing an approach for producing plant bioactive natural compounds in a photosynthetic microorganism by functionally coupling a biosynthetic enzyme to photosystem I. This enables driving of the enzymatic reactions with electrons extracted from the photosynthetic electron transport chain. As a proof of concept, we have genetically fused the soluble catalytic domain of the cytochrome P450 CYP79A1, originating from the endoplasmic reticulum membranes of Sorghum bicolor, to a photosystem I subunit in the cyanobacterium Synechococcus sp. PCC 7002, thereby targeting it to the thylakoids. The engineered enzyme showed light-driven activity both in vivo and in vitro, demonstrating the possibility to achieve light-driven biosynthesis of high-value plant specialized metabolites in cyanobacteria.

Biomimetic membranes and methods of making biomimetic membranes

Science.gov (United States)

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

SDS-facilitated in vitro formation of a transmembrane B-type cytochrome is mediated by changes in local pH

DEFF Research Database (Denmark)

Weber, M.; Schneider, D.; Prodöhl, A.

2011-01-01

cytochrome b(559)', which can be efficiently assembled in vitro from a heme-binding PsbF homo-dimer by combining free heme with the apo-cytochrome b(559)'. Unfolding of the protein dissolved in the mild detergent dodecyl maltoside may be induced by addition of SDS, which at high concentrations leads to dimer...... dissociation. Surprisingly, absorption spectroscopy reveals that heme binding and cytochrome formation at pH 8.0 are optimal at intermediate SDS concentrations. Stopped-flow kinetics revealed that genuine conformational changes are involved in heme binding at these SDS concentrations. GPS (Global Protein...... folding State mapping) NMR measurements showed that optimal heme binding is intimately related to a change in the degree of histidine protonation. In the absence of SDS, the pH curve for heme binding is bell-shaped with an optimum at around pH 6-7. At alkaline pH values, the negative electrostatic...

Periplasmic Cytochrome c(3) of Desulfovibrio vulgaris Is Directly Involved in H2-Mediated Metal but Not Sulfate Reduction

International Nuclear Information System (INIS)

Elias, Dwayne A.; Suflita, Joseph M.; McInerney, Michael J.; Krumholz, Lee R.

2004-01-01

Kinetic parameters and the role of cytochrome c3 in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (Km 220 uM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H2 and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H2 and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H2, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate or pyruvate-reduced, but not H2-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H2 was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H2 was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c3 is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate

Gel layer formation on membranes in Membrane Bioreactors

NARCIS (Netherlands)

Van den Brink, P.F.H.

2014-01-01

The widespread application of membrane bioreactors (MBRs) for municipal wastewater treatment is hampered by membrane fouling. Fouling increases energy demand, reduces process performance and creates the need for more frequent (chemical) membrane cleaning or replacement. Membrane fouling in MBRs is

Nanodisc-solubilized membrane protein library reflects the membrane proteome.

Science.gov (United States)

Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

2013-05-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

Science.gov (United States)

Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

2017-01-01

Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

Mapping of redox state of mitochondrial cytochromes in live cardiomyocytes using Raman microspectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda A; Treiman, Marek; Brazhe, Alexey R

2012-01-01

This paper presents a nonivasive approach to study redox state of reduced cytochromes [Formula: see text], [Formula: see text] and [Formula: see text] of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach ...

Flux Enhancement in Membrane Distillation Using Nanofiber Membranes

Directory of Open Access Journals (Sweden)

T. Jiříček

2016-01-01

Full Text Available Membrane distillation (MD is an emerging separation technology, whose largest application potential lies in the desalination of highly concentrated solutions, which are out of the scope of reverse osmosis. Despite many attractive features, this technology is still awaiting large industrial application. The main reason is the lack of commercially available membranes with fluxes comparable to reverse osmosis. MD is a thermal separation process driven by a partial vapour pressure difference. Flux, distillate purity, and thermal efficiency are always in conflict, all three being strictly connected with pore size, membrane hydrophobicity, and thickness. The world has not seen the ideal membrane yet, but nanofibers may offer a solution to these contradictory requirements. Membranes of electrospun PVDF were tested under various conditions on a direct contact (DCMD unit, in order to determine the optimum conditions for maximum flux. In addition, their performance was compared to commonly available PTFE, PE, and PES membranes. It was confirmed that thinner membranes have higher fluxes and a lower distillate purity and also higher energy losses via conduction across the membrane. As both mass and heat transfer are connected, it is best to develop new membranes with a target application in mind, for the specific membrane module and operational conditions.

Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

Science.gov (United States)

Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

2017-02-01

Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

Betulin induces cytochrome c release and apoptosis in colon cancer cells via NOXA.

Science.gov (United States)

Zhou, Zhiyuan; Zhu, Chenfang; Cai, Zhongfang; Zhao, Feng; He, Liu; Lou, Xiaolou; Qi, Xiaoliang

2018-05-01

Betulin is a common triterpene that can be readily obtained from various plants, particularly birch trees, in their natural environment. Specific tumor cells are sensitive to betulin, whereas healthy cells are not. Betulin was observed to stimulate programmed cell death of various cancer cell lines; however, the precise molecular mechanism of action of betulin remains unknown. The present study used colon cancer cells, in which mass apoptosis triggered by betulin was identified, and the apoptotic process was demonstrated to occur via the activation of caspase-3 and -9 pathways. In addition, release of cytochrome c was detected. Furthermore, the pro-apoptotic member of the Bcl-2 protein family, NOXA, was induced following treatment with betulin, and the downregulation of NOXA markedly suppressed the release of cytochrome c and apoptosis in colon cancer cells. Conversely, the overexpression of NOXA further enhanced betulin-induced apoptosis. The present study therefore offers novel insights into the mechanism of action of the natural compound betulin against tumors.

Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes

International Nuclear Information System (INIS)

Paggi, Damian Alvarez; Martin, Diego F.; Kranich, Anja; Hildebrandt, Peter; Marti, Marcelo A.; Murgida, Daniel H.

2009-01-01

In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes.

Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes

Energy Technology Data Exchange (ETDEWEB)

Paggi, Damian Alvarez; Martin, Diego F. [Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina); Kranich, Anja; Hildebrandt, Peter [Institut fuer Chemie, Technische Universitaet Berlin, Str. des 17, Juni 135, Sekr. PC14, D-10623 Berlin (Germany); Marti, Marcelo A. [Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina)], E-mail: marcelo@qi.fcen.uba.ar; Murgida, Daniel H. [Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina)], E-mail: dhmurgida@qi.fcen.uba.ar

2009-09-01

In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes.

Covalent Modification of Cytochrome C by Reactive Metabolites of Furan

OpenAIRE

Phillips, Martin B.; Sullivan, Mathilde M.; Villalta, Peter W.; Peterson, Lisa A.

2013-01-01

Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivit...

Fabrication of electrospun nanofibrous membranes for membrane distillation application

KAUST Repository

Francis, Lijo

2013-02-01

Nanofibrous membranes of Matrimid have been successfully fabricated using an electrospinning technique under optimized conditions. Nanofibrous membranes are found to be highly hydrophobic with a high water contact angle of 130°. Field emission scanning electron microscopy and pore size distribution analysis revealed the big pore size structure of electrospun membranes to be greater than 2 μm and the pore size distribution is found to be narrow. Flat sheet Matrimid membranes were fabricated via casting followed by phase inversion. The morphology, pore size distribution, and water contact angle were measured and compared with the electrospun membranes. Both membranes fabricated by electrospinning and phase inversion techniques were tested in a direct contact membrane distillation process. Electrospun membranes showed high water vapor flux of 56 kg/m2-h, which is very high compared to the casted membrane as well as most of the fabricated and commercially available highly hydrophobic membranes. ©2013 Desalination Publications.

Successful recombinant production of Allochromatium vinosum cytochrome c' requires coexpression of cmm genes in heme-rich Escherichia coli JCB712

International Nuclear Information System (INIS)

Evers, Toon H.; Merkx, Maarten

2005-01-01

Cytochrome c' from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN - . This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available. Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography. Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c'. Characterization using circular dichroism, UV-vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization

Enzymes activities involving bacterial cytochromes incorporated in clays

International Nuclear Information System (INIS)

Lojou, E.; Giudici-Orticoni, M.Th.; Bianco, P.

2005-01-01

With the development of bio electrochemistry, researches appeared on the enzymes immobilization at the surface of electrodes for the realization of bioreactors and bio sensors. One of the main challenges is the development of host matrix able to immobilize the protein material preserving its integrity. In this framework the authors developed graphite electrodes modified by clay films. These electrodes are examined for two enzyme reactions involving proteins of sulfate-reduction bacteria. Then in the framework of the hydrogen biological production and bioreactors for the environmental pollution de-pollution, the electrochemical behavior of the cytochrome c3 in two different clays deposed at the electrode is examined

A new liver function test using the asialoglycoprotein-receptor system on the liver cell membrane, 3

International Nuclear Information System (INIS)

Hazama, Hiroshi; Kawa, Soukichi; Kubota, Yoshitsugu

1986-01-01

We evaluated the vilidity of a new liver function test using liver scintigraphy based on the asialoglycoprotein (ASGP) receptor system on the liver cell membrane in rats with galactosamine-induced acute liver disorder and those with carbon tetra-chloride-induced chronic liver disorder. Neoglycoprotein (GHSA) produced by combining human serum albumin with 32 galactose units was labeled with 99m Tc and administered (50 μg/100 g body weight) to rats with acute or chronic liver disorder. Clearance curves were produced based on liver scintigrams and analysed using the two-compartment model to obtain parameters. In acute liver disorder, the prolongation of 99m Tc-GHSA clearance and the decrease in ASGP receptor activities correlated well to the increase in serum GOT and the decrease in the esterified to total cholesterol ratio (E/T ratio); in chronic liver disorder, they correlated significantly to the increase in the content of liver hydroxyproline (Hyp) which increased in proportion to the severity of liver fibrosis studied histologically, and to the decrease in the contents of cytochrome P-450 and cytochrome b 5 in liver microsomes. Significant correlation was observed between the prolongation of 99m Tc-GHSA clearance and the decrease in ASGP receptor activities in both acute and chronic liver disorders. These findings indicate that the measurement of 99m Tc-GHSA clearance can be a new liver function test sensitively reflecting the severity of liver damage. (author)

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.

Science.gov (United States)

Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B; Erman, James E

2014-01-03

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0. Copyright © 2013 Elsevier Inc. All rights reserved.

An overview on the allelic variant of CYP2D6 genotype

African Journals Online (AJOL)

Yomi

plants, fungi, slime molds, bacteria and archaea. More than 7700 distinct CYP sequences are known. ... SIGNIFICANCE OF CYTOCHROME P450 IN HUMANS. Human CYPs are primarily membrane-associated proteins, ..... Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6. Genomics.

Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

Science.gov (United States)

Ho, J; Smith, S; Roh, H K

2014-01-01

A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus microplus Embryos

Directory of Open Access Journals (Sweden)

Carlos Logullo

2011-06-01

Full Text Available The physiological roles of polyphosphates (polyP recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 µg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5’-triphosphate synthesis in hard tick embryos.

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus) microplus Embryos

Science.gov (United States)

Campos, Eldo; Façanha, Arnoldo R.; Costa, Evenilton P.; Fraga, Amanda; Moraes, Jorge; da Silva Vaz, Itabajara; Masuda, Aoi; Logullo, Carlos

2011-01-01

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi) and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5′-triphosphate synthesis in hard tick embryos. PMID:21747692

Molecular characterization of cytochrome P450 1B1 and effect of ...

African Journals Online (AJOL)

CYP1B which belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to arylhydrocarbon receptors (AhR) ligands. In this study, a new complementary DNA (cDNA) of the CYP1B subfamily ...

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS.

Science.gov (United States)

Tengölics, Roland; Mészáros, Lívia; Győri, E; Doffkay, Zsolt; Kovács, Kornél L; Rákhely, Gábor

2014-10-01

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled. Copyright © 2014 Elsevier B.V. All rights reserved.

Membrane order in the plasma membrane and endocytic recycling compartment.

Science.gov (United States)

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

Science.gov (United States)

Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

2015-03-10

Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

Electrochemistry and biosensing activity of cytochrome c immobilized on a mesoporous interface assembled from carbon nanospheres

International Nuclear Information System (INIS)

Wang, Y.; Bian, X.; Liao, L.; Zhu, J.; Guo, K.; Kong, J.; Liu, B.

2012-01-01

We report on an amperometric biosensor for hydrogen peroxide. It is obtained via layer-by-layer assembly of ordered mesoporous carbon nanospheres and poly(diallyldimethylammonium) on the surface of an indium tin oxide (ITO) glass electrode and subsequent adsorption of cytochrome c. UV-vis absorption spectroscopy was applied to characterize the process of forming the assembled layers. Cyclic voltammetry revealed a direct and quasi-reversible electron transfer between cytochrome c and the surface of the modified ITO electrode. The surface-controlled electron transfer has an apparent heterogeneous electron-transfer rate constant (k s ) of 5.9 ± 0.2 s -1 in case of the 5-layer electrode. The biosensor displays good electrocatalytic response to the reduction of H 2O 2, and the amperometric signal increase steadily with the concentration of H 2 O 2 in the range from 5 μM to 1.5 mM. The detection limit is 1 μM at pH 7.4. The apparent Michaelis-Menten constant (K m ) of the sensor is 0.53 mM. We assume that the observation of a direct electron transfer of cytochrome c on mesoporous carbon nanospheres may form the basis for a feasible approach for durable and reliable detection of H 2 O 2 . (author)

Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

Science.gov (United States)

Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

2017-04-01

Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

Prediction of activation energies for hydrogen abstraction by cytochrome p450

DEFF Research Database (Denmark)

Olsen, Lars; Rydberg, Patrik; Rod, Thomas Holm

2006-01-01

We have estimated the activation energy for hydrogen abstraction by compound I in cytochrome P450 for a diverse set of 24 small organic substrates using state-of-the-art density functional theory (B3LYP). We then show that these results can be reproduced by computationally less demanding methods,...... of the less demanding methods are applied to study the CYP3A4 metabolism of progesterone and dextromethorphan....

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

Directory of Open Access Journals (Sweden)

Jun Wu

2016-09-01

Full Text Available Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1 and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

In situ determination of the reduction levels of cytochromes b and c in growing bacteria : a case study with N2-fixing Azorhizobium caulinodans

NARCIS (Netherlands)

Pronk, A.F.; Boogerd, F C; Stoof, C.; Oltmann, L F; Stouthamer, A.H.; van Verseveld, H W

1993-01-01

The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth

Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans

NARCIS (Netherlands)

Bloemen, L. J.; Monster, A. C.; Kezic, S.; Commandeur, J. N.; Veulemans, H.; Vermeulen, N. P.; Wilmer, J. W.

2001-01-01

To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100

Expression of cytochrome P450 genes in CD34(+) hematopoietic stem and progenitor cells

Czech Academy of Sciences Publication Activity Database

Souček, P.; Anzenbacher, P.; Skoumalová, I.; Dvořák, Michal

2005-01-01

Roč. 23, č. 9 (2005), s. 1417-1422 ISSN 1066-5099 Institutional research plan: CEZ:AV0Z50520514 Keywords : CD34+ stem/progenitor cells * cytochrome P450 isoforms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.094, year: 2005

Soybean meal fermented by Aspergillus awamori increases the cytochrome P-450 content of the liver microsomes of mice.

Science.gov (United States)

Kishida, T; Ataki, H; Takebe, M; Ebihara, K

2000-04-01

The effect of soybean meal fermented by Aspergillus awamori on the acute lethality of acetaldehyde, pentobarbital sleeping time, and cytochrome P-450 content of the hepatic microsomes was studied in mice. Most of the daidzin and genistin in soybean meal (SBM) were converted into the respective aglycones, daidzein and genistein, by fermentation. In experiment 1, mice were fed isonitrogenic test diets with one of the following five protein sources for 28 d: casein, SBM, fermented and hot-air-dried SBM (FSBM-HD), fermented and freeze-dried SBM (FSBM-FD), or methanol-extracted FSBM-FD (FSMB-FD-R). The acute lethality of acetaldehyde in mice fed the FSBM-FD diet was significantly lower than that in mice fed the SBM, FSBM-HD, or FSBM-FD-R diet. In experiments 2 and 3, mice were fed isonitrogenic test diets with one of the following four protein sources for 28 d: casein, SBM, FSBM-FD, and FSBM-FD-R. The pentobarbital sleeping time was significantly shorter and the cytochrome P-450 content was significantly higher in the mice fed the FSBM-FD diet than the respective value in mice fed the other test diets. In experiment 4, mice were fed one of eight diets which contained different levels of aglycone obtained by varying the proportion of FSBM-FD and FSBM-FD-R, for 28 d. The cytochrome P-450 content in hepatic microsomes increased as the dietary level of isoflavonoid aglycones increased, but there was a saturation phenomenon. These results suggest that soy isoflavonoid aglycones are more potent inducers of cytochrome P-450 than isoflavonoid glycosides.

Mode of Antifungal Drugs Interaction with Cytochrome P- 450

Directory of Open Access Journals (Sweden)

M- Mahmodian

1991-07-01

Full Text Available Computer was used to identify the interactions of substrates and antifungal drugs with the enzyme, Cytochrome P-450; and then Molplot.bas computer program was applied to get three dimensional figures of 5-hydroxy camphor.oxidation products of camphor analogues, and antifungal drugs.Cartesian characteristics of atoms building molecules, are taken from Buildz. for program, which can calculate X,Y,Z coordinates of atoms by Zmatrix data. The other program which can calculate X,Y,Z coordinates, using fractional characteristics, is the Coord, for program that, gives our cartesian characteristics of the atoms of molecule, then by using these data, we obtain three dimensional figures and distance between active atoms in compounds under consideration. Results show that distance between two oxygen atoms in 5-exo-hydroxy- camphor and the other compounds obtained from oxidation of camphor analogues, with the distance of two oxygen atoms in antifungal compounds under discussion are equal. Therefore, we can conclude that, the antifungal molecule also interacts with enzyme's active site, by its own sites, in a similar manner to the 5-hydroxy camphor molecule, which is:"n1. Nitrogen atom (N of Imidazole and Triazole ring in antifungal molecule with Iron atom in heam molecule belonging to Cytochrome P-450 enzyme, are coordinated."n2. The other atoms such as : 0,S or N in structure of the antifungal drug are coordinated with hydrogen atom of hydroxyl group belong ing to Tyr-96 in the structure of enzyme, forming hydrogen bonding.

Giant plasma membrane vesicles: models for understanding membrane organization.

Science.gov (United States)

Levental, Kandice R; Levental, Ilya

2015-01-01

The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of sex hormones on relative quantities of multiple species of cytochrome P-450 in rat liver microsomes

International Nuclear Information System (INIS)

Fujita, S.; Peisach, J.; Chevion, M.; Hebrew Univ., Jerusalem

1981-01-01

EPR spectra of rat liver microsomes from male, female and hormonally-treated castrated hepatectomized rats were studied. The spectra, especially in the region of gsub(max) suggested a heterogeneity of local environments of the low spin ferric heme indicative of multiple structures for cytochrome P-450. Certain features in the spectrum correlated with sexual differences. It is suggested that the changes in the relative amplitudes of the EPR features represent differences in the relative abundance of the individual proteins in the mixture that, in turn, are related to the sexual differences of metabolic patterns for reactions catalyzed by cytochrome P-450. (author)

An assay of optimal cytochrome c oxidase activity in fish gills.

Science.gov (United States)

Hu, Yau-Chung; Chung, Meng-Han; Lee, Tsung-Han

2018-07-15

Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50-100 μg fish gill homogenate in conjunction with 75-100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish. Copyright © 2018 Elsevier Inc. All rights reserved.

Structure and physical properties of bio membranes and model membranes

International Nuclear Information System (INIS)

Tibor Hianik

2006-01-01

Bio membranes belong to the most important structures of the cell and the cell organelles. They play not only structural role of the barrier separating the external and internal part of the membrane but contain also various functional molecules, like receptors, ionic channels, carriers and enzymes. The cell membrane also preserves non-equilibrium state in a cell which is crucial for maintaining its excitability and other signaling functions. The growing interest to the bio membranes is also due to their unique physical properties. From physical point of view the bio membranes, that are composed of lipid bilayer into which are incorporated integral proteins and on their surface are anchored peripheral proteins and polysaccharides, represent liquid s crystal of smectic type. The bio membranes are characterized by anisotropy of structural and physical properties. The complex structure of bio membranes makes the study of their physical properties rather difficult. Therefore several model systems that mimic the structure of bio membranes were developed. Among them the lipid monolayers at an air-water interphase, bilayer lipid membranes, supported bilayer lipid membranes and liposomes are most known. This work is focused on the introduction into the physical word of the bio membranes and their models. After introduction to the membrane structure and the history of its establishment, the physical properties of the bio membranes and their models are stepwise presented. The most focus is on the properties of lipid monolayers, bilayer lipid membranes, supported bilayer lipid membranes and liposomes that were most detailed studied. This lecture has tutorial character that may be useful for undergraduate and graduate students in the area of biophysics, biochemistry, molecular biology and bioengineering, however it contains also original work of the author and his co-worker and PhD students, that may be useful also for specialists working in the field of bio membranes and model

Low-temperature kinetic measurements of microsecond freeze-hyperquench (MHQ) cytochrome oxidase monitored by UV-visible spectroscopy with a newly designed cuvette.

Science.gov (United States)

Wiertz, F G M; de Vries, S

2006-02-01

A special cuvette was designed to measure optical changes of MHQ (microsecond freeze-hyperquench) powder samples at temperatures below approx. 250 K. Reduced cytochrome c oxidase from Paracoccus denitrificans was reacted with O(2) for 100 micros, frozen as a powder and transferred to the cuvette. Subsequently, cytochrome oxidase was allowed to react further following stepwise increments of the temperature from 100 K up to 250 K while recording spectra between 300 and 700 nm. The temperature was raised only when no further changes in the spectra could be detected. The experiment yielded spectra of the A, P(M), F and O intermediate states. This demonstrated that the catalytic cycle of cytochrome oxidase at low temperature is similar to that at room temperature and so verifies the suitability of this method for the study of enzymes with high catalytic-centre activity.

Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

Directory of Open Access Journals (Sweden)

T. Jiříček

2017-01-01

Full Text Available Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest flux was achieved with the thinnest membranes and the best energy efficiency was achieved with the thickest membranes. All membranes had salt retention above 99%. Nanotechnology offers the potential to find modern solutions for desalination of waste waters, by introducing new materials with revolutionary properties, but new membranes must be developed according to the target application.

Structure and function of cytochrome c2 in electron transfer complexes with the photosynthetic reaction center of Rhodobacter sphaeroides: optical linear dichroism and EPR.

Science.gov (United States)

Drepper, F; Mathis, P

1997-02-11

The photosynthetic reaction center (RC) and its secondary electron donor the water-soluble cytochrome (cyt) c2 from the purple bacterium Rhodobacter sphaeroides have been used in cross-linked and non-cross-linked complexes, oriented in compressed gels or partially dried multilayers, to study the respective orientation of the primary donor P (BChl dimer) and of cyt c2. Three methods were used: (i) Polarized optical absorption spectra at 295 and 10 K were measured and the linear dichroism of the two individual transitions (Qx, Qy), which are nearly degenerate within the alpha-band of reduced cyt c2, was determined. Attribution of the polarization directions to the molecular axes within the heme plane yielded the average cyt orientation in the complexes. (ii) Time-resolved flash absorption measurements using polarized light allowed determination of the orientation of cyt c2 in complexes which differ in their kinetics of electron transfer. (iii) EPR spectroscopy of ferricyt c2 in cross-linked RC-cyt c2 complexes was used to determine the angle between the heme and the membrane plane. The results suggest the following structural properties for the docking of cyt c2 to the RC: (i) In cross-linked complexes, the two cytochromes displaying half-lives of 0.7 and 60 micros for electron transfer to P+ are similarly oriented (difference plane is parallel to the symmetry axis of the RC (0 degrees +/- 10 degrees). Moreover, the Qy transition, which is assumed to be polarized within the ring III-ring I direction of the heme plane, makes an angle of 56 degrees +/- 1 degree with the symmetry axis. (iii) The dichroism spectrum for the fast phase (0.7 micros) for the non-cross-linked cyt c2-RC complex suggests an orientation similar to that of cross-linked cyt c2, but the heme plane is tilted about 20 degrees closer to the membrane. An alternative model is that two or more bound states of cyt c2 with heme plane tilt angles between 0 degrees and 30 degrees allow the fast electron

Novel extrahepatic cytochrome P450s

International Nuclear Information System (INIS)

Karlgren, Maria; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

2005-01-01

The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis

Retrospective, multicentric study of 180 children with cytochrome C oxidase deficiency

Czech Academy of Sciences Publication Activity Database

Böhm, M.; Pronicka, E.; Karczmarewicz, E.; Pronicki, M.; Piekutowska-Abramczuk, D.; Sykut-Cegielska, J.; Mierzewska, H.; Hansíková, H.; Veselá, K.; Tesařová, M.; Houšťková, H.; Houštěk, Josef; Zeman, J.

2006-01-01

Roč. 59, č. 1 (2006), s. 21-26 ISSN 0031-3998 R&D Projects: GA ČR(CZ) GA303/03/0749; GA MŠk(CZ) 1M0520; GA MZd(CZ) NR8065 Grant - others:Framework Programme(XE) LSHM-CT-2004-503116; GA-(XE) GLG1-CT-2002-90358 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondria * disease * cytochrome c oxidase Subject RIV: FG - Pediatrics Impact factor: 2.619, year: 2006

Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding

OpenAIRE

Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.

2009-01-01

The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three protein forms which were assigned to a low-temperature and a high-temperature His-Met intermediate species and a bis-histidinate form (although the presence of a His-Lys form cannot be excluded). The muc...

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

Science.gov (United States)

Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

1994-01-01

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

Active site intermediates in the reduction of O(2) by cytochrome oxidase, and their derivatives.

Science.gov (United States)

Wikström, Mårten

2012-04-01

The mechanism of dioxygen activation and reduction in cell respiration, as catalysed by cytochrome c oxidase, has a long history. The work by Otto Warburg, David Keilin and Britton Chance defined the dioxygen-binding heme iron centre, viz. das Atmungsferment, or cytochrome a(3). Chance brought the field further in the mid-1970's by ingenious low-temperature studies that for the first time identified the primary enzyme-substrate (ES) Michaelis complex of cell respiration, the dioxygen adduct of heme a(3), which he termed Compound A. Further work using optical, resonance Raman, EPR, and other sophisticated spectroscopic techniques, some of which with microsecond time resolution, has brought us to the situation today, where major principles of how O(2) reduction occurs in respiration are well understood. Nonetheless, some questions have remained open, for example concerning the precise structures, catalytic roles, and spectroscopic properties of the breakdown products of Compound A that have been called P, F (for peroxy and ferryl), and O (oxidised). This nomenclature has been known to be inadequate for some time already, and an alternative will be suggested here. In addition, the multiple forms of P, F and O states have been confusing, a situation that we endeavour to help clarifying. The P and F states formed artificially by reacting cytochrome oxidase with hydrogen peroxide are especially scrutinised, and some novel interpretations will be given that may account for previously unexplained observations. Copyright © 2011 Elsevier B.V. All rights reserved.

Fouling in Membrane Distillation, Osmotic Distillation and Osmotic Membrane Distillation

Directory of Open Access Journals (Sweden)

Mourad Laqbaqbi

2017-03-01

Full Text Available Various membrane separation processes are being used for seawater desalination and treatment of wastewaters in order to deal with the worldwide water shortage problem. Different types of membranes of distinct morphologies, structures and physico-chemical characteristics are employed. Among the considered membrane technologies, membrane distillation (MD, osmotic distillation (OD and osmotic membrane distillation (OMD use porous and hydrophobic membranes for production of distilled water and/or concentration of wastewaters for recovery and recycling of valuable compounds. However, the efficiency of these technologies is hampered by fouling phenomena. This refers to the accumulation of organic/inorganic deposits including biological matter on the membrane surface and/or in the membrane pores. Fouling in MD, OD and OMD differs from that observed in electric and pressure-driven membrane processes such electrodialysis (ED, membrane capacitive deionization (MCD, reverse osmosis (RO, nanofiltration (NF, ultrafiltration (UF, microfiltration (MF, etc. Other than pore blockage, fouling in MD, OD and OMD increases the risk of membrane pores wetting and reduces therefore the quantity and quality of the produced water or the concentration efficiency of the process. This review deals with the observed fouling phenomena in MD, OD and OMD. It highlights different detected fouling types (organic fouling, inorganic fouling and biofouling, fouling characterization techniques as well as various methods of fouling reduction including pretreatment, membrane modification, membrane cleaning and antiscalants application.

Molecular phylogeny of some avian species using Cytochrome b gene sequence analysis

Science.gov (United States)

Awad, A; Khalil, S. R; Abd-Elhakim, Y. M

2015-01-01

Veritable identification and differentiation of avian species is a vital step in conservative, taxonomic, forensic, legal and other ornithological interventions. Therefore, this study involved the application of molecular approach to identify some avian species i.e. Chicken (Gallus gallus), Muskovy duck (Cairina moschata), Japanese quail (Coturnix japonica), Laughing dove (Streptopelia senegalensis), and Rock pigeon (Columba livia). Genomic DNA was extracted from blood samples and partial sequence of the mitochondrial cytochrome b gene (358 bp) was amplified and sequenced using universal primers. Sequences alignment and phylogenetic analyses were performed by CLC main workbench program. The obtained five sequences were deposited in GenBank and compared with those previously registered in GenBank. The similarity percentage was 88.60% between Gallus gallus and Coturnix japonica and 80.46% between Gallus gallus and Columba livia. The percentage of identity between the studied species and GenBank species ranged from 77.20% (Columba oenas and Anas platyrhynchos) to 100% (Gallus gallus and Gallus sonneratii, Coturnix coturnix and Coturnix japonica, Meleagris gallopavo and Columba livia). Amplification of the partial sequence of mitochondrial cytochrome b gene proved to be practical for identification of an avian species unambiguously. PMID:27175180

Flow and fouling in membrane filters: Effects of membrane morphology

Science.gov (United States)

Sanaei, Pejman; Cummings, Linda J.

2015-11-01

Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

Helical Propensity Affects the Conformational Properties of the Denatured State of Cytochrome c'.

Science.gov (United States)

Danielson, Travis A; Bowler, Bruce E

2018-01-23

Changing the helical propensity of a polypeptide sequence might be expected to affect the conformational properties of the denatured state of a protein. To test this hypothesis, alanines at positions 83 and 87 near the center of helix 3 of cytochrome c' from Rhodopseudomonas palustris were mutated to serine to decrease the stability of this helix. A set of 13 single histidine variants in the A83S/A87S background were prepared to permit assessment of the conformational properties of the denatured state using histidine-loop formation in 3 M guanidine hydrochloride. The data are compared with previous histidine-heme loop formation data for wild-type cytochrome c'. As expected, destabilization of helix 3 decreases the global stabilities of the histidine variants in the A83S/A87S background relative to the wild-type background. Loop stability versus loop size data yields a scaling exponent of 2.1 ± 0.2, similar to the value of 2.3 ± 0.2 obtained for wild-type cytochrome c'. However, the stabilities of all histidine-heme loops, which contain the helix 3 sequence segment, are increased in the A83S/A87S background compared to the wild-type background. Rate constants for histidine-heme loop breakage are similar for the wild-type and A83S/A87S variants. However, for histidine-heme loops that contain the helix 3 sequence segment, the rate constants for loop formation increase in the A83S/A87S background compared to the wild-type background. Thus, residual helical structure appears to stiffen the polypeptide chain slowing loop formation in the denatured state. The implications of these results for protein folding mechanisms are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Pulse Radiolysis Studies of Temperature Dependent Electron Transfers among Redox Centers in ba(3)-Cytochrome c Oxidase from Thermus thermophilus

DEFF Research Database (Denmark)

Farver, Ole; Wherland, Scot; Antholine, William E

2010-01-01

The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the Cu(A)(r) site to the low-spin heme-(a)b(o) site, i.e., Cu(A)(r) + heme-a(b)(o) ......The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the Cu(A)(r) site to the low-spin heme-(a)b(o) site, i.e., Cu(A)(r) + heme...... in cytochrome ba(3) had no effect on the rate of this reaction whereas the II-Met160Leu Cu(A)-mutation was slower by an amount corresponding to a decreased driving force of ∼0.06 eV. The structures support the presence of a common, electron-conducting "wire" between Cu(A) and heme-a(b). The transfer...

Oxidative modification of methionine80 in cytochrome c by reaction with peroxides.

Science.gov (United States)

Nugraheni, Ari Dwi; Ren, Chunguang; Matsumoto, Yorifumi; Nagao, Satoshi; Yamanaka, Masaru; Hirota, Shun

2018-05-01

The Met80-heme iron bond of cytochrome c (cyt c) is cleaved by the interaction of cyt c with cardiolipin (CL) in membranes. The Met80 dissociation enhances the peroxidase activity of cyt c and triggers cyt c release from mitochondrion to the cytosol at the early stage of apoptosis. This paper demonstrates the selective oxidation of Met80 for the reaction of ferric cyt c with a peroxide, meta-chloroperbenzoic acid (mCPBA), in the presence of CL-containing liposomes by formation of a ferryl species (Compound I). After the reaction of cyt c with mCPBA in the presence of 1,2-dioloeyl-sn-glycero-3-phosphocholine (DOPC) liposomes containing CL, the electrospray ionization mass spectrum of the peptide fragments, obtained by digestion of cyt c with lysyl endopeptidase, exhibited a peak at m/z = 795.45; whereas, this peak was not observed for the peptide fragments obtained after the reaction in the presence of DOPC liposomes not containing CL. According to the tandem mass spectrum of the m/z = 795.45 peptide fragment, Met80 was modified with a 16 Da mass increase. The purified Met80-modified cyt c exhibited a peroxidase activity more than 5-fold higher than that of the unmodified protein. Transient absorption bands around 650 nm were generated by the reactions with mCPBA for ferric wild-type cyt c in the presence of CL-containing DOPC liposomes and ferric Y67F cyt c in the absence of liposomes. The formation and decomposition rates of the 650-nm absorption species increased and decreased, respectively, by increasing the mCPBA concentration in the reaction, indicating transient formation of Compound I. Copyright © 2018 Elsevier Inc. All rights reserved.

A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast.

NARCIS (Netherlands)

Blakely, E.L.; Mitchell, A.L.; Fisher, N.; Meunier, B.; Nijtmans, L.G.J.; Schaefer, A.M.; Jackson, M.J.; Turnbull, D.M.; Taylor, R.W.

2005-01-01

Whereas the majority of disease-related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical

Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c.

Science.gov (United States)

Imai, Mizue; Saio, Tomohide; Kumeta, Hiroyuki; Uchida, Takeshi; Inagaki, Fuyuhiko; Ishimori, Koichiro

2016-01-22

Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the μs-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO. Copyright © 2015 Elsevier Inc. All rights reserved.

Evidence for induction of cytochrome P-450I in patients with tropical chronic pancreatitis.

Science.gov (United States)

Chaloner, C; Sandle, L N; Mohan, V; Snehalatha, C; Viswanathan, M; Braganza, J M

1990-06-01

Theophylline kinetics, as an in vivo probe for the potentially toxic cytochrome P-450I pathway of drug metabolism, were studied in 11 healthy volunteers and 11 patients with calcific chronic pancreatitis at Madras, South India. Theophylline clearance was faster in the patients than controls [median 69 (range 39-114) vs 45 (33-56) ml h-1 kg-1, p = 0.003]. In keeping with this finding, detailed social histories identified a higher exposure level in the patients to xenobiotics that are inducers of cytochrome P-450I and/or yield reactive metabolites upon processing thereby (score 7, 4-11 vs 3, 2-9, p = 0.002). However, the concentration of D-glucaric acid in urine, as a marker of phase II conjugating pathways of drug metabolism, was similar in patients and controls. This pattern of drug metabolism could predispose to oxidant stress: hence micronutrient antioxidant supplements may have therapeutic (or even prophylactic) value in tropical chronic pancreatitis.

Cytochrome C effect on gamma-ray efficiency on barley seeds at different metabolic states

International Nuclear Information System (INIS)

Yankulov, M.

1981-01-01

Radiobiological studies of the effect of gamma-rays on the barley seeds were performed. It was shown that the different metabolic states of the seeds do not modify the effect of the independent treatment with cytochrome C, while the action of the gamma-rays is markedly modified. With the increase in the preliminary seed soaking time in H 2 O, the total lethality in the case of irradiated treatments rises from 54.10% to 91.00% and that of sterility to 13.13 and 57.44% for 12 and 72 hrs, respectively. The preliminary and post-irradiation treatment of seeds with cytochrome C markedly reduces the effect of gamma-rays, calculated by the criteria of general lethality and sterility, the trend towards an increase in the sensitivity with the increase in the extention of the preliminary soaking time of seeds in water being preserved. Preliminary soaking in the seeds in water also modifies the mutagenic effect of gamma-rays to a considerable extent. (author)

Site heteroplasmy in the mitochondrial cytochrome b gene of the sterlet sturgeon Acipenser ruthenus

Directory of Open Access Journals (Sweden)

Andreea Dudu

2012-01-01

Full Text Available Sturgeons are fish species with a complex biology. They are also characterized by complex aspects including polyploidization and easiness of hybridization. As with most of the Ponto-Caspian sturgeons, the populations of Acipenser ruthenus from the Danube have declined drastically during the last decades. This is the first report on mitochondrial point heteroplasmy in the cytochrome b gene of this species. The 1141 bp sequence of the cytb gene in wild sterlet sturgeon individuals from the Lower Danube was determined, and site heteroplasmy evidenced in three of the 30 specimens collected. Two nucleotide sequences were identified in these heteroplasmic individuals. The majority of the heteroplasmic sites are synonymous and do not modify the sequence of amino acids in cytochrome B protein. To date, several cases of point heteroplasmy have been reported in animals, mostly due to paternal leakage of mtDNA. The presence of specific point heteroplasmic sites might be interesting for a possible correlation with genetically distinct groups in the Danube River.

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450_(BM3) demethylases

OpenAIRE

Lewis, Jared C.; Bastian, Sabine; Bennett, Clay S.; Fu, Yu; Mitsuda, Yuuichi; Chen, Mike M.; Greenberg, William A.; Wong, Chi-Huey; Arnold, Frances H.

2009-01-01

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450_(BM3)) demethylases that provides a highly efficient means to access valuable intermediate...

Influence of some anti-inflammatory drugs on the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content

Energy Technology Data Exchange (ETDEWEB)

Mostafa, M.H.; Sheweita, S.A.; Abdel-Moneam, N.M. (Alexandria Univ. (Egypt))

1990-06-01

The metabolism of benzo({alpha})pyrene is mediated by the mixed function oxidase system including the cytochrome P450-dependent aryl hydrocarbon hydroxylase. The data of the present study revealed the ability of various commonly used anti-inflammatory drugs to alter the activity of this enzyme system, where all the tested drugs, namely phenyl butazone, ketoprofen, piroxicam, and acetaminophen, caused an increase in both the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content whether administered as a single dose or as a repeated dose for 6 consecutive days. The percentage of change for all drugs except phenyl butazone was proportional to the duration of drug administration. On the other hand, pyrazole which is chemically related to phenyl butazone, had no significant effect when administered as a single dose but caused a decrease in both studied parameters when administered as a repeated dose for 6 consecutive days. The mechanisms by which these commonly used drugs modify the aryl hydrocarbon hydroxylase activity and the cytochrome p450 content are discussed in the text.

Fundamentals of membrane bioreactors materials, systems and membrane fouling

CERN Document Server

Ladewig, Bradley

2017-01-01

This book provides a critical, carefully researched, up-to-date summary of membranes for membrane bioreactors. It presents a comprehensive and self-contained outline of the fundamentals of membrane bioreactors, especially their relevance as an advanced water treatment technology. This outline helps to bring the technology to the readers’ attention, and positions the critical topic of membrane fouling as one of the key impediments to its more widescale adoption. The target readership includes researchers and industrial practitioners with an interest in membrane bioreactors.

Role of Glycol Chitosan-incorporated Ursolic Acid Nanoparticles in ...

African Journals Online (AJOL)

and apoptosis via mitochondrial pathway due to decrease in membrane potential and release of cytochrome C, as ... triggering of apoptosis as determined by DNA fragmentation ... used to transfer proteins onto a PVDF membrane which was ...

Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P-450 in Rhodococcus strains.

OpenAIRE

Shao, Z Q; Behki, R

1996-01-01

A cytochrome P-450 system in Rhodococcus strains, encoded by thcB, thcC, and thcD, participates in the degradation of thiocarbamates and several other pesticides. The regulation of the system was investigated by fusing a truncated lacZ in frame to thcB, the structural gene for the cytochrome P-450 monooxygenase. Analysis of the thcB-lacZ fusion showed that the expression of thcB was 10-fold higher in the presence of the herbicide EPTC (s-ethyl dipropylthiocarbamate). Similar enhancement of th...

Cytochrome P450 humanised mice

Directory of Open Access Journals (Sweden)

Gonzalez Frank J

2004-05-01

Full Text Available Abstract Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s. These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach.

Cytochrome P450 humanised mice

Science.gov (United States)

2004-01-01

Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

Adaptive silicone-membrane lenses: planar vs. shaped membrane

CSIR Research Space (South Africa)

Schneider, F

2009-08-01

Full Text Available Engineering, Georges-Koehler-Allee 102, Freiburg 79110, Germany florian.schneider@imtek.uni-freiburg.de ABSTRACT We compare the performance and optical quality of two types of adaptive fluidic silicone-membrane lenses. The membranes feature either a...-membrane lenses: planar vs. shaped membrane Florian Schneider1,2, Philipp Waibel2 and Ulrike Wallrabe2 1 CSIR, Materials Science and Manufacturing, PO Box 395, Pretoria 0001, South Africa 2 University of Freiburg – IMTEK, Department of Microsystems...

Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect

Directory of Open Access Journals (Sweden)

Nikola Kovářová

2016-06-01

Full Text Available This paper describes data related to a research article entitled “Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects” [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1−/− and control (SURF1+/+ mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX, to reversible inhibition of mitochondrial translation in SURF1−/− mouse and SURF1 patient fibroblast cell lines.

Degradation of Polypropylene Membranes Applied in Membrane Distillation Crystallizer

Directory of Open Access Journals (Sweden)

Marek Gryta

2016-03-01

Full Text Available The studies on the resistance to degradation of capillary polypropylene membranes assembled in a membrane crystallizer were performed. The supersaturation state of salt was achieved by evaporation of water from the NaCl saturated solutions using membrane distillation process. A high feed temperature (363 K was used in order to enhance the degradation effects and to shorten the test times. Salt crystallization was carried out by the application of batch or fluidized bed crystallizer. A significant membrane scaling was observed regardless of the method of realized crystallization. The SEM-EDS, DSC, and FTIR methods were used for investigations of polypropylene degradation. The salt crystallization onto the membrane surface accelerated polypropylene degradation. Due to a polymer degradation, the presence of carbonyl groups on the membranes’ surface was identified. Besides the changes in the chemical structure a significant mechanical damage of the membranes, mainly caused by the internal scaling, was also found. As a result, the membranes were severely damaged after 150 h of process operation. A high level of salt rejection was maintained despite damage to the external membrane surface.

Membrane fusion

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

Introducing Membrane Charge and Membrane Potential to T Cell Signaling

Directory of Open Access Journals (Sweden)

Yuanqing Ma

2017-11-01

Full Text Available While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Polyazole hollow fiber membranes for direct contact membrane distillation

KAUST Repository

Maab, Husnul; Alsaadi, Ahmad Salem; Francis, Lijo; Livazovic, Sara; Ghaffour, NorEddine; Amy, Gary L.; Nunes, Suzana Pereira

2013-01-01

Porous hollow fiber membranes were fabricated from fluorinated polyoxadiazole and polytriazole by a dry-wet spinning method for application in desalination of Red Sea water by direct contact membrane distillation (DCMD). The data were compared with commercially available hollow fiber MD membranes prepared from poly(vinylidene fluoride). The membranes were characterized by electron microscopy, liquid entry pressure (LEP), and pore diameter measurements. Finally, the hollow fiber membranes were tested for DCMD. Salt selectivity as high as 99.95% and water fluxes as high as 35 and 41 L m -2 h-1 were demonstrated, respectively, for polyoxadiazole and polytriazole hollow fiber membranes, operating at 80 C feed temperature and 20 C permeate. © 2013 American Chemical Society.

Polyazole hollow fiber membranes for direct contact membrane distillation

KAUST Repository

Maab, Husnul

2013-08-07

Porous hollow fiber membranes were fabricated from fluorinated polyoxadiazole and polytriazole by a dry-wet spinning method for application in desalination of Red Sea water by direct contact membrane distillation (DCMD). The data were compared with commercially available hollow fiber MD membranes prepared from poly(vinylidene fluoride). The membranes were characterized by electron microscopy, liquid entry pressure (LEP), and pore diameter measurements. Finally, the hollow fiber membranes were tested for DCMD. Salt selectivity as high as 99.95% and water fluxes as high as 35 and 41 L m -2 h-1 were demonstrated, respectively, for polyoxadiazole and polytriazole hollow fiber membranes, operating at 80 C feed temperature and 20 C permeate. © 2013 American Chemical Society.

Proton-stimulated Cl-HCO3 antiport by basolateral membrane vesicles of lobster hepatopancreas

International Nuclear Information System (INIS)

Ahearn, G.A.; Grover, M.L.; Tsuji, R.T.; Clay, L.P.

1987-01-01

Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium 36 Cl uptake experiments at a variety of transmembrane osmotic gradients. 36 Cl uptake into vesicles preloaded with HCO 3 was significantly greater than into vesicles lacking HCO 3 . This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO 3 exchange was potential sensitive and stimulated by an electrically negative vesicle interior. 36 Cl influx (4-s exposures) into HCO 3 -loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and apparent diffusion. 36 Cl influx was a hyperbolic function of both internal [HCO 3 ] and internal [Cl]. The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO 3 being strongly preferred. 36 Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO 3 antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO 3 to power anion exchange and transfer protons against a concentration gradient

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

Effect of Flavonoids on Glutathione Level, Lipid Peroxidation and Cytochrome P450 CYP1A1 Expression in Human Laryngeal Carcinoma Cell Lines

Directory of Open Access Journals (Sweden)

Lidija Vuković

2007-01-01

Full Text Available Flavonoids are phytochemicals exhibiting a wide range of biological activities, among which are antioxidant activity, the ability to modulate activity of several enzymes or cell receptors and possibility to interfere with essential biochemical pathways. Using human laryngeal carcinoma HEp2 cells and their drug-resistant CK2 subline, we examined the effect of five flavonoids, three structurally related flavons (quercetin, fisetin, and myricetin, one flavonol (luteolin and one glycosilated flavanone (naringin for: (i their ability to inhibit mitochondrial dehydrogenases as an indicator of cytotoxic effect, (ii their influence on glutathione level, (iii antioxidant/prooxidant effects and influence on cell membrane permeability, and (iv effect on expression of cytochrome CYP1A1. Cytotoxic action of the investigated flavonoids after 72 hours of treatment follows this order: luteolin>quercetin>fisetin>naringin>myricetin. Our results show that CK2 were more resistant to toxic concentrations of flavonoids as compared to parental cells. Quercetin increased the total GSH level in both cell lines. CK2 cells are less perceptible to lipid peroxidation and damage caused by free radicals. Quercetin showed prooxidant effect in both cell lines, luteolin only in HEp2 cells, whereas other tested flavonoids did not cause lipid peroxidation in the tested cell lines. These data suggest that the same compound, quercetin, can act as a prooxidant, but also, it may prevent damage in cells caused by free radicals, due to the induction of GSH, by forming less harmful complex. Quercetin treatment damaged cell membranes in both cell lines. Fisetin caused higher cell membrane permeability only in HEp2 cells. However, these two compounds did not enhance the damage caused by hydrogen peroxide. Quercetin, naringin, myricetin and fisetin increased the expression of CYP1A1 in both cell lines, while luteolin decreased basal level of CYP1A1 only in HEp2 cells. In conclusion, small

A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

Science.gov (United States)

Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

2015-09-01

Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. Copyright © 2015 Elsevier Inc. All rights reserved.

Stability of cytochromes P450 and phase II conjugation systems in precision-cut rat lung slices cultured up to 72 h.

Science.gov (United States)

Umachandran, Meera; Ioannides, Costas

2006-07-05

The objective of the present study was to evaluate the stability of cytochrome P450 enzymes and of the conjugation enzyme systems epoxide hydrolase, glucuronosyl transferase, sulphotransferase and glutathione S-transferase in precision-cut rat lung slices incubated in RPMI media for different time periods up to 72 h. Moreover, the effect of culturing of lung slices on total glutathione levels and glutathione reductase was also investigated. Monitoring of cytochrome P450 activity was achieved using established diagnostic probes, but when activity in the lung was low the maintenance of the various enzymes in culture was determined immunologically using Western blotting. The dealkylation of pentoxyresorufin declined markedly during the first 4h of incubation but in the case of ethoxyresorufin loss of activity was more gradual and less severe. Western blot analysis revealed that the rate of decrease in cytochrome P450 apoprotein levels was isoform-specific with CYP2E1 being the most stable and CYP3A the least stable. Generally, phase II activities, especially cytosolic sulphotransferase, were relatively more stable throughout the incubation period compared with cytochromes P450. Finally, glutathione reductase activity and total glutathione levels were maintained throughout the 72 h incubation. The present studies indicate that xenobiotic-metabolising enzymes in precision-cut rat lung slices decline in culture, but the rate of loss differs and depends on the nature of the enzyme.

The effects of selected flavonoids on cytochromes P450 in rat liver and small intestine

Czech Academy of Sciences Publication Activity Database

Křížková, J.; Burdová, K.; Stiborová, M.; Křen, Vladimír; Hodek, P.

2009-01-01

Roč. 2, č. 3 (2009), s. 201-204 ISSN 1337-6853 R&D Projects: GA ČR GD305/09/H008 Institutional research plan: CEZ:AV0Z50200510 Keywords : flavonoids * cytochrome p450 * small intestine Subject RIV: EE - Microbiology, Virology

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

DEFF Research Database (Denmark)

Bavishi, Krutika; Laursen, Tomas; Martinez, Karen Laurence

2016-01-01

Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially...... was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions...

Single-molecule Mapping of Long-range Electron Transfer for a Cytochrome b562 Variant

DEFF Research Database (Denmark)

Della Pia, Eduardo Antonio; Chi, Qijin; Jones, D. Dafydd

2011-01-01

Cytochrome b562 was engineered to introduce a cysteine residue at a surface-exposed position to facilitate direct self-assembly on a Au(111) surface. The confined protein exhibited reversible and fast electron exchange with a gold substrate over a distance of 20 Å between the heme redox center an...

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c