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.

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.

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.

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

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.

Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol

NARCIS (Netherlands)

Bessems, J.G.M.; Koppele, J.M. te; Dijk, P.A. van; Stee, L.L.P. van; Commandeur, J.N.M.; Vermeulen, N.P.E.

1996-01-01

1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -C(H)3, -C2H5, -iC3H7) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K(s,app) varied

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

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 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

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.

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...

Magnetite Compensates for the Lack of a Pilin-Associated c-Type Cytochrome in Extracellular Electron Exchange

DEFF Research Database (Denmark)

Liu, Fanghua; Rotaru, Amelia-Elena; Shrestha, Pravin

2015-01-01

investigation revealed that magnetite attached to the electrically conductive pili of Geobacter species in a manner reminiscent of the association of the multi-heme c-type cytochrome OmcS with the pili of Geobacter sulfurreducens. Magnetite conferred extracellular electron capabilities on an Omc...

Ruthenium-modified cytochrome c: temperature dependence of the rate of intramolecular electron transfer

International Nuclear Information System (INIS)

Isied, S.S.; Kuehn, C.; Worosila, G.

1984-01-01

The ruthenium-modified horse heart cytochrome c, Ru(III)-cyt c(III), where the ruthenium is bound to the histidines-33 residue has been synthesized and characterized by ruthenium analysis, UV-vis and CD spectra, and differential pulse polarography and cyclic voltammetry. The intermediate Ru(III)-cyt c(III) has been generated by pulse-radioanalysis with use of four different radicals, CO 2 -., (CH 3 )COH., (CH 2 OH) 3 CCHOH, and -OCCH(OH)C(OH)CO 2 -. The rate of intramolecular electron transfer within the Ru(III)-cyt c(III) complex and its temperature dependence were determined over a 40 0 C temperature range with the CO 2 -. radical. At 25 0 C, these values are k/sub u/=53 +/- s/sup -1/ (pH 7.01 M phosphate buffer, 0.1 M NaHCO 2 ), ΔH/sup +/=3.5 +/- 0.2 kcal mol/sup -1/, and ΔS/sup +/=-39 +/- 1 eu

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.

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

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.

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

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

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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.

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

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

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

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.

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

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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.

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

Science.gov (United States)

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.

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

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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.

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 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.

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.

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

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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.

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

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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.

Sex-dependent alteration of cardiac cytochrome P450 gene expression by doxorubicin in C57Bl/6 mice.

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Grant, Marianne K O; Seelig, Davis M; Sharkey, Leslie C; Zordoky, Beshay N

2017-01-01

There is inconclusive evidence about the role of sex as a risk factor for doxorubicin (DOX)-induced cardiotoxicity. Recent experimental studies have shown that adult female rats are protected against DOX-induced cardiotoxicity. However, the mechanisms of this sexual dimorphism are not fully elucidated. We have previously demonstrated that DOX alters the expression of several cytochrome P450 (CYP) enzymes in the hearts of male rats. Nevertheless, the sex-dependent effect of DOX on the expression of CYP enzymes is still not known. Therefore, in the present study, we determined the effect of acute DOX exposure on the expression of CYP genes in the hearts of both male and female C57Bl/6 mice. Acute DOX cardiotoxicity was induced by a single intraperitoneal injection of 20 mg/kg DOX in male and female adult C57Bl/6 mice. Cardiac function was assessed 5 days after DOX exposure by trans-thoracic echocardiography. Mice were euthanized 1 day or 6 days after DOX or saline injection. Thereafter, the hearts were harvested and weighed. Heart sections were evaluated for pathological lesions. Total RNA was extracted and expression of natriuretic peptides, inflammatory and apoptotic markers, and CYP genes was measured by real-time PCR. Adult female C57Bl/6 mice were protected from acute DOX-induced cardiotoxicity as they show milder pathological lesions, less inflammation, and faster recovery from DOX-induced apoptosis and DOX-mediated inhibition of beta-type natriuretic peptide. Acute DOX exposure altered the gene expression of multiple CYP genes in a sex-dependent manner. In 24 h, DOX exposure caused male-specific induction of Cyp1b1 and female-specific induction of Cyp2c29 and Cyp2e1. Acute DOX exposure causes sex-dependent alteration of cardiac CYP gene expression. Since cardiac CYP enzymes metabolize several endogenous compounds to biologically active metabolites, sex-dependent alteration of CYP genes may play a role in the sexual dimorphism of acute DOX

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.

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

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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.

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)

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens.

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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.

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

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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

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

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....

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...

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.

Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans

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Harry R Beller

2013-08-01

Full Text Available Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV and Fe(II oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II oxidation, namely (a whole-genome transcriptional studies [using FeCO3, Fe2+, and U(IV oxides as electron donors under denitrifying conditions], (b Fe(II oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c random transposon-mutagenesis studies with screening for Fe(II oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II oxidation. The non-defective mutants included the c1-cytochrome subunit of the cytochrome bc1 complex (complex III, which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II oxidation and to current concepts of reverse electron transfer. A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV oxidation, nor have other c-type cytochromes yet been implicated in the process.

Early Events, Kinetic Intermediates and the Mechanism of Protein Folding in Cytochrome c

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David S. Kliger

2009-04-01

Full Text Available Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t £ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-ms conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 ms timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.

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

Science.gov (United States)

Soto, Iliana C; Barrientos, Antoni

2016-02-20

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

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

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...

Stability enhancement of cytochrome c through heme deprotonation and mutations.

Science.gov (United States)

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

2009-01-01

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

Unexpected dependence on pH of NO release from Paracoccus pantotrophus cytochrome cd1

International Nuclear Information System (INIS)

Sam, Katharine A.; Tolland, John D.; Fairhurst, Shirley A.; Higham, Christopher W.; Lowe, David J.; Thorneley, Roger N.F.; Allen, James W.A.; Ferguson, Stuart J.

2008-01-01

A previous study of nitrite reduction by Paracoccus pantotrophus cytochrome cd 1 at pH 7.0 identified early reaction intermediates. The c-heme rapidly oxidised and nitrite was reduced to NO at the d 1 -heme. A slower equilibration of electrons followed, forming a stable complex assigned as 55% cFe(III)d 1 Fe(II)-NO and 45% cFe(II)d 1 Fe(II)-NO + . No catalytically competent NO release was observed. Here we show that at pH 6.0, a significant proportion of the enzyme undergoes turnover and releases NO. An early intermediate, which was previously overlooked, is also identified; enzyme immediately following product release is a candidate. However, even at pH 6.0 a considerable fraction of the enzyme remains bound to NO so another component is required for full product release. The kinetically stable product formed at the end of the reaction differs significantly at pH 6.0 and 7.0, as does its rate of formation; thus the reaction is critically dependent on pH

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.

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'.

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.

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.

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

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

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.

Chiral Selectivity in Inter-reactant Recognition and Electron Transfer of the Oxidation of Horse Heart Cytochrome c by Trioxalatocobaltate(III)

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Nazmutdinov, Renat R.; Bronshtein, Michael D.; Zinkicheva, Tamara T.

2016-01-01

We have studied electron transfer between cytochrome c and the chiral transition-metal complex pair Λ- and Δ-[Co(Ox)3]3− (Ox2− = oxalate) via strong ion-pair formation. Chirality was found in both ion-pair formation and electron transfer, with the Λ enantiomer the more strongly bound and faster r...... reacting. Investigations of the chirality using electron-transfer theory combined with quantum-chemical and statistical-mechanical calculations showed that chirality is solely in inter-reactant interaction and electronic overlap.......We have studied electron transfer between cytochrome c and the chiral transition-metal complex pair Λ- and Δ-[Co(Ox)3]3− (Ox2− = oxalate) via strong ion-pair formation. Chirality was found in both ion-pair formation and electron transfer, with the Λ enantiomer the more strongly bound and faster...

MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression.

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Jonathan J Wilksch

2011-08-01

Full Text Available Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae

Transcriptome Profiling of Tomato Uncovers an Involvement of Cytochrome P450s and Peroxidases in Stigma Color Formation

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Yan Zhang

2017-05-01

Full Text Available Stigma is a crucial structure of female reproductive organ in plants. Stigma color is usually regarded as an important trait in variety identification in some species, but the molecular mechanism of stigma color formation remains elusive. Here, we characterized a tomato mutant, yellow stigma (ys, that shows yellow rather than typical green color in the stigma. Analysis of pigment contents revealed that the level of flavonoid naringenin chalcone was increased in the ys stigma, possibly as a result of higher accumulation of p-coumaric acid, suggesting that naringenin chalcone might play a vital role in yellow color control in tomato stigma. To understand the genes and gene networks that regulate tomato stigma color, RNA-sequencing (RNA-Seq analyses were performed to compare the transcriptomes of stigmas between ys mutant and wild-type (WT. We obtained 507 differentially expressed genes, in which, 84 and 423 genes were significantly up-regulated and down-regulated in the ys mutant, respectively. Two cytochrome P450 genes, SlC3H1 and SlC3H2 which encode p-coumarate 3-hydroxylases, and six peroxidase genes were identified to be dramatically inhibited in the yellow stigma. Further bioinformatic and biochemical analyses implied that the repression of the two SlC3Hs and six PODs may indirectly lead to higher naringenin chalcone level through inhibiting lignin biosynthesis, thereby contributing to yellow coloration in tomato stigma. Thus, our data suggest that two SlC3Hs and six PODs are involved in yellow stigma formation. This study provides valuable information for dissecting the molecular mechanism of stigma color control in tomato.Statement: This study reveals that two cytochrome P450s (SlC3H1 and SlC3H2 and six peroxidases potentially regulate the yellow stigma formation by indirectly enhancing biosynthesis of yellow-colored naringenin chalcone in the stigma of tomato.

Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.

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Yuki Ishii

Full Text Available Knockout serum replacement (KOSR is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

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

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.

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

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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.

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.

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.

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

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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.

Therapeutic assessment of cytochrome C for the prevention of obesity through endothelial cell-targeted nanoparticulate system.

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Hossen, Md Nazir; Kajimoto, Kazuaki; Akita, Hidetaka; Hyodo, Mamoru; Ishitsuka, Taichi; Harashima, Hideyoshi

2013-03-01

Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t1/2 (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.

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

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.

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

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"....

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

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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.

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

NARCIS (Netherlands)

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

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

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.

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.

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

Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

Science.gov (United States)

Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

2001-06-01

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

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.

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

Science.gov (United States)

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.

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.

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.

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.

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

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

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

Science.gov (United States)

Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor

2009-02-01

PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers

Directory of Open Access Journals (Sweden)

Feifel Sven C

2011-12-01

Full Text Available Abstract Background For bioanalytical systems sensitivity and biomolecule activity are critical issues. The immobilization of proteins into multilayer systems by the layer-by-layer deposition has become one of the favorite methods with this respect. Moreover, the combination of nanoparticles with biomolecules on electrodes is a matter of particular interest since several examples with high activities and direct electron transfer have been found. Our study describes the investigation on silica nanoparticles and the redox protein cytochrome c for the construction of electro-active multilayer architectures, and the electron transfer within such systems. The novelty of this work is the construction of such artificial architectures with a non-conducting building block. Furthermore a detailed study of the size influence of silica nanoparticles is performed with regard to formation and electrochemical behavior of these systems. Results We report on interprotein electron transfer (IET reaction cascades of cytochrome c (cyt c immobilized by the use of modified silica nanoparticles (SiNPs to act as an artificial matrix. The layer-by-layer deposition technique has been used for the formation of silica particles/cytochrome c multilayer assemblies on electrodes. The silica particles are characterized by dynamic light scattering (DLS, Fourier transformed infrared spectroscopy (FT-IR, Zeta-potential and transmission electron microscopy (TEM. The modified particles have been studied with respect to act as an artificial network for cytochrome c and to allow efficient interprotein electron transfer reactions. We demonstrate that it is possible to form electro-active assemblies with these non-conducting particles. The electrochemical response is increasing linearly with the number of layers deposited, reaching a cyt c surface concentration of about 80 pmol/cm2 with a 5 layer architecture. The interprotein electron transfer through the layer system and the

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

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.

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

International Nuclear Information System (INIS)

Peschek, G.A.; Wastyn, M.; Trnka, M.; Molitor, V.; Fry, I.V.; Packer, L.

1989-01-01

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [ 35 S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min -1 (mg of protein) -1 , respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa 3 -type enzyme from the properties of its redox-active and EDTA-resistant Cu 2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa 3 -type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa 3 -type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme

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.

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

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.

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.

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...

Ultrafast hydrogen exchange reveals specific structural events during the initial stages of folding of cytochrome c.

Science.gov (United States)

Fazelinia, Hossein; Xu, Ming; Cheng, Hong; Roder, Heinrich

2014-01-15

Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.

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.

The Membrane Modulates Internal Proton Transfer in Cytochrome c Oxidase

DEFF Research Database (Denmark)

Öjemyr, Linda Nasvik; Ballmoos, Christoph von; Faxén, Kristina

2012-01-01

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O-2 reduction...... DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.......-glycerol) (DOPG). In addition, a small Change in the internal Cu-A-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic...

Stability enhancement of cytochrome c through heme deprotonation and mutations

OpenAIRE

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

2009-01-01

The chemical denaturation of Pseudomonas aeruginosa cytochrome c551 variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0 – 4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond form...

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.

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.

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.

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.

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

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

Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

Science.gov (United States)

Uno, Tomohide; Nakano, Ryosuke; Kanamaru, Kengo; Takenaka, Shinji; Uno, Yuichi; Imaishi, Hiromasa

2017-11-01

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids. Copyright © 2017 John Wiley & Sons, Ltd.

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

Toxin formation by Clostridium botulinum type B in radurized fish

International Nuclear Information System (INIS)

Suhadi, F.; Thayib, S.S.

1981-01-01

The relation between maximum storage life and earliest toxin formation by proteolytic and nonproteolytic strains of C. botulinum type B in irradiated and unirradiated raw fish was determinated. The fish species used were Rastrelliger sp., Euthynnus sp. and Scomberomorus sp. Uninoculated fish samples held under the same treatment conditions were evaluated for the estimation of storage life by untrained panelist. The results showed that a storage temperature at or lower than 5.6 0 C is recommended in order to avoid botulism hazard caused by nonproteolytic type B. When the samples were inoculated with spores of proteolytic strains, no toxic samples were found during the storage life in all treatments with storage temperatures at or lower than 10.2 0 C. Toxin formation by proteolytic strains of C. botulinum type B in boiled (''Pindang'') chub mackerel (Rastrelliger sp.) under storage at ambient temperatures (27-31 0 C) was also determinated. The results showed that in the samples which were inoculated before the process of ''Pindang'', the earliest toxin formations were detected after the samples were spoiled regardless of the irradiation dose, strain and inoculum level; while in control unsalted samples, toxin was detected before or after the samples were spoiled, depending on the strain and inoculum level. Salt content in ordinary ''Pindang'' fish plays a major role both in extension of the storage life and the delay in toxin formation. When the samples were inoculated after the process of ''Pindang'', toxin was detected before or after the samples were spoiled, depending on the strain, salt content, irradiation dose and inoculum level. Irradiation does not prevent the toxin formation in ''Pindang'' fish if the samples are heavily contaminated with proteolytic strains of C. botulinum type B after cooking. (author)

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.

Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

Science.gov (United States)

Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

2018-01-01

The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

In situ detection of microbial c-type cytochrome based on intrinsic peroxidase-like activity using screen-printed carbon electrode.

Science.gov (United States)

Wen, Junlin; He, Daigui; Yu, Zhen; Zhou, Shungui

2018-08-15

C-type cytochromes (c-cyts) facilitate microbial extracellular electron transfer and play critical roles in biogeochemical cycling, bioelectricity generation and bioremediation. In this study, a simple and effective method has been developed to detect microbial c-cyts by means of peroxidase mimetic reaction on screen-printed carbon electrode (SPCE). To this end, bacteria cells were immobilized onto the working electrode surface of SPCE by a simple drop casting. After introducing 3,3',5,5'-tetramethylbenzidine (TMB) solution, microbial c-cyts with peroxidase-like activity catalyze the oxidation of TMB in the presence of hydrogen peroxide. The oxidized TMB was electrochemically determined and the current signal was employed to calculate the c-cyts content. This electrochemical method is highly sensitive for microbial c-cyts with a low detection limit of 40.78 fmol and a wide detection range between 51.70 fmol and 6.64 pmol. Moreover, the proposed technique can be universally expanded to detect c-cyts in other bacteria species such as Fontibacter ferrireducens, Pseudomonas aeruginosa, Comamonas guangdongensis and Escherichia coli. Furthermore, the proposed method confers an in situ facile and quantitative c-cyts detection without any destructive sample preparations, complex electrode modifications and expensive enzyme- or metal particle- based signal amplification. The suggested method advances an intelligent strategy for in situ quantification of microbial c-cyts and consequently holds promising application potential in microbiology and environmental science. Copyright © 2018 Elsevier B.V. All rights reserved.

The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7).

Science.gov (United States)

Charizanis, C; Juhnke, H; Krems, B; Entian, K D

1999-10-01

In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.

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

SMU.940 regulates dextran-dependent aggregation and biofilm formation in Streptococcus mutans.

Science.gov (United States)

Senpuku, Hidenobu; Yonezawa, Hideo; Yoneda, Saori; Suzuki, Itaru; Nagasawa, Ryo; Narisawa, Naoki

2018-02-01

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence-stimulating peptide. Eight competence-stimulating peptide-dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran-dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild-type. GbpC is known to be involved in the dextran-dependent aggregation of S. mutans. An SMU.940-gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran-dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran-dependent aggregation and biofilm formation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In Situ Spectral Kinetics of Cr(VI) Reduction by c-Type Cytochromes in A Suspension of Living Shewanella putrefaciens 200

Science.gov (United States)

Liu, Tongxu; Li, Xiaomin; Li, Fangbai; Han, Rui; Wu, Yundang; Yuan, Xiu; Wang, Ying

2016-07-01

Although c-type cytochromes (c-Cyts) mediating metal reduction have been mainly investigated with in vitro purified proteins of dissimilatory metal reducing bacteria, the in vivo behavior of c-Cyts is still unclear given the difficulty in measuring the proteins of intact cells. Here, c-Cyts in living Shewanella putrefaciens 200 (SP200) was successfully quantified using diffuse-transmission UV/Vis spectroscopy due to the strong absorbance of hemes, and the in situ spectral kinetics of Cr(VI) reduction by c-Cyts were examined over time. The reduced product Cr(III) observed on the cell surface may play a role in inhibiting the Cr(VI) reduction and reducing the cell numbers with high concentrations (>200 μM) of Cr(VI) evidenced by the 16S rRNA analysis. A brief kinetic model was established with two predominant reactions, redox transformation of c-Cyts and Cr(VI) reduction by reduced c-Cyts, but the fitting curves were not well-matched with c-Cyts data. The Cr(III)-induced inhibitory effect to the cellular function of redox transformation of c-Cyts was then added to the model, resulting in substantially improved the model fitting. This study provides a case of directly examining the reaction properties of outer-membrane enzyme during microbial metal reduction processes under physiological conditions.

Menadione enhances oxyradical formation in earthworm extracts: vulnerability of earthworms to quinone toxicity

International Nuclear Information System (INIS)

Osman, A.M.; Besten, P.J. den; Noort, P.C.M. van

2003-01-01

NAD(P)H-cytochrome c reductase activities have been determined in the earthworms, L. rubellus and A. chlorotica, extracts. Menadione (0.35 mM, maximum concentration tested) was found to stimulate the rates of NADPH- and NADH-dependent cytochrome c reduction by three- and twofold, respectively. Superoxide dismutase (SOD) inhibited completely this menadione-mediated stimulation, suggesting that ·O 2 - is involved in the redox cycling of menadione. However, SOD had no effect on the basal activity (activity in the absence of quinone) in the case of NADH-dependent cytochrome c reduction, whereas it partially inhibited the basal activity of NADPH-cytochrome c reduction. This indicates direct electron transfer in the former case and the formation of superoxide anion in the latter. DT-diaphorase, measured as the dicumarol-inhibitable part of menadione reductase activity, was not detectable in the earthworms' extracts. In contrast, it was found that DT-diaphorase represents about 70% of the menadione reductase activities in the freshwater mussel, Dreissena polymorpha. The results of this work suggest that earthworms, compared with mussels, could be more vulnerable to oxidative stress from quinones due to lack, or very low level of DT-diaphorase, an enzyme considered to play a significant role in the detoxification of quinones. On the contrary, mussels have efficient DT-diaphorase, which catalyzes two-electron reduction of menadione directly to hydroquinone, thus circumventing the formation of semiquinone

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

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

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

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

One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.

Science.gov (United States)

Boerma, Jan Simon; Vermeulen, Nico P E; Commandeur, Jan N M

2014-01-25

Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2'-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway.

Science.gov (United States)

Alecu, Irina; Othman, Alaa; Penno, Anke; Saied, Essa M; Arenz, Christoph; von Eckardstein, Arnold; Hornemann, Thorsten

2017-01-01

The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic β-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

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

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner.

Science.gov (United States)

Mukherjee, Madhumati; Chaudhari, Snehal N; Balachandran, Riju S; Vagasi, Alexandra S; Kipreos, Edward T

2017-12-15

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

A flavonoid isolated from Streptomyces sp. (ERINLG-4) induces apoptosis in human lung cancer A549 cells through p53 and cytochrome c release caspase dependant pathway.

Science.gov (United States)

Balachandran, C; Sangeetha, B; Duraipandiyan, V; Raj, M Karunai; Ignacimuthu, S; Al-Dhabi, N A; Balakrishna, K; Parthasarathy, K; Arulmozhi, N M; Arasu, M Valan

2014-12-05

The aim of this study was to investigate the anticancer activity of a flavonoid type of compound isolated from soil derived filamentous bacterium Streptomyces sp. (ERINLG-4) and to explore the molecular mechanisms of action. Cytotoxic properties of ethyl acetate extract was carried out against A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxic properties of isolated compound were investigated in A549 lung cancer cell line, COLO320DM cancer cell line and Vero cells. The compound showed potent cytotoxic properties against A549 lung cancer cell line and moderate cytotoxic properties against COLO320DM cancer cell line. Isolated compound showed no toxicity up to 2000 μg/mL in Vero cells. So we have chosen the A549 lung cancer cell line for further anticancer studies. Intracellular visualization was done by using a laser scanning confocal microscope. Apoptosis was measured using DNA fragmentation technique. Treatment of the A549 cancer cells with isolated compound significantly reduced cell proliferation, increased formation of fragmented DNA and apoptotic body. Activation of caspase-9 and caspase-3 indicated that compound may be inducing intrinsic and extrinsic apoptosis pathways. Bcl-2, p53, pro-caspases, caspase-3, caspase-9 and cytochrome c release were detected by western blotting analysis after compound treatment (123 and 164 μM). The activities of pro-caspases-3, caspase-9 cleaved to caspase-3 and caspase-9 gradually increased after the addition of isolated compound. But Bcl-2 protein was down regulated after treatment with isolated compound. Molecular docking studies showed that the compound bound stably to the active sites of caspase-3 and caspase-9. These results strongly suggest that the isolated compound induces apoptosis in A549 cancer cells via caspase activation through cytochrome c release from mitochondria. The present results might provide helpful suggestions for the design of

Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements

International Nuclear Information System (INIS)

Sakamoto, Koichi; Kamiya, Masakatsu; Uchida, Takeshi; Kawano, Keiichi; Ishimori, Koichiro

2010-01-01

Research highlights: → The dynamic parameters for the backbone dynamics in Cyt c were determined. → The backbone mobility of Cyt c is highly restricted due to the covalently bound heme. → The backbone mobility of Cyt c is more restricted upon the oxidation of the heme. → The redox-dependent dynamics are shown in the backbone of Cyt c. → The backbone dynamics of Cyt c would regulate the electron transfer from Cyt c. -- Abstract: Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D 15 N NMR relaxation experiments. 15 N T 1 and T 2 values and 1 H- 15 N NOEs of uniformly 15 N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S 2 ), the effective correlation time for internal motion (τ e ), the 15 N exchange broadening contributions (R ex ) for each residue, and the overall correlation time (τ m ) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S 2 value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S 2 values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.

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.

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.

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...

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.

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...

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.

Relative Propensities of Cytochrome c Oxidase and Cobalt Corrins for Reaction with Cyanide and Oxygen: Implications for Amelioration of Cyanide Toxicity.

Science.gov (United States)

Yuan, Quan; Pearce, Linda L; Peterson, Jim

2017-12-18

In aqueous media at neutral pH, the binding of two cyanide molecules per cobinamide can be described by two formation constants, K f1 = 1.1 (±0.6) × 10 5 M -1 and K f2 = 8.5 (±0.1) × 10 4 M -1 , or an overall cyanide binding constant of ∼1 × 10 10 M -2 . In comparison, the cyanide binding constants for cobalamin and a fully oxidized form of cytochrome c oxidase, each binding a single cyanide anion, were found to be 7.9 (±0.5) × 10 4 M -1 and 1.6 (±0.2) × 10 7 M -1 , respectively. An examination of the cyanide-binding properties of cobinamide at neutral pH by stopped-flow spectrophotometry revealed two kinetic phases, rapid and slow, with apparent second-order rate constants of 3.2 (±0.5) × 10 3 M -1 s -1 and 45 (±1) M -1 s -1 , respectively. Under the same conditions, cobalamin exhibited a single slow cyanide-binding kinetic phase with a second-order rate constant of 35 (±1) M -1 s -1 . All three of these processes are significantly slower than the rate at which cyanide is bound by complex IV during enzyme turnover (>10 6 M -1 s -1 ). Overall, it can be understood from these findings why cobinamide is a measurably better cyanide scavenger than cobalamin, but it is unclear how either cobalt corrin can be antidotal toward cyanide intoxication as neither compound, by itself, appears able to out-compete cytochrome c oxidase for available cyanide. Furthermore, it has also been possible to unequivocally show in head-to-head comparison assays that the enzyme does indeed have greater affinity for cyanide than both cobalamin and cobinamide. A plausible resolution of the paradox that both cobalamin and cobinamide clearly are antidotal toward cyanide intoxication, involving the endogenous auxiliary agent nitric oxide, is suggested. Additionally, the catalytic consumption of oxygen by the cobalt corrins is demonstrated and, in the case of cobinamide, the involvement of cytochrome c when present. Particularly in the case of cobinamide, these oxygen-dependent

Menadione enhances oxyradical formation in earthworm extracts: vulnerability of earthworms to quinone toxicity

Energy Technology Data Exchange (ETDEWEB)

Osman, A.M.; Besten, P.J. den; Noort, P.C.M. van

2003-10-08

NAD(P)H-cytochrome c reductase activities have been determined in the earthworms, L. rubellus and A. chlorotica, extracts. Menadione (0.35 mM, maximum concentration tested) was found to stimulate the rates of NADPH- and NADH-dependent cytochrome c reduction by three- and twofold, respectively. Superoxide dismutase (SOD) inhibited completely this menadione-mediated stimulation, suggesting that {center_dot}O{sub 2}{sup -} is involved in the redox cycling of menadione. However, SOD had no effect on the basal activity (activity in the absence of quinone) in the case of NADH-dependent cytochrome c reduction, whereas it partially inhibited the basal activity of NADPH-cytochrome c reduction. This indicates direct electron transfer in the former case and the formation of superoxide anion in the latter. DT-diaphorase, measured as the dicumarol-inhibitable part of menadione reductase activity, was not detectable in the earthworms' extracts. In contrast, it was found that DT-diaphorase represents about 70% of the menadione reductase activities in the freshwater mussel, Dreissena polymorpha. The results of this work suggest that earthworms, compared with mussels, could be more vulnerable to oxidative stress from quinones due to lack, or very low level of DT-diaphorase, an enzyme considered to play a significant role in the detoxification of quinones. On the contrary, mussels have efficient DT-diaphorase, which catalyzes two-electron reduction of menadione directly to hydroquinone, thus circumventing the formation of semiquinone.

Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

International Nuclear Information System (INIS)

Nall, B.T.; Zuniga, E.H.

1990-01-01

Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5- 13 C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degree C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in sloe exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters. Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c. For oxidized iso-2, five resonances are observed at high temperatures, suggesting flip rates for all five tyrosines sufficient to average static chemical shift differences. At lower temperatures, there is evidence of intermediate and slow flipping for some of the rings

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

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

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...

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.

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...

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

Science.gov (United States)

Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

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

pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

Takeda, Kouta [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan); Matsumura, Hirotoshi; Ishida, Takuya [Department of Biomaterial Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Yoshida, Makoto [Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509 (Japan); Igarashi, Kiyohiko; Samejima, Masahiro [Department of Biomaterial Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Ohno, Hiroyuki [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan); Nakamura, Nobuhumi, E-mail: nobu1@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan)

2016-08-26

A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of L-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters. - Highlights: • pH dependencies of activity were different for the reduction of cyt c and DCPIP. • DET-based bioelectrocatalysis of CcPDH was observed. • The similar pH-dependent profile was found with cyt c and electrode. • The present results suggested that IET reaction of CcPDH shows pH dependence.

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.

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).

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

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.

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.

Controlled adsorption of cytochrome c to nanostructured gold surfaces

International Nuclear Information System (INIS)

Gomes, Inês; Feio, Maria J.; Santos, Nuno C.; Eaton, Peter; Serro, Ana Paula; Saramago, Benilde; Pereira, Eulália; Franco, Ricardo

2012-01-01

Controlled electrostatic physisorption of horse heart cytochrome c (Cyt c) onto nanostructured gold surfaces was investigated using Quartz-Crystal Microbalance measurements in planar gold surfaces with or without functionalization using a self-assembled monolayer (SAM) of the alkanethiol mercaptoundecanoic acid (MUA). MUA is a useful functionalization ligand for gold surfaces, shedding adsorbed biomolecules from the excessive electron density of the metal. A parallel analysis was conducted in the corresponding curved surfaces of 15 nm gold nanoparticles (AuNPs), using zeta-potential and UV– visible spectroscopy. Atomic Force Microscopy of both types of functionalized gold surfaces with a MUA SAM, allowed for visualization of Cyt c deposits on the nanostructured gold surface. The amount of Cyt c adsorbed onto the gold surface could be controlled by the solution pH. For the assays conducted at pH 4.5, when MUA SAM- functionalized planar gold surfaces are positive or neutral, and Cyt c has a positive net charge, only 13 % of the planar gold surface area was coated with protein. In contrast, at pH 7.4, when MUA SAM-functionalized planar gold surfaces and Cyt c have opposite charges, a protein coverage of 28 % could be observed implying an adsorption process strongly governed by electrostatic forces. Cyt c adsorption on planar and curved gold surfaces are found to be greatly favored by the presence of a MUA-capping layer. In particular, on the AuNPs, the binding constant is three times larger than the binding constant obtained for the original citrate-capped AuNPs.

Controlled adsorption of cytochrome c to nanostructured gold surfaces

Energy Technology Data Exchange (ETDEWEB)

Gomes, Ines [Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, REQUIMTE, Departamento de Quimica (Portugal); Feio, Maria J. [Faculdade de Ciencias da Universidade do Porto, REQUIMTE, Departamento de Quimica e Bioquimica (Portugal); Santos, Nuno C. [Faculdade de Medicina da Universidade de Lisboa, Instituto de Medicina Molecular (Portugal); Eaton, Peter [Faculdade de Ciencias da Universidade do Porto, REQUIMTE, Departamento de Quimica e Bioquimica (Portugal); Serro, Ana Paula; Saramago, Benilde [Centro de Quimica Estrutural, Instituto Superior Tecnico (Portugal); Pereira, Eulalia [Faculdade de Ciencias da Universidade do Porto, REQUIMTE, Departamento de Quimica e Bioquimica (Portugal); Franco, Ricardo, E-mail: ricardo.franco@fct.unl.pt [Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, REQUIMTE, Departamento de Quimica (Portugal)

2012-12-15

Controlled electrostatic physisorption of horse heart cytochrome c (Cyt c) onto nanostructured gold surfaces was investigated using Quartz-Crystal Microbalance measurements in planar gold surfaces with or without functionalization using a self-assembled monolayer (SAM) of the alkanethiol mercaptoundecanoic acid (MUA). MUA is a useful functionalization ligand for gold surfaces, shedding adsorbed biomolecules from the excessive electron density of the metal. A parallel analysis was conducted in the corresponding curved surfaces of 15 nm gold nanoparticles (AuNPs), using zeta-potential and UV- visible spectroscopy. Atomic Force Microscopy of both types of functionalized gold surfaces with a MUA SAM, allowed for visualization of Cyt c deposits on the nanostructured gold surface. The amount of Cyt c adsorbed onto the gold surface could be controlled by the solution pH. For the assays conducted at pH 4.5, when MUA SAM- functionalized planar gold surfaces are positive or neutral, and Cyt c has a positive net charge, only 13 % of the planar gold surface area was coated with protein. In contrast, at pH 7.4, when MUA SAM-functionalized planar gold surfaces and Cyt c have opposite charges, a protein coverage of 28 % could be observed implying an adsorption process strongly governed by electrostatic forces. Cyt c adsorption on planar and curved gold surfaces are found to be greatly favored by the presence of a MUA-capping layer. In particular, on the AuNPs, the binding constant is three times larger than the binding constant obtained for the original citrate-capped AuNPs.

Formation of M23C6-type precipitates and chromium-depleted zones in austenite stainless steel

International Nuclear Information System (INIS)

Kaneko, Kenji; Fukunaga, Tatsuya; Yamada, Kazuhiro; Nakada, Nobuo; Kikuchi, Masao; Saghi, Zineb; Barnard, Jon S.; Midgley, Paul A.

2011-01-01

Graphical abstract: Precipitate formation during the in situ annealing experiment at 650 o C. -- Formation of M 23 C 6 carbides and chromium-depleted zones in commercially available type 304L stainless steel were investigated by in situ transmission electron microscopy and analytical transmission electron microscopy. It was found that each individual small M 23 C 6 carbide starts to grow with a clear orientation relationship with the matrix, and film-like carbide was subsequently observed at the interfaces with asymmetric Cr-depleted zones. From these experimental results, a model describing the precipitation of M 23 C 6 and the formation of the Cr-depleted zone was proposed.

Multifunctional Cytochrome c: Learning New Tricks from an Old Dog.

Science.gov (United States)

Alvarez-Paggi, Damián; Hannibal, Luciana; Castro, María A; Oviedo-Rouco, Santiago; Demicheli, Veronica; Tórtora, Veronica; Tomasina, Florencia; Radi, Rafael; Murgida, Daniel H

2017-11-08

Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein.

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.

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

Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

OpenAIRE

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 compari...

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

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.

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

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.

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

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.

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.

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...

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

Polyethylene glycol promotes autoxidation of cytochrome c.

Science.gov (United States)

Sato, Wataru; Uchida, Takeshi; Saio, Tomohide; Ishimori, Koichiro

2018-06-01

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Tissue- and Condition-Specific Isoforms of Mammalian Cytochrome c Oxidase Subunits: From Function to Human Disease

Directory of Open Access Journals (Sweden)

Christopher A. Sinkler

2017-01-01

Full Text Available Cytochrome c oxidase (COX is the terminal enzyme of the electron transport chain and catalyzes the transfer of electrons from cytochrome c to oxygen. COX consists of 14 subunits, three and eleven encoded, respectively, by the mitochondrial and nuclear DNA. Tissue- and condition-specific isoforms have only been reported for COX but not for the other oxidative phosphorylation complexes, suggesting a fundamental requirement to fine-tune and regulate the essentially irreversible reaction catalyzed by COX. This article briefly discusses the assembly of COX in mammals and then reviews the functions of the six nuclear-encoded COX subunits that are expressed as isoforms in specialized tissues including those of the liver, heart and skeletal muscle, lung, and testes: COX IV-1, COX IV-2, NDUFA4, NDUFA4L2, COX VIaL, COX VIaH, COX VIb-1, COX VIb-2, COX VIIaH, COX VIIaL, COX VIIaR, COX VIIIH/L, and COX VIII-3. We propose a model in which the isoforms mediate the interconnected regulation of COX by (1 adjusting basal enzyme activity to mitochondrial capacity of a given tissue; (2 allosteric regulation to adjust energy production to need; (3 altering proton pumping efficiency under certain conditions, contributing to thermogenesis; (4 providing a platform for tissue-specific signaling; (5 stabilizing the COX dimer; and (6 modulating supercomplex formation.

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

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

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

Science.gov (United States)

Meyer, Thomas; Melin, Frédéric; Richter, Oliver-M H; Ludwig, Bernd; Kannt, Aimo; Müller, Hanne; Michel, Hartmut; Hellwig, Petra

2015-02-27

Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The antibiotic tiamulin is a potent inducer and inhibitor of cytochrome P4503A via the formation of a stable metabolic intermediate complex. Studies in primary hepatocyte cultures and liver microsomes of the pig.

Science.gov (United States)

Witkamp, R F; Nijmeijer, S M; Monshouwer, M; Van Miert, A S

1995-05-01

Tiamulin is a semisynthetic antibiotic frequently used in agricultural animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds that are simultaneously administered. To explain this, it has been suggested that tiamulin selectively inhibits oxidative drug metabolism via the formation of a cytochrome P450 metabolic intermediate complex. The aim of the present study was to provide further support for this hypothesis. When hepatic microsomes and cultured primary pig hepatocytes were incubated with tiamulin, a maximum in the absorbance spectrum at 455 nm was observed, which disappeared after adding KFe(CN)6. When hepatocytes were incubated with tiamulin for 72 hr, cytochrome P450 content and cytochrome P4503A apoprotein levels were increased. Tiamulin strongly inhibited and concentration dependently inhibited the hydroxylation rate of testosterone at the 6 beta-position in both microsomes and hepatocytes, and the microsomal N-demethylation rate of ethylmorphine. Other testosterone hydroxylations were inhibited to a lesser extent or not affected. The relative inhibition of the hydroxylation of testosterone at the 6 beta-position was more pronounced in microsomes from rifampicin- and triacetyloleandomycin-treated pigs. The results indicate that cytochrome P450 complex formation can at least partly explain the interactions observed with tiamulin. Tiamulin seems to be a strong, probably selective, inhibitor of the cytochrome P4503A subfamily and an interesting tool for further research.

Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

Science.gov (United States)

Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Di Rocco, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco

2009-10-15

The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

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

Interaction of rocuronium with human liver cytochromes P450.

Science.gov (United States)

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

2015-02-01

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 microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

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 ...

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...

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy.

Science.gov (United States)

Hallmann, Kerstin; Kudin, Alexei P; Zsurka, Gábor; Kornblum, Cornelia; Reimann, Jens; Stüve, Burkhard; Waltz, Stephan; Hattingen, Elke; Thiele, Holger; Nürnberg, Peter; Rüb, Cornelia; Voos, Wolfgang; Kopatz, Jens; Neumann, Harald; Kunz, Wolfram S

2016-02-01

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release

Directory of Open Access Journals (Sweden)

Zhang YueMei

2005-02-01

Full Text Available Abstract Background Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic, and Bcl-2 (anti-apoptotic genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. Results In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or

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

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

Science.gov (United States)

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.

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.

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.

Conservative treatment of bone tissue metabolic disorders among patients with vitamin D-dependent rickets type II with genetic abnormality of type I collagen formation

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S.M. Martsyniak

2017-08-01

Full Text Available Background. The purpose of the article is to determine the effect of conservative therapy on genetically caused disorders of bone tissue metabolism in patients with vitamin D-dependent rickets type II and genetic abnormality of type I collagen formation (VDDR(COL1. Materials and methods. At the premises of consulting and outpatient department of SI “Institute of Traumatology and Orthopaedics of the NAMS of Ukraine”, 13 patients having VDDR type II and genetic damage of type I collagen formation were examined and treated. The medical treatment was conducted in four stages. The first stage included full examination of patients (calcium and phosphorus levels in the blood serum and their urinary excretion, as well as determination of calcidiol and calcitriol serum levels, indicators of parathyroid hormone and osteocalcin, and a marker of bone formation P1NP and osteoresorption b-CTx. At this stage, children were obligated to undergo a genetic test to detect changes (polymorphism in alleles of receptors to vitamin D and type I collagen. Besides genetic tests, examinations at the other stages were conducted in full. Results. The study has shown the following. The genetically caused abnormality of reception to vitamin D results into substantial accumulation of vitamin D active metabolite in the blood serum. When combined with gene­tic abnormality of type I collagen formation, it significantly affected bone formation and destruction processes that causes development of osteomalacia (parathormone — vitamin D — osteocalcin system. The comprehensive study of vitamin D metabolism and biochemical vitals of bone tissue in patients having VDDR (COL1 brought us to understanding of some issues related to pathogenesis and nature of osteomalacia and, in future, osteoporotic changes on different levels, ensured us to express these changes by corresponding indices in the biochemical research and, finally, to develop appropriate schemes for the treatment of

In vitro complex formation and inhibition of hepatic cytochrome P450 activity by different macrolides and tiamulin in goats and cattle

NARCIS (Netherlands)

Zweers-Zeilmaker, W.M.; Miert, A.S.J.P.A.M. van; Horbach, G.J.; Witkamp, R.F.

1998-01-01

In humans, clinically relevant drug–drug interactions occur with some macrolide antibiotics via the formation of stable metabolic intermediate (MI) complexes with enzymes of the cytochrome P4503A (CYP3A) subfamily. The formation of such complexes can result in a decreased biotransformation rate of

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

Sulfite oxidase activity of cytochrome c: Role of hydrogen peroxide

Directory of Open Access Journals (Sweden)

Murugesan Velayutham

2016-03-01

Full Text Available In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+cyt c in the absence and presence of H2O2. Electron paramagnetic resonance (EPR spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO were performed with sulfite, Fe3+cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3- was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different.

Science.gov (United States)

Zaidi, Sobia; Haque, Md Anzarul; Ubaid-Ullah, Shah; Prakash, Amresh; Hassan, Md Imtaiyaz; Islam, Asimul; Batra, Janendra K; Ahmad, Faizan

2017-05-01

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

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.

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

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors.

Science.gov (United States)

Guan, Xiaoqian; Chen, Shuai; Voon, Chia Pao; Wong, Kam-Bo; Tikkanen, Mikko; Lim, Boon L

2018-01-01

Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP + oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

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...

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.

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

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.

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

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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.

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

Directory of Open Access Journals (Sweden)

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

In situ monitoring of stacking fault formation and its carrier lifetime mediation in p-type 4H-SiC

Energy Technology Data Exchange (ETDEWEB)

Chen, Bin, E-mail: chenbinmse@gmail.com; Chen, Jun; Yao, Yuanzhao; Sekiguchi, Takashi [National Institute for Materials Science, Tsukuba, Ibaraki 305-0044 (Japan); Matsuhata, Hirofumi; Okumura, Hajime [National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568 (Japan)

2014-07-28

Using the fine control of an electron beam (e-beam) in scanning electron microscopy with the capabilities of both electrical and optical imaging, the stacking fault (SF) formation together with its tuning of carrier lifetime was in situ monitored and investigated in p-type 4H-SiC homoepitaxial films. The SFs were formed through engineering basal plane dislocations with the energy supplied by the e-beam. The e-beam intensity required for the SF formation in the p-type films was ∼100 times higher than that in the n-type ones. The SFs reduced the minority-carrier lifetime in the p-type films, which was opposite to that observed in the n-type case. The reason for the peculiar SF behavior in the p-type 4H-SiC is discussed with the cathodoluminescence results.

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.

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.

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.

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.

Graphene-cyclodextrin-cytochrome c layered assembly with improved electron transfer rate and high supramolecular recognition capability.

Science.gov (United States)

Gong, Cheng-Bin; Guo, Cong-Cong; Jiang, Dan; Tang, Qian; Liu, Chang-Hua; Ma, Xue-Bing

2014-06-01

This study aimed to develop a new graphene-based layered assembly, named graphene-cyclodextrin-cytochrome c with improved electron transfer rate. This assembly has combined high conductivity of graphene nanosheets (GNs), selectively binding properties and electronegativity of cyclodextrins (CDs), as well as electropositivity of cytochrome c (Cyt c). This assembly can also mimic the confined environments of the intermembrane space of mitochondria. A β-cyclodextrin (β-CD) functionalized GN (GN-CD) assembly was initially prepared by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide with hydrazine hydrate in the presence of β-CD. Cyt c was then intercalated to the GN-CD assembly to form a layered self-assembled structure, GN-CD-Cyt c, through electrostatic interaction. Compared with GNs and GN-CD, GN-CD-Cyt c assembly displayed improved electron transfer rate and high supramolecular recognition capability toward six probe molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

Relationship between CNS metabolism and cytoarchitecture: a review of 14C-deoxyglucose studies with correlation to cytochrome oxidase histochemistry

International Nuclear Information System (INIS)

Di Rocco, R.J.; Kageyama, G.H.; Wong-Riley, M.T.

1989-01-01

Since the inception of the 14 C-deoxyglucose method and its extension to in vivo imaging of regional cerebral glucose metabolism in humans by positron emission tomography, uncertainty has persisted concerning the type of work to which regional metabolism is coupled, as well as the distribution of this work within the neuron. 14 C-deoxyglucose studies indicate that functionally-coupled neural metabolism is more apparent in axon terminals and perhaps dendrites than neuronal perikarya. Moreover, it appears that most of the metabolism in axon terminals is accounted for by Na+-K+-ATPase activity. Nevertheless, cytochrome oxidase histochemistry reveals the presence of intensely reactive mitochondria in soma-dendrite regions opposite presynaptic axon terminals, thereby indicating that continuous temporal and spatial summation of postsynaptic graded potentials is associated with increased metabolism. While the situation concerning the relative postsynaptic metabolic prices of EPSP's and IPSP's remains uncertain, the presence of elevated levels of cytochrome oxidase activity within certain classes of presynaptic terminals indicates that active excitation and inhibition is associated with increases in presynaptic metabolism. This observation has been confirmed in 14 C-deoxyglucose studies. Nevertheless, studies of neonatal hippocampus indicate that, before metabolic activity shifts to dendritic and telodendritic regions of electrophysiological activity, metabolism is high in somal foci of biosynthesis. 51 references

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

Temperature-dependent thermal and thermoelectric properties of n -type and p -type S c1 -xM gxN

Science.gov (United States)

Saha, Bivas; Perez-Taborda, Jaime Andres; Bahk, Je-Hyeong; Koh, Yee Rui; Shakouri, Ali; Martin-Gonzalez, Marisol; Sands, Timothy D.

2018-02-01

Scandium Nitride (ScN) is an emerging rocksalt semiconductor with octahedral coordination and an indirect bandgap. ScN has attracted significant attention in recent years for its potential thermoelectric applications, as a component material in epitaxial metal/semiconductor superlattices, and as a substrate for defect-free GaN growth. Sputter-deposited ScN thin films are highly degenerate n -type semiconductors and exhibit a large thermoelectric power factor of ˜3.5 ×10-3W /m -K2 at 600-800 K. Since practical thermoelectric devices require both n- and p-type materials with high thermoelectric figures-of-merit, development and demonstration of highly efficient p-type ScN is extremely important. Recently, the authors have demonstrated p-type S c1 -xM gxN thin film alloys with low M gxNy mole-fractions within the ScN matrix. In this article, we demonstrate temperature dependent thermal and thermoelectric transport properties, including large thermoelectric power factors in both n- and p-type S c1 -xM gxN thin film alloys at high temperatures (up to 850 K). Employing a combination of temperature-dependent Seebeck coefficient, electrical conductivity, and thermal conductivity measurements, as well as detailed Boltzmann transport-based modeling analyses of the transport properties, we demonstrate that p-type S c1 -xM gxN thin film alloys exhibit a maximum thermoelectric power factor of ˜0.8 ×10-3W /m -K2 at 850 K. The thermoelectric properties are tunable by adjusting the M gxNy mole-fraction inside the ScN matrix, thereby shifting the Fermi energy in the alloy films from inside the conduction band in case of undoped n -type ScN to inside the valence band in highly hole-doped p -type S c1 -xM gxN thin film alloys. The thermal conductivities of both the n- and p-type films were found to be undesirably large for thermoelectric applications. Thus, future work should address strategies to reduce the thermal conductivity of S c1 -xM gxN thin-film alloys, without affecting

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.

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...

Densities and Kinematic Viscosities for the Systems Benzene + Methyl Formate, Benzene + Ethyl Formate, Benzene + Propyl Formate, and Benzene + Butyl Formate

DEFF Research Database (Denmark)

Emmerling, Uwe; Rasmussen, Peter

1998-01-01

a Redlich-Kister type of expression with temperature-independent parameters and the data for the systems benzene + ethyl formate, benzene + propyl formate, and benzene + butyl formate with temperature-dependent parameters. The viscosities have furthermore been compared to values predicted by means of the GC......Densities and kinematic viscosities have been measured for the system benzene + methyl formate at 20°C and for the systems benzene + ethyl formate, benzene + propyl formate, and benzene + butyl formate from 20°C to 50°C. The results for the system benzene + methyl formate have been correlated using...

Visualizing changes in electron distribution in coupled chains of cytochrome bc(1) by modifying barrier for electron transfer between the FeS cluster and heme c(1).

Science.gov (United States)

Cieluch, Ewelina; Pietryga, Krzysztof; Sarewicz, Marcin; Osyczka, Artur

2010-02-01

Cytochrome c(1) of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of cytochrome bc(1) by modifying heme c(1) redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of cytochrome c(1) which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme c(1) back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme c(1) and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated cytochrome bc(1) revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of cytochrome bc(1) exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites

Design-dependent gauge factors of highly doped n-type 4H-SiC piezoresistors

International Nuclear Information System (INIS)

Akiyama, T; Briand, D; De Rooij, N F

2012-01-01

This paper presents the experimentally obtained gauge factor (GF) of 4H-SiC piezoresistors, fabricated out of the n-type epitaxial layer and characterized on millimeter-size SiC cantilever beams at room temperature. It was found that the GF is dependent on the piezoresistor's length and width. Piezoresistors narrower than approximately 30 µm showed a width-dependent GF. The highest GF of 20.8 was obtained with a single element piezoresistor in transverse orientation. In longitudinal orientation, the highest GF was −10, which was obtained with a clustered piezoresistor with plural identical elements. Essential factors to consider for the design of optimum 4H-SiC piezoresistors for a Wheatstone bridge configuration are presented. (paper)

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

International Nuclear Information System (INIS)

Ahluwalia, Unnati; Deep, Shashank

2012-01-01

Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

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

Characterization of cytochrome c as marker for retinal cell degeneration by uv/vis spectroscopic imaging

Science.gov (United States)

Hollmach, Julia; Schweizer, Julia; Steiner, Gerald; Knels, Lilla; Funk, Richard H. W.; Thalheim, Silko; Koch, Edmund

2011-07-01

Retinal diseases like age-related macular degeneration have become an important cause of visual loss depending on increasing life expectancy and lifestyle habits. Due to the fact that no satisfying treatment exists, early diagnosis and prevention are the only possibilities to stop the degeneration. The protein cytochrome c (cyt c) is a suitable marker for degeneration processes and apoptosis because it is a part of the respiratory chain and involved in the apoptotic pathway. The determination of the local distribution and oxidative state of cyt c in living cells allows the characterization of cell degeneration processes. Since cyt c exhibits characteristic absorption bands between 400 and 650 nm wavelength, uv/vis in situ spectroscopic imaging was used for its characterization in retinal ganglion cells. The large amount of data, consisting of spatial and spectral information, was processed by multivariate data analysis. The challenge consists in the identification of the molecular information of cyt c. Baseline correction, principle component analysis (PCA) and cluster analysis (CA) were performed in order to identify cyt c within the spectral dataset. The combination of PCA and CA reveals cyt c and its oxidative state. The results demonstrate that uv/vis spectroscopic imaging in conjunction with sophisticated multivariate methods is a suitable tool to characterize cyt c under in situ conditions.

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.

Role of Met80 and Tyr67 in the low-pH conformational equilibria of cytochrome c.

Science.gov (United States)

Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Bellei, Marzia; Di Rocco, Giulia; Salewski, Johannes; Hildebrandt, Peter; Sola, Marco

2012-07-31

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.

Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

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

2012-03-15

Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, {Delta}{chi}. For most nuclei, the PCSs back-calculated from the {Delta}{chi} tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches-clustered around G41, N52, and A81-exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

International Nuclear Information System (INIS)

Volkov, Alexander N.; Vanwetswinkel, Sophie; Van de Water, Karen; Nuland, Nico A. J. van

2012-01-01

Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches—clustered around G41, N52, and A81—exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

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)

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

Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

Science.gov (United States)

Dumoulin, Alexandre; Ter-Avetisyan, Gohar; Schmidt, Hannes; Rathjen, Fritz G

2018-04-24

Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade

Directory of Open Access Journals (Sweden)

Alexandre Dumoulin

2018-04-01

Full Text Available Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP, the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα. In the absence of any one of these components, neurons in dorsal root ganglia (DRG and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

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

Identification of rabbit cytochromes P450 2C1 and 2C2 as arachidonic acid epoxygenases.

Science.gov (United States)

Laethem, R M; Koop, D R

1992-12-01

Microsomes prepared from COS-1 cells transiently expressing rabbit cytochromes P450 2C1 and 2C2 catalyzed the metabolism of arachidonic acid to predominantly 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) when microsomal epoxide hydrolase activity was inhibited by 0.2 mM 1,2-epoxy-3,3,3-trichloropropane. P450 2C2 catalyzed the formation of 11,12-EET and 14,15-EET at a ratio of 3.0 and also produced 19-hydroxyeicosatetraenoic acid (19-HETE). The 11,12-EET, 14,15-EET, and 19-HETE represented 48.3, 15.9, and 12.8%, respectively, of the total metabolites formed. P450 2C1 produced a similar but distinct ratio of 11,12-EET to 14,15-EET (2.0) and did not produce any detectable 19-HETE. The 11,12-EET and 14,15-EET represented 63.0 and 31.1%, respectively, of the total metabolites formed. The 8,9- and 5,6-EETs were not detected with either enzyme. The ratio of the 11,12-EET to 14,15-EET was 1.5 with P450 2CAA, a P450 arachidonic acid epoxygenase (P450 2CAA) that had an amino-terminal sequence identical to that of P450 2C2 [J. Biol. Chem. 267:5552-5559 (1992)]. P450 2C1, 2C2, and 2CAA metabolized lauric acid. The ratio of omega-1- to omega-hydroxylated laurate was 3.6, 3.4, and 2.4 for P450 2CAA, P450 2C2, and P450 2C1, respectively. Purified P450 2CAA had a slightly greater apparent molecular weight than expressed P450 2C2 on sodium dodecyl sulfate-polyacrylamide gels. The results clearly establish that rabbit P450 2C1 and 2C2 are arachidonic acid epoxygenases, and they suggest that P450 2CAA and 2C2 are very similar but may not be identical isoforms.

Synthesis of cytochrome c oxidase 1 (SCO1) inhibits insulin sensitivity by decreasing copper levels in adipocytes.

Science.gov (United States)

Wei, Xiang-Bo; Guo, Liang; Liu, Yang; Zhou, Shui-Rong; Liu, Yuan; Dou, Xin; Du, Shao-Yue; Ding, Meng; Peng, Wan-Qiu; Qian, Shu-Wen; Huang, Hai-Yan; Tang, Qi-Qun

2017-09-23

Dysregulation of insulin signaling leads to type 2 diabetes mellitus (T2DM) and other metabolic disorders. Obesity is an important contributor to insulin resistance, and although the understanding of this relationship has improved in recent years, the mechanism of obesity-induced insulin resistance is not completely understood. Disorders of copper metabolism tend to accompany the development of obesity, which increases the risk of insulin resistance. Synthesis of cytochrome c oxidase 1 (SCO1) functions in the assembly of cytochrome c oxidase (COX) and cellular copper homeostasis. However, the role of SCO1 in the regulation of metabolism remains unknown. Here, we found that obese mice had higher expression of SCO1 and lower levels of copper in white adipose tissue (WAT) than did the control mice. Overexpression of SCO1 in adipocytes was associated with copper deficiency. Copper increased insulin sensitivity by decreasing the level of phosphatase and tensin homolog (PTEN) protein. Ectopic expression of SCO1 led to insulin resistance and was accompanied by a decrease in intracellular copper level, and addition of copper abolished the inhibitory effect of SCO1 on insulin sensitivity. Our results demonstrated a novel role of SCO1 in modulating insulin sensitivity via the regulation of copper concentration in WAT and suggested a potential therapeutic target for T2DM. Copyright © 2017. Published by Elsevier Inc.

Cytochrome C Dynamics at Gold and Glassy Carbon Surfaces Monitored by in Situ Scanning Tunnel Microscopy

DEFF Research Database (Denmark)

Andersen, Jens Enevold Thaulov; Møller, Per; Pedersen, Marianne Vind

1995-01-01

We have investigated the absorption of cytochrome c on gold and glassy carbon substrates by in situ scanning tunnel microscopy under potentiostatic control of both substrate and tip. Low ionic strength and potential ranges where no Faradaic current flows were used. Cyt c aggregates into flat...

Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

Directory of Open Access Journals (Sweden)

Carlo Travaglini-Allocatelli

2013-01-01

Full Text Available Cytochromes c (Cyt c are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i heme translocation and delivery, (ii apoCyt thioreductive pathway, and (iii apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria.

Graphene–cyclodextrin–cytochrome c layered assembly with improved electron transfer rate and high supramolecular recognition capability

Energy Technology Data Exchange (ETDEWEB)

Gong, Cheng-Bin; Guo, Cong-Cong; Jiang, Dan; Tang, Qian, E-mail: qiantang@swu.edu.cn; Liu, Chang-Hua; Ma, Xue-Bing

2014-06-01

This study aimed to develop a new graphene-based layered assembly, named graphene–cyclodextrin–cytochrome c with improved electron transfer rate. This assembly has combined high conductivity of graphene nanosheets (GNs), selectively binding properties and electronegativity of cyclodextrins (CDs), as well as electropositivity of cytochrome c (Cyt c). This assembly can also mimic the confined environments of the intermembrane space of mitochondria. A β-cyclodextrin (β-CD) functionalized GN (GN–CD) assembly was initially prepared by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide with hydrazine hydrate in the presence of β-CD. Cyt c was then intercalated to the GN–CD assembly to form a layered self-assembled structure, GN–CD–Cyt c, through electrostatic interaction. Compared with GNs and GN–CD, GN–CD–Cyt c assembly displayed improved electron transfer rate and high supramolecular recognition capability toward six probe molecules. - Highlights: • A new tertiary layered assembly named GN–CD–Cyt c was prepared. • Compared with GNs and GN–CD, GN–CD–Cyt c shows improved electron transfer rate. • GN–CD–Cyt c displays high supramolecular recognition capability.

Graphene–cyclodextrin–cytochrome c layered assembly with improved electron transfer rate and high supramolecular recognition capability

International Nuclear Information System (INIS)

Gong, Cheng-Bin; Guo, Cong-Cong; Jiang, Dan; Tang, Qian; Liu, Chang-Hua; Ma, Xue-Bing

2014-01-01

This study aimed to develop a new graphene-based layered assembly, named graphene–cyclodextrin–cytochrome c with improved electron transfer rate. This assembly has combined high conductivity of graphene nanosheets (GNs), selectively binding properties and electronegativity of cyclodextrins (CDs), as well as electropositivity of cytochrome c (Cyt c). This assembly can also mimic the confined environments of the intermembrane space of mitochondria. A β-cyclodextrin (β-CD) functionalized GN (GN–CD) assembly was initially prepared by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide with hydrazine hydrate in the presence of β-CD. Cyt c was then intercalated to the GN–CD assembly to form a layered self-assembled structure, GN–CD–Cyt c, through electrostatic interaction. Compared with GNs and GN–CD, GN–CD–Cyt c assembly displayed improved electron transfer rate and high supramolecular recognition capability toward six probe molecules. - Highlights: • A new tertiary layered assembly named GN–CD–Cyt c was prepared. • Compared with GNs and GN–CD, GN–CD–Cyt c shows improved electron transfer rate. • GN–CD–Cyt c displays high supramolecular recognition capability

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)

Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation

DEFF Research Database (Denmark)

Schroll, C.; Barken, Kim Bundvig; Krogfelt, K.A.

2010-01-01

nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation. Results: Isogenic fimbriae mutants of the clinical K. pneumoniae...... of planktonic cells. Type 1 fimbriae did not influence biofilm formation and the expression of type 1 fimbriae was found to be down-regulated in biofilm forming cells. In contrast, expression of type 3 fimbriae was found to strongly promote biofilm formation. Conclusion: By use of well defined isogenic mutants...... we found that type 3 fimbriae, but not type 1 fimbriae, strongly promote biofilm formation in K. pneumoniae C3091. As the vast majority of clinical K. pneumoniae isolates express type 3 fimbriae, this fimbrial adhesin may play a significant role in development of catheter associated K. pneumoniae...

Titration Behavior of Residues at the Entrance of the D-Pathway of Cytochrome c Oxidase from Paracoccus denitrificans Investigated by Continuum Electrostatic Calculations

International Nuclear Information System (INIS)

Olkhova, Elena; Helms, Volkhard H.; Michel, Hartmut

2005-01-01

Continuum electrostatic calculations were employed to investigate the titration curves of the fully oxidized state of wild type and several variants of cytochrome c oxidase from Paracoccus denitrificans (N131D, N131C, N131V, and D124N) for different values of the dielectric constant of the protein. The effects of the mutations at the entrance of the D-proton transfer pathway were found to be quite localized to their immediate surroundings. The results can be well interpreted in the light of the available biochemical and structural data and help understanding the effects of mutations on proton conductivity. The mutations of aspartic acid Asp-I-124 to a neutral residue resulted in a decreased pKa value of His-I-28 suggesting that the mutation of His-I-28 may have a significant influence on the coupling of electron and proton transfer in cytochrome c oxidase. We also investigated the effect of the mutations N131D, N131C, and N131V on the residue Glu-I-278 in terms of its pKa value and electrostatic interaction energies

Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation

OpenAIRE

Jobson, Richard W.; Nielsen, Rasmus; Laakkonen, Liisa; Wikström, Mårten; Albert, Victor A.

2004-01-01

Much recent attention in the study of adaptation of organismal form has centered on developmental regulation. As such, the highly conserved respiratory machinery of eukaryotic cells might seem an unlikely target for selection supporting novel morphologies. We demonstrate that a dramatic molecular evolutionary rate increase in subunit I of cytochrome c oxidase (COX) from an active-trapping lineage of carnivorous plants is caused by positive Darwinian selection. Bladderworts (Utricularia) trap ...

Formation mechanism of SiC in C-Si system by ion irradiation

International Nuclear Information System (INIS)

Hishita, Shunichi; Aizawa, Takashi; Suehara, Shigeru; Haneda, Hajime

2003-01-01

The irradiation effects of 2 MeV He + , Ne + , and Ar + ions on the film structure of the C-Si system were investigated with RHEED and XPS. The ion dose dependence of the SiC formation was kinetically analyzed. The SiC formation at moderate temperature was achieved by 2 MeV ion irradiation when the thickness of the initial carbon films was appropriate. The evolution process of the SiC film thickness consisted of the 3 stages. The first stage was the steep increase of the SiC, and was governed by the inelastic collision. The second was the gentle increase of the SiC, and was governed by the diffusion. The last was the decrease of the SiC, and was caused by the sputtering. The formation mechanism of the SiC was discussed. (author)

Cox1 mutation abrogates need for Cox23 in cytochrome c oxidase biogenesis

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Richard Dela Cruz

2016-06-01

Full Text Available Cox23 is a known conserved assembly factor for cytochrome c oxidase, although its role in cytochrome c oxidase (CcO biogenesis remains unresolved. To gain additional insights into its role, we isolated spontaneous suppressors of the respiratory growth defect in cox23∆ yeast cells. We recovered independent colonies that propagated on glycerol/lactate medium for cox23∆ cells at 37°C. We mapped these mutations to the mitochondrial genome and specifically to COX1 yielding an I101F substitution. The I101F Cox1 allele is a gain-of-function mutation enabling yeast to respire in the absence of Cox23. CcO subunit steady-state levels were restored with the I101F Cox1 suppressor mutation and oxygen consumption and CcO activity were likewise restored. Cells harboring the mitochondrial genome encoding I101F Cox1 were used to delete genes for other CcO assembly factors to test the specificity of the Cox1 mutation as a suppressor of cox23∆ cells. The Cox1 mutant allele fails to support respiratory growth in yeast lacking Cox17, Cox19, Coa1, Coa2, Cox14 or Shy1, demonstrating its specific suppressor activity for cox23∆ cells.

Calcium transport in vesicles energized by cytochrome oxidase

Energy Technology Data Exchange (ETDEWEB)

Rosier, Randy N. [Univ. of Rochester, NY (United States)

1979-01-01

Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K⁺ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K⁺ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

Precipitation of cytochromes c with Na2CdI4

International Nuclear Information System (INIS)

Zhuravleva, D.V.; Kulish, M.A.; Mironov, A.F.

1996-01-01

Using cytochrome c from horse heart and the yeast Candida valida as examples, it was shown that a complex anion, cadmium tetraiodide (CdI 4 2- ), precipitated proteins from aqueous solutions at the reagent concentrations below 50 mM. The composition and pH value of the solution, as well as the starting protein concentration, considerably influenced the precipitation. The results suggest that this reagent acts on the protein by a mechanism similar to the salting-out process. The ability to act at small concentrations is the advantage of CdI 4 2- over conventional agents

Geometrical analysis of cytochrome c unfolding

Science.gov (United States)

Urie, Kristopher G.; Pletneva, Ekaterina; Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

2011-01-01

A geometrical model has been developed to study the unfolding of iso-1 cytochrome c. The model draws on the crystallographic data reported for this protein. These data were used to calculate the distance between specific residues in the folded state, and in a sequence of extended states defined by n = 3, 5, 7, 9, 11, 13, and 15 residue units. Exact calculations carried out for each of the 103 residues in the polypeptide chain demonstrate that different regions of the chain have different unfolding histories. Regions where there is a persistence of compact structures can be identified, and this geometrical characterization is fully consistent with analyses of time-resolved fluorescence energy-transfer (TrFET) data using dansyl-derivatized cysteine side-chain probes at positions 39, 50, 66, 85, and 99. The calculations were carried out assuming that different regions of the polypeptide chain unfold synchronously. To test this assumption, lattice Monte Carlo simulations were performed to study systematically the possible importance of asynchronicity. Calculations show that small departures from synchronous dynamics can arise if displacements of residues in the main body of the chain are much more sluggish than near-terminal residues.

A new BODIPY/nanoparticle/Ni affinity system for binding of cytochrome c

Energy Technology Data Exchange (ETDEWEB)

Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Kursunlu, Ahmed Nuri [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Arslan, Gulsin [Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey)

2015-09-15

Highlights: • BODIPY was synthesized, and then attached to magnetic nanoparticles. • Ni(II) ions were chelated on prepared material. • The binding of cytochrome c to obtained material was studied. - Abstract: In this study, 3,5-{Bis[4,4-difluoro, 8-(2,6-diethyl, 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)]}benzoylchloride (BODIPY) was synthesized for the improving of a new immobilized metal affinity supporting material. Firstly, the synthesized BODIPY was immobilized on iron oxide superparamagnetic nanoparticles (SPIONs) and then, Ni(II) ions were chelated with the active terminals of BODIPY on nanoparticles surfaces to prepare an immobilized metal affinity (IMA) adsorbent for protein adsorption. The amount of BODIPY coated on SPIONs was about 29.7 μM at 10 mg nanoparticles. 738 μmol of Ni(II) ions were loaded to 10 mg of the SPIONs/BODIPY. The binding amount of cytochrome c was found to be 170 μg to the SPIONs/BODIPY/Ni at pH 7.4. The binding amount of the molecules on SPIONs was analyzed by using UV–vis, fluorescence and atomic absorption spectroscopy. The characterization of the prepared surfaces was performed by FT-IR, SEM and TEM.

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c

NARCIS (Netherlands)

Monari, S.; Millo, D.; Ranieri, A.; di Rocco, G.; van der Zwan, G.; Gooijer, C.; Peressini, S.; Tavagnacco, C.; Hildebrandt, P.; Borsari, M.

2010-01-01

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements,

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

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.

A novel bi-protein bio-interphase of cytochrome c and glucose oxidase: Electron transfer and electrocatalysis

International Nuclear Information System (INIS)

Song, Yonghai; Liu, Hongyu; Wang, Yu; Wang, Li

2013-01-01

Graphical abstract: Glucose oxidase (GOD) and cytochrome c (Cyt c) were co-entrapped in the poly(diallyldimethylammonium chloride)–graphene nanosheets–gold nanoparticles (PDDA–Gp–AuNPs) nanocomposites modified glassy carbon electrode. Electron transfer and electrocatalysis of the novel bi-protein bio-interphase were investigated. The bio-interphase developed here not only successfully achieved DET of GOD, but also showed great potential for the fabrication of novel glucose biosensors with linear response up to 18 mM. Highlights: ► A bio-interphase composed of cytochrome c and glucose oxidase was developed. ► The electron transfer in the bio-interphase was investigated. ► Electrocatalytic performances of bio-interphase were explored. ► The bio-interphase exhibited good electrocatalytic response glucose. - Abstract: Glucose oxidase (GOD) and cytochrome c (Cyt c) were co-entrapped in the poly(diallyldimethylammonium chloride)–graphene nanosheets–gold nanoparticles (PDDA–Gp–AuNPs) hybrid nanocomposites modified glassy carbon electrode to prepare a novel bi-protein bio-interphase. Electron transfer and electrocatalysis of the bi-protein bio-interphase were investigated in detail. The results showed that the PDDA–Gp–AuNPs nanocomposites accelerated the electron transfer between proteins and electrode. The bi-protein exhibited effective direct electron transfer (DET) reaction with an apparent rate constant (k s ) of 2.36 s −1 . The optimal molar ratio and total amount of Cyt c and GOD in the bio-interphase for DET of GOD was estimated to be about 3:1 and 1.40 nmol, respectively. The bi-protein bio-interphase could be used to detect glucose based on the consumption of O 2 with the oxidation of glucose catalyzed by GOD. The resulted biosensor exhibits wide linear range from 2.0 to 18.0 mM. Thus, this study not only successfully achieved DET of GOD, but also constructed a novel biosensor for glucose detection

Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

Science.gov (United States)

Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

2006-01-01

Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

In vitro kinetics of P700+ reduction of Thermosynechococcus elongatus trimeric Photosystem I complexes by recombinant cytochrome c 6 using a Joliot-type LED spectrophotometer.

Science.gov (United States)

Nguyen, Khoa; Vaughn, Michael; Frymier, Paul; Bruce, Barry D

2017-01-01

The reduction rate of photo-oxidized Photosystem I (PSI) with various natural and artificial electron donors have been well studied by transient absorption spectroscopy. The electron transfer rate from various donors to P 700 + has been measured for a wide range of photosynthetic organisms encompassing cyanobacteria, algae, and plants. PSI can be a limiting component due to tedious extraction and purification methods required for this membrane protein. In this report, we have determined the in vivo, intracellular cytochrome c 6 (cyt c 6 )/PSI ratio in Thermosynechococcus elongatus (T.e.) using quantitative Western blot analysis. This information permitted the determination of P 700 + reduction kinetics via recombinant cyt c 6 in a physiologically relevant ratio (cyt c 6 : PSI) with a Joliot-type, LED-driven, pump-probe spectrophotometer. Dilute PSI samples were tested under varying cyt c 6 concentration, temperature, pH, and ionic strength, each of which shows similar trends to the reported literature utilizing much higher PSI concentrations with laser-based spectrophotometer. Our results do however indicate kinetic differences between actinic light sources (laser vs. LED), and we have attempted to resolve these effects by varying our LED light intensity and duration. The standardized configuration of this spectrophotometer will also allow a more uniform kinetic analysis of samples in different laboratories. We can conclude that our findings from the LED-based system display an added total protein concentration effect due to multiple turnover events of P 700 + reduction by cyt c 6 during the longer illumination regime.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Science.gov (United States)

Marroquin-Guzman, Margarita; Wilson, Richard A

2015-04-01

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Directory of Open Access Journals (Sweden)

Margarita Marroquin-Guzman

2015-04-01

Full Text Available Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

Prognostic relevance of cytochrome C oxidase in primary glioblastoma multiforme.

Directory of Open Access Journals (Sweden)

Corinne E Griguer

Full Text Available Patients with primary glioblastoma multiforme (GBM have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%, and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.

A putative novel nuclear-encoded subunit of the cytochrome c oxidase complex in trypanosomatids

Czech Academy of Sciences Publication Activity Database

Maslov, D. A.; Zíková, Alena; Kyselová, Iveta; Lukeš, Julius

2002-01-01

Roč. 125, 1-2 (2002), s. 113-125 ISSN 0166-6851 R&D Projects: GA ČR GA204/00/1212 Grant - others:NIH(US) AI40634 Institutional research plan: CEZ:AV0Z6022909 Keywords : cytochrome c oxidase * mitochondrion * kinetoplast Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.911, year: 2002

Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

Science.gov (United States)

Singh, Upendra Kumar; Patel, Rajan

2018-05-25

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C 8 mim][Cl], and 1-decyl-3-methylimidazolium chloride [C 10 mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C 8 mim] + and [C 10 mim] + cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C 10 mim] + cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C 8 mim] + cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C 10 mim][Cl], and this was correlated with the fast and medium lifetimes (τ 1 and τ 2 ) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C 10 mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C 10 mim] + cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.

Isolation and characterization of cAMP-free and cAMP-bound forms of bovine heart type II cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Cobb, C.E.

1986-01-01

Bovine heart type II cAMP-dependent protein kinase holoenzyme (cAMP-PK) was purified to homogeneity as determined by denaturing SDS-PAGE. An HPLC-DEAE purification step resolved two distinct peaks of cAMP-dependent kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. They had the same Stoke's radii and had very similar sedimentation coefficients. As determined by densitometric scanning of SDS-PAGE brands, by their mobility on denaturing PAGE, and by the ratios of equilibrium [ 3 H] cAMP binding to maximal kinase activity, the subunit stoichiometry of the two peaks was the same. In a cAMP assay it was found that Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Dissociation assays indicated that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP, and Peak 2 could be partially converted to Peak 1 by aging. The interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2

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.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

Science.gov (United States)

Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

2017-01-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

Science.gov (United States)

Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

2017-06-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T 4 /g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation. Copyright © 2017 Elsevier Inc. All rights reserved.

The production of ammonia by multiheme cytochromes C.

Science.gov (United States)

Simon, Jörg; Kroneck, Peter M H

2014-01-01

The global biogeochemical nitrogen cycle is essential for life on Earth. Many of the underlying biotic reactions are catalyzed by a multitude of prokaryotic and eukaryotic life forms whereas others are exclusively carried out by microorganisms. The last century has seen the rise of a dramatic imbalance in the global nitrogen cycle due to human behavior that was mainly caused by the invention of the Haber-Bosch process. Its main product, ammonia, is a chemically reactive and biotically favorable form of bound nitrogen. The anthropogenic supply of reduced nitrogen to the biosphere in the form of ammonia, for example during environmental fertilization, livestock farming, and industrial processes, is mandatory in feeding an increasing world population. In this chapter, environmental ammonia pollution is linked to the activity of microbial metalloenzymes involved in respiratory energy metabolism and bioenergetics. Ammonia-producing multiheme cytochromes c are discussed as paradigm enzymes.

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.

de Toni-Fanconi-Debré syndrome with Leigh syndrome revealing severe muscle cytochrome c oxidase deficiency

NARCIS (Netherlands)

Ogier, H.; Lombes, A.; Scholte, H. R.; Poll-The, B. T.; Fardeau, M.; Alcardi, J.; Vignes, B.; Niaudet, P.; Saudubray, J. M.

1988-01-01

We describe a patient with severe muscle cytochrome c oxidase deficiency who had de Toni-Fanconi-Debré syndrome and acute neurologic deterioration resembling Leigh syndrome, without clear evidence of muscle abnormality. Metabolic investigations revealed elevated cerebrospinal fluid lactate values

Redox reactions of cytochrome c in isolated mitochondria exposed to blue or red lasers using resonance Raman spectroscopy

Science.gov (United States)

Denton, Michael L.; Gonzalez, Cherry C.; Noojin, Gary D.; Yakovlev, Vladislav V.

2018-02-01

Resonance Raman spectroscopy of cytochrome c was used to follow reduction/oxidation (redox) states of isolated mitochondria in response to blue or red laser exposure. Mitochondria were isolated from hTERT-RPE1 cells and were kept in a buffer formulation known to be conducive to electron transport chain (ETC) activity. Using either pyruvate or succinate as substrates for ETC, we found differences in the redox responses of cytochrome c for different exposure laser irradiance and excitation wavelength. We anticipate that the proposed new method will be valuable in the study of metabolic processes in mitochondria in response to low level laser exposure, and thus aid in elucidating the mechanism(s) of photobiomodulation.

Protein alkylation, transcriptional responses and cytochrome c release during acrolein toxicity in A549 cells: influence of nucleophilic culture media constituents.

Science.gov (United States)

Thompson, Colin A; Burcham, Philip C

2008-06-01

Acrolein is a toxic combustion product that elicits apoptotic and/or necrotic cell death depending on the conditions under which exposure occurs. As a strong electrophile, side-reactions with nucleophilic media constituents seem likely to accompany study of its toxicity in vitro, but these reactions are poorly characterized. We have thus examined the effect of media composition on the toxicity of acrolein in A549 cells. Cells were exposed to acrolein in either Dulbecco's buffered saline (DBS) or F12 supplemented with various concentrations of fetal bovine serum. Cell viability was assessed using the MTT assay, while heme oxygenase-1 (HO-1) and cytoplasmic cytochrome c were measured as respective markers of transcriptional response and apoptosis. Protein damage was evaluated using the protein carbonyl assay. Compared to F12 media (with or without serum), maximal cell death as evaluated using the MTT assay, as well as adduction of intracellular proteins, occurred when cells were exposed to acrolein in DBS. In contrast, cytochrome c release was maximal in cells exposed to acrolein in serum-containing F12, conditions which inhibited protein modification and overt cell death. These findings highlight the need for careful attention to experimental conditions when conducting in vitro toxicological studies of reactive substances.

Differences in renal metabolism of insulin and cytochrome c

International Nuclear Information System (INIS)

Herrman, J.; Simmons, R.E.; Frank, B.H.; Rabkin, R.

1988-01-01

Kidneys degrade small proteins such as cytochrome c (CYT c) by the classic lysosomal pathway. However, because alternate routes for the transport and degradation of protein hormones have been identified in other tissues, the authors set out to determine whether extralysosomal sites might participate in the renal degradation of insulin. First, they compared the effect of the lysosomal inhibitor NH 4 Cl on insulin and CYT c degradation by isolated perfused rat kidneys. After kidneys were loaded with radiolabeled proteins to allow for absorption and transport to lysosomes, degradation was measured in the presence or absence of inhibitors. Next they followed the subcellular distribution of 125 I-labeled insulin in kidneys exposed to 125 I-labeled insulin in vivo or when isolated and perfused. Under both circumstances the distribution of insulin on a linear sucrose gradient differed from that of the lysosomal enzyme N-acetyl-β-glucosaminidase. In contrast, [ 14 CH 3 ]CYT c, injected in vivo, distributed over a density similar to the lysosomal marker. Thus important differences exist between the renal metabolism of CYT c, which proceeds in lysosomes, and the renal metabolism of insulin. These include rate of degradation, sensitivity to NH 4 Cl, and subcellular sites of localization. Accordingly, they suggest that insulin degradation may occur, at least in part, in a different compartment from the classic lysosomal site of protein degradation

Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

International Nuclear Information System (INIS)

Rettie, A.E.; Boberg, M.; Rettenmeier, A.W.; Baillie, T.A.

1988-01-01

The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction

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

Science.gov (United States)

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

2009-01-01

The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 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 c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 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 bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

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

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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

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

Science.gov (United States)

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography.

Science.gov (United States)

Ali, Faiz; Cheong, Won Jo

2015-10-01

A silica capillary of 50 μm internal diameter and 500 mm length (416 mm effective length) was chemically modified with 4-(trifluoromethoxy) phenyl isocyanate in the presence of dibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with the terminal halogen of the bound ligand to incorporate the initiator moiety, and in situ polymerization was performed using a monomer mixture of styrene, N-phenylacrylamide, and methacrylic acid. The resultant open tubular capillary column immobilized with the copolymer layer was used for the separation of tryptic digest of cytochrome C in capillary electrochromatography. The sample was well eluted and separated into many components. The elution patterns of tryptic digest of cytochrome C were studied with respect to pH and water content in the mobile phase. This preliminary study demonstrates that open tubular capillary electrochromatography columns with a modified copolymer layer composed of proper nonpolar and polar units fabricated by reversible addition-fragmentation transfer polymerization can be useful as separation media for proteomic analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits

Czech Academy of Sciences Publication Activity Database

Gnipová, Anna; Panicucci, Brian; Paris, Zdeněk; Verner, Zdeněk; Horváth, A.; Lukeš, Julius; Zíková, Alena

2012-01-01

Roč. 184, č. 2 (2012), s. 90-98 ISSN 0166-6851 R&D Projects: GA AV ČR KJB500960901; GA ČR GA204/09/1667 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma * RNA interference * Mitochondrion * Respiratory complexes * Cytochrome c oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112001065#

On the relationship between the snowflake type aloft and the surface precipitation types at temperatures near 0 °C

Science.gov (United States)

Sankaré, Housseyni; Thériault, Julie M.

2016-11-01

Winter precipitation types can have major consequences on power outages, road conditions and air transportation. The type of precipitation reaching the surface depends strongly on the vertical temperature of the atmosphere, which is often composed of a warm layer aloft and a refreezing layer below it. A small variation of the vertical structure can lead to a change in the type of precipitation near the surface. It has been shown in previous studies that the type of precipitation depends also on the precipitation rate, which is directly linked to the particle size distribution and that a difference as low as 0.5 °C in the vertical temperature profile could change the type of precipitation near the surface. Given the importance of better understanding the formation of winter precipitation type, the goal of this study is to assess the impact of the snowflake habit aloft on the type of precipitation reaching the surface when the vertical temperature is near 0 °C. To address this, a one dimensional cloud model coupled with a bulk microphysics scheme was used. Four snowflake types (dendrite, bullet, column and graupel) have been added to the scheme. The production of precipitation at the surface from these types of snow has been compared to available observations. The results showed that the thickness of the snow-rain transition is four times deeper when columns and graupel only fall through the atmosphere compared to dendrites. Furthermore, a temperature of the melting layer that is three (four) times warmer is required to completely melt columns and graupel (dendrites). Finally, the formation of freezing rain is associated with the presence of lower density snowflakes (dendrites) aloft compared to the production of ice pellets (columns). Overall, this study demonstrated that the type of snowflakes has an impact on the type of precipitation reaching the surface when the temperature is near 0 °C.

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

Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo.

Science.gov (United States)

Blöcker, Dagmar; Bachmeyer, Christoph; Benz, Roland; Aktories, Klaus; Barth, Holger

2003-05-13

The binding component (C2II) of the binary Clostridium botulinum C2 toxin mediates transport of the actin ADP-ribosylating enzyme component (C2I) into the cytosol of target cells. C2II (80 kDa) is activated by trypsin cleavage, and proteolytically activated C2II (60 kDa) oligomerizes to heptamers in solution. Activated C2II forms channels in lipid bilayer membranes which are highly cation selective and voltage-gated. A role for this channel in C2I translocation across the cell membrane into the cytosol is discussed. Amino acid residues 303-331 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which likely facilitates membrane insertion and channel formation by creating two antiparallel beta-strands. Some of the residues are in strategic positions within the putative C2II channel, in particular, glutamate 307 (E307) localized in its center and glycine 316 (G316) localized on the trans side of the membrane. Here, single-lysine substitutions of these amino acids and the double mutant E307K/G316K of C2II were analyzed in vivo and in artificial lipid bilayer experiments. The pH dependence of C2I transport across cellular membranes was altered, and a pH of properties of C2II were substantially changed by the mutations, as evidenced by reduced cation selectivity. Interestingly, the voltage dependence of wild-type C2II was completely lost for the E307K mutant, which means that E307 is responsible for voltage gating. Chloroquine blocked the E307K mutant channel and intoxication of Vero cells by mutant C2II and C2I, indicating that chloroquine binding does not involve E307. Overall, the voltage gating and cation selectivity of the C2II channel do not play an important role in translocation of C2I into the cytosol.

Electrochemistry of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17).

Science.gov (United States)

Martin, Lisandra L; Kubeil, Clemens; Simonov, Alexandr N; Kuznetsov, Vladimir L; Corbin, C Jo; Auchus, Richard J; Conley, Alan J; Bond, Alan M; Rodgers, Raymond J

2017-02-05

Within the superfamily of cytochrome P450 enzymes (P450s), there is a small class which is functionally employed for steroid biosynthesis. The enzymes in this class appear to have a small active site to accommodate the steroid substrates specifically and snuggly, prior to the redox transformation or hydroxylation to form a product. Cytochrome P450c17 is one of these and is also a multi-functional P450, with two activities, the first 17α-hydroxylation of pregnenolone is followed by a subsequent 17,20-lyase transformation to dehydroepiandrosterone (DHEA) as the dominant pathways to cortisol precursors or androgens in humans, respectively. How P450c17 regulates these two redox reactions is of special interest. There is a paucity of direct electrochemical studies on steroidogenic P450s, and in this mini-review we provide an overview of these studies with P450c17. Historical consideration as to the difficulties in obtaining reliable electrochemistry due to issues of handling proteins on an electrode, together with advances in the electrochemical techniques are addressed. Recent work using Fourier transformed alternating current voltammetry is highlighted as this technique can provide both catalytic information simultaneously with the underlying redox transfer with the P450 haem. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

Conformational control of the binding of diatomic gases to cytochrome c'.

Science.gov (United States)

Manole, Andreea; Kekilli, Demet; Svistunenko, Dimitri A; Wilson, Michael T; Dobbin, Paul S; Hough, Michael A

2015-06-01

The cytochromes c' (CYTcp) are found in denitrifying, methanotrophic and photosynthetic bacteria. These proteins are able to form stable adducts with CO and NO but not with O2. The binding of NO to CYTcp currently provides the best structural model for the NO activation mechanism of soluble guanylate cyclase. Ligand binding in CYTcps has been shown to be highly dependent on residues in both the proximal and distal heme pockets. Group 1 CYTcps typically have a phenylalanine residue positioned close to the distal face of heme, while for group 2, this residue is typically leucine. We have structurally, spectroscopically and kinetically characterised the CYTcp from Shewanella frigidimarina (SFCP), a protein that has a distal phenylalanine residue and a lysine in the proximal pocket in place of the more common arginine. Each monomer of the SFCP dimer folds as a 4-alpha-helical bundle in a similar manner to CYTcps previously characterised. SFCP exhibits biphasic binding kinetics for both NO and CO as a result of the high level of steric hindrance from the aromatic side chain of residue Phe 16. The binding of distal ligands is thus controlled by the conformation of the phenylalanine ring. Only a proximal 5-coordinate NO adduct, confirmed by structural data, is observed with no detectable hexacoordinate distal NO adduct.

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

Science.gov (United States)

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.

Theoretical analysis of the domain-swapped dimerization of cytochrome c: An MD and 3D-RISM approach

Science.gov (United States)

Yoshida, Norio; Higashi, Masahiro; Motoki, Hideyoshi; Hirota, Shun

2018-01-01

The structural stability of a cytochrome c domain-swapped dimer compared with that of the monomer was investigated by molecular dynamics (MD) simulations and by three-dimensional reference interaction site model (3D-RISM) theory. The structural fluctuation and structural energy of cytochrome c were treated by MD simulations, and the solvation thermodynamics was treated by 3D-RISM theory. The domain-swapped dimer state is slightly less stable than the monomer state, which is consistent with experimental observations; the total free energy difference is calculated as 25 kcal mol-1. The conformational change and translational/rotational entropy change contribute to the destabilization of the dimer, whereas the hydration and vibrational entropy contribute to the stabilization. Further analyses on the residues located at the hinge loop for swapping were conducted, and the results reveal details at the molecular level of the structural and interaction changes upon dimerization.

Essential role of RSK2 in c-Fos-dependent osteosarcoma development.

Science.gov (United States)

David, Jean-Pierre; Mehic, Denis; Bakiri, Latifa; Schilling, Arndt F; Mandic, Vice; Priemel, Matthias; Idarraga, Maria Helena; Reschke, Markus O; Hoffmann, Oskar; Amling, Michael; Wagner, Erwin F

2005-03-01

Inactivation of the growth factor-regulated S6 kinase RSK2 causes Coffin-Lowry syndrome in humans, an X-linked mental retardation condition associated with progressive skeletal abnormalities. Here we show that mice lacking RSK2 develop a progressive skeletal disease, osteopenia due to impaired osteoblast function and normal osteoclast differentiation. The phenotype is associated with decreased expression of Phex, an endopeptidase regulating bone mineralization. This defect is probably not mediated by RSK2-dependent phosphorylation of c-Fos on serine 362 in the C-terminus. However, in the absence of RSK2, c-Fos-dependent osteosarcoma formation is impaired. The lack of c-Fos phosphorylation leads to reduced c-Fos protein levels, which are thought to be responsible for decreased proliferation and increased apoptosis of transformed osteoblasts. Therefore, RSK2-dependent stabilization of c-Fos is essential for osteosarcoma formation in mice and may also be important for human osteosarcomas.

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

Cloning and tissue expression of cytochrome P450 1B1 and 1C1 ...

African Journals Online (AJOL)

Cytochrome P450 1 (CYP1) is widely used as an indicator of exposure to environmental contaminants. In the study, two full-length complementary DNAs encode for CYP1B1 and CYP1C1 were cloned from medaka liver exposed to 500 ppb β-naphthoflavone for 24 h. CYP1B1, having 1984 bp, contains an open reading ...

Identification of a cytochrome P4502E1/Bid/C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice.

Science.gov (United States)

Sebastian, Becky M; Roychowdhury, Sanjoy; Tang, Hui; Hillian, Antoinette D; Feldstein, Ariel E; Stahl, Gregory L; Takahashi, Kazue; Nagy, Laura E

2011-10-14

Chronic, heavy alcohol exposure results in inflammation in adipose tissue, insulin resistance, and liver injury. Here we have identified a CYP2E1/Bid/C1q-dependent pathway that is activated in response to chronic ethanol and is required for the development of inflammation in adipose tissue. Ethanol feeding for 25 days to wild-type (C57BL/6J) mice increased expression of multiple markers of adipose tissue inflammation relative to pair-fed controls independent of increased body weight or adipocyte size. Ethanol feeding increased the expression of CYP2E1 in adipocytes, but not stromal vascular cells, in adipose tissue and Cyp2e1(-/-) mice were protected from adipose tissue inflammation in response to ethanol. Ethanol feeding also increased the number of TUNEL-positive nuclei in adipose tissue of wild-type mice but not in Cyp2e1(-/-) or Bid (-/-) mice. Apoptosis contributed to adipose inflammation, as the expression of multiple inflammatory markers was decreased in mice lacking the Bid-dependent apoptotic pathway. The complement protein C1q binds to apoptotic cells, facilitating their clearance and activating complement. Making use of C1q-deficient mice, we found that activation of complement via C1q provided the critical link between CYP2E1/Bid-dependent apoptosis and onset of adipose tissue inflammation in response to chronic ethanol. In summary, chronic ethanol increases CYP2E1 activity in adipose, leading to Bid-mediated apoptosis and activation of complement via C1q, finally resulting in adipose tissue inflammation. Taken together, these data identify a novel mechanism for the development of adipose tissue inflammation that likely contributes to the pathophysiological effects of ethanol.

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.

Cytochrome c oxidase inhibition by calcium at physiological ionic composition of the medium: Implications for physiological significance of the effect.

Science.gov (United States)

Vygodina, Tatiana V; Mukhaleva, Elizaveta; Azarkina, Natalia V; Konstantinov, Alexander A

2017-12-01

Cytochrome c oxidase (CcO) from mammalian mitochondria binds Ca 2+ and Na + in a special cation binding site. Binding of Ca 2+ brings about partial inhibition of the enzyme while Na + competes with Ca 2+ for the binding site and protects the enzyme from the inhibition [Vygodina, T., Kirichenko, A. and Konstantinov, A.A. (2013). Direct Regulation of Cytochrome c oxidase by Calcium Ions. PLoS One 8(9): e74436]. In the original studies, the inhibition was found to depend significantly on the ionic composition of the buffer. Here we describe inhibition of CcO by Ca 2+ in media containing the main ionic components of cytoplasm (150mM KCl, 12mM NaCl and 1mM MgCl 2 ). Under these conditions, Ca 2+ inhibits CcO with effective K i of 20-26μM, that is an order of magnitude higher than determined earlier in the absence of Na + . At physiological value of ionic strength, the inhibition can be observed at any turnover number of CcO, rather than only at low TN (calcium matches closely the known value of "K m " for Ca 2+ -induced activation of the mitochondrial calcium uniporter. The inhibition of CcO by Ca 2+ is proposed to modulate mitochondrial Ca 2+ -uptake via the mitochondrial calcium uniporter, promote permeability transition pore opening and induce reduction of Mia40 in the mitochondrial intermembrane space. Copyright © 2017 Elsevier B.V. All rights reserved.

A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms

Science.gov (United States)

Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros

2015-01-01

Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

A C-type lectin from Bothrops jararacussu venom disrupts Staphylococcal biofilms.

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Raphael Contelli Klein

Full Text Available Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16 showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model.

12(R)-hydroxyicosatetraenoic acid: a cytochrome P450-dependent arachidonate metabolite that inhibits Na+, K+-ATPase in the cornea

International Nuclear Information System (INIS)

Schwartzman, M.L.; Balazy, M.; Masferrer, J.; Abraham, N.G.; McGiff, J.C.; Murphy, R.C.

1987-01-01

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na + , K + -ATPase from the corneal epithelium in a dose-dependent manner. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive- and negative-ionization modes was carried out on derivatized compound C. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from [ 2 H 8 ]arachidonate were retained in the structure. Compound C was characterized as a 12-hydroxyicosatetraenoic acid. However, only 12(R) isomer was found to be an inhibitor of the Na + , K + -ATPase from the corneal epithelium, suggesting that the biologically active compound C was 12(R)-hydroxyy-5,8,10,14-icosatetraenoic acid. Such an inhibitor of Na + , K + -ATPase synthesized in the cornea may have an important role in regulating ocular transparency and aqueous human secretion

Direct electron transfer of Cytochrome c at mono-dispersed and negatively charged perylene-graphene matrix.

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Zhang, Nan; Lv, Xiangyu; Ma, Weiguang; Hu, Yuwei; Li, Fenghua; Han, Dongxue; Niu, Li

2013-03-30

Mono-dispersed 3,4,9,10-perylene tetracarboxylic acid (PTCA) functionalized graphene sheets (PTCA-graphene) were fabricated by a chemical route and dispersed well in aqueous solution. PTCA-graphene with plenty of -COOH groups as electrostatic absorbing sites were beneficial to the loading of Cytochrome c (Cyt c). Cyt c, which was tightly immobilized on the PTCA-graphene modified glassy carbon electrode, maintained its natural conformation. Direct electron transfer of Cyt c and the electro-catalytic activity towards the reduction of H2O2 were also achieved. It has been substantiated that PTCA-graphene is a preferable biocompatible matrix for Cyt c. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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

2014-08-19

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

Influence of cytochrome 2C19 allelic variants on on-treatment platelet reactivity evaluated by five different platelet function tests.

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Gremmel, Thomas; Kopp, Christoph W; Moertl, Deddo; Seidinger, Daniela; Koppensteiner, Renate; Panzer, Simon; Mannhalter, Christine; Steiner, Sabine

2012-05-01

The antiplatelet effect of clopidogrel has been linked to cytochrome P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of function variants were found to have a gene-dose effect on clopidogrel metabolism. However, genotyping is only one aspect of predicting response to clopidogrel and several platelet function tests are available to measure platelet response. Patients and methods We studied the influence of CYP2C19 allelic variants on on-treatment platelet reactivity as assessed by light transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay, multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined with the Infiniti® CYP450 2C19+ assay and categorized into four metabolizer states (ultrarapid, extensive, intermediate, poor). Platelet reactivity increased linearly from ultrarapid to poor metabolizers using the VerifyNow P2Y12 assay (P = 0.04), the VASP assay (P = 0.02) and the Impact-R (P = 0.04). The proportion of patients with high on-treatment residual platelet reactivity (HRPR) identified by LTA, the VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status decreased, while no such relationship could be identified for results of MEA and Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17) was an independent predictor of HRPR in LTA and the VASP assay while it did not reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the Impact-R. Depending on the type of platelet function test differences in the association of on-treatment platelet reactivity with CYP2C19 carrier status are observed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Using an SU-8 Photoresist Structure and Cytochrome C Thin Film Sensing Material for a Microbolometer

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Guo-Dung John Su

2012-11-01

Full Text Available There are two critical parameters for microbolometers: the temperature coefficient of resistance (TCR of the sensing material, and the thermal conductance of the insulation structure. Cytochrome c protein, having a high TCR, is a good candidate for infrared detection. We can use SU-8 photoresist for the thermal insulation structure, given its low thermal conductance. In this study, we designed a platform structure based on a SU-8 photoresist. We fabricated an infrared sensing pixel and recorded a high TCR for this new structure. The SU-8 photoresist insulation structure was fabricated using the exposure dose method. We experimentally demonstrated high values of TCR from 22%/K to 25.7%/K, and the measured noise was 1.2 × 10–8 V2/Hz at 60 Hz. When the bias current was 2 μA, the calculated voltage responsivity was 1.16 × 105 V/W. This study presents a new kind of microbolometer based on cytochrome c protein on top of an SU-8 photoresist platform that does not require expensive vacuum deposition equipment.

Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects

Czech Academy of Sciences Publication Activity Database

Kovářová, Nikola; Pecina, Petr; Nůsková, Hana; Vrbacký, Marek; Zeviani, M.; Mráček, Tomáš; Viscomi, C.; Houštěk, Josef

2016-01-01

Roč. 1862, č. 4 (2016), s. 705-715 ISSN 0925-4439 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MZd(CZ) NT12370 Institutional support: RVO:67985823 Keywords : cytochrome c oxidase * respiratory supercomplexes * leigh syndrome * SURF1−/− mouse knockout * doxycycline * pulse-chase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.476, year: 2016

Designing new catalytic C-C and C-N bond formations promoted by organoactinides

International Nuclear Information System (INIS)

Eisen, M.S.; Straub, T.; Haskel, A.

1998-01-01

Organoactinides of the type Cp 2 * AcMe 2 (Cp * =C 5 Me 5 ; Ac=Th; U) are active catalytic precursors for the oligomerization of terminal alkynes HC≡CR (R=alkyl, aryl, SiMe 3 ). The regioselectivity and the extent of oligomerization depend strongly on the alkyne substituent R, whereas the catalytic reactivity is similar for both organoactinides. Reaction with tert-butylacetylene yields regioselectively the E-2,4-disubstituted 1-buten-3-yne dimer whereas trimethylsilylacetylene is regioselective trimerized to the E,E-1,4,6-tris(trimethylsilyl)-1,3-hexa diene-5-yne, with small amounts (3-5%) of the corresponding E-2,4-disubstituted 1-buten-3-yne dimer. Oligomerization with less bulky alkyl and aryl substituted alkynes produces a mixture of higher oligomers with no regioselectivity. Using the Cp 2 * ThMe 2 catalyst, we have recently developed a strategic method to control the extent and in some cases the regioselectivity of the catalyzed oligomerization of nonbulky terminal alkynes to dimers and/or trimers. The metallocene catalytic precursors ensure the selective synthesis of small oligomers by the addition of specific amines. Catalytic ''tailoring'' to dimer and trimers can be achieved by using small or bulky amines, respectively. Kinetic and mechanistic data for the controlling experiments argue that the turnover-limiting step involves the acetylide actinide complex formation with the rapid insertion of the alkyne and protonolysis by the amine. The analog Cp 2 * UMe 2 in the presence of primary amines induce the selective C-N bond formation, producing enamines which are tautomerized to the corresponding imines. (orig.)

Alterations of sirtuins in mitochondrial cytochrome c-oxidase deficiency.

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Arne Björn Potthast

Full Text Available Sirtuins are NAD+ dependent deacetylases, which regulate mitochondrial energy metabolism as well as cellular response to stress. The NAD/NADH-system plays a crucial role in oxidative phosphorylation linking sirtuins and the mitochondrial respiratory chain. Furthermore, sirtuins are able to directly deacetylate and activate different complexes of the respiratory chain. This prompted us to analyse sirtuin levels in skin fibroblasts from patients with cytochrome c-oxidase (COX deficiency and to test the impact of different pharmaceutical activators of sirtuins (SRT1720, paeonol to modulate sirtuins and possibly respiratory chain enzymes in patient cells in vitro.We assayed intracellular levels of sirtuin 1 and the mitochondrial sirtuins SIRT3 and SIRT4 in human fibroblasts from patients with COX- deficiency. Furthermore, sirtuins were measured after inhibiting complex IV in healthy control fibroblasts by cyanide and after incubation with activators SRT1720 and paeonol. To determine the effect of sirtuin inhibition at the cellular level we measured total cellular acetylation (control and patient cells, with and without treatment by Western blot.We observed a significant decrease in cellular levels of all three sirtuins at the activity, protein and transcriptional level (by 15% to 50% in COX-deficient cells. Additionally, the intracellular concentration of NAD+ was reduced in patient cells. We mimicked the biochemical phenotype of COX- deficiency by incubating healthy fibroblasts with cyanide and observed reduced sirtuin levels. A pharmacological activation of sirtuins resulted in normalized sirtuin levels in patient cells. Hyper acetylation was also reversible after treatment with sirtuin activators. Pharmacological modulation of sirtuins resulted in altered respiratory chain complex activities.We found inhibition of situins 1, 3 and 4 at activity, protein and transcriptional levels in fibroblasts from patient with COX-deficiency. Pharmacological

Synthesis of ¹³C-lidocaine as a probe of breath test for the evaluation of cytochrome P450 activity.

Science.gov (United States)

Mitome, Hidemichi; Sugiyama, Erika; Sato, Hitoshi; Akira, Kazuki

2014-01-01

(13)C-Labeled lidocaine, 2-di[1-(13)C]ethylamino-N-(2,6-dimethylphenyl)acetamide (1), was synthesized from [1-(13)C]acetic acid in six steps, as a probe for a breath test to evaluate in vivo cytochrome P450 activity. The measurement of (13)CO2 in breath was successfully performed following oral administration of (13)C-lidocaine 1 to mice.

Spectroscopic and electrochemical characterization of cytochrome c encapsulated in a bio sol-gel matrix.

Science.gov (United States)

Deriu, Daniela; Pagnotta, Sara Emanuela; Santucci, Roberto; Rosato, Nicola

2008-08-01

Sol-gel technique represents a remarkably versatile method for protein encapsulation. To enhance sol-gel biocompatibility, systems envisaging the presence of calcium and phosphates in the sol-gel composition were recently prepared and investigated. Unfortunately, the low pH at which solutions were prepared (pH proteins, because the acidic environment induces protein denaturation. In this paper we apply a new protocol based on the introduction of calcium nitrate to the inorganic phase, with formation of a binary bioactive system. In this case protein encapsulation results versatile and secure, being achieved at a pH close to neutrality (pH 6.0); also, the presence of calcium is expected to enhance system biocompatibility. To determine the properties of the salt-doped sol-gel and the influence exerted on entrapped biosystems, the structural and functional properties of embedded cytochrome c have been investigated. Data obtained indicate that the salt-doped sol-gel induces no significant change in the structure and the redox properties of the embedded protein; also, the matrix increases protein stability. Interestingly, the presence of calcium nitrate appears determinant for refolding of the acid-denatured protein. This is of interest in the perspective of future applications in biosensoristic area.

Deletion map of CYC1 mutants and its correspondence to mutationally altered iso-1-cytochromes c of yeast

International Nuclear Information System (INIS)

Sherman, F.; Jackson, M.; Liebman, S.W.; Schweingruber, A.M.; Stewart, J.W.

1975-01-01

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants

Tissue-specific cytochrome c oxidase assembly defects due to mutations in SCO2 and SURF1

Czech Academy of Sciences Publication Activity Database

Stibůrek, L.; Veselá, K.; Hansíková, H.; Pecina, Petr; Tesařová, M.; Černá, L.; Houštěk, Josef; Zeman, J.

2005-01-01

Roč. 392, č. 3 (2005), s. 625-632 ISSN 0264-6021 R&D Projects: GA ČR(CZ) GA303/03/0749; GA MŠk(CZ) 1M0520; GA ČR(CZ) GP303/03/D132 Grant - others:GA UK(CZ) 17/2004/C; Priority 1(XE) LSHMCT-2004-503116 Institutional research plan: CEZ:AV0Z50110509 Keywords : assembly pathway * cytochrome c oxidase * SURF1 Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.224, year: 2005

Influence of paraoxonase-1 Q192R and cytochrome P450 2C19 polymorphisms on clopidogrel response

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Li L

2012-02-01

Full Text Available Rolf P Kreutz1,2, Perry Nystrom2, Yvonne Kreutz2, Jia Miao2, Zeruesenay Desta2, Jeffrey A Breall1, Lang Li2, ChienWei Chiang2, Richard Kovacs1, David A Flockhart2, Yan Jin21Krannert Institute of Cardiology, 2Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, IN, USABackground: The metabolic activation of clopidogrel is a two-step process. It has been suggested that paraoxonase-1 (PON1 is a rate-limiting enzyme in the conversion of 2-oxo-clopidogrel to an active thiol metabolite. Conflicting results have been reported in regard to (1 the association of a common polymorphism of PON1 (Q192R with reduced rates of coronary stent thrombosis in patients taking clopidogrel and (2 its effects on platelet inhibition in patient populations of European descent. Methods: Blood samples from 151 subjects of mixed racial background with established coronary artery disease and who received clopidogrel were analyzed. Platelet aggregation was determined with light transmittance aggregometry and VerifyNow® P2Y12 assay. Genotyping for cytochrome P450 2C19 (CYP2C19*2 and *3 and PON1 (Q192R polymorphisms was performed.Results: Carriers of CYP2C19*2 alleles exhibited lower levels of platelet inhibition and higher on-treatment platelet aggregation than noncarriers. There was no significant difference in platelet aggregation among PON1 Q192R genotypes. Homozygous carriers of the wild-type variant of PON1 (QQ192 had similar on-treatment platelet reactivity to carriers of increased-function variant alleles during maintenance clopidogrel dosing, as well as after administration of a clopidogrel 600 mg loading dose.Conclusion: CYP2C19*2 allele is associated with impaired platelet inhibition by clopidogrel and high on-treatment platelet aggregation. PON1 (Q192R polymorphism does not appear to be a significant determinant of clopidogrel response.Keywords: PON1, platelet, aggregation, cytochrome P450 enzymes

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

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.

In vitro RBE-LET dependence for multiple particle types

DEFF Research Database (Denmark)

Sørensen, Brita Singers; Overgaard, Jens; Bassler, Niels

2011-01-01

Background. In vitro RBE values for various high LET radiation types have been determined for many different cell types. Occasionally it is criticized that RBE for a given endpoint cannot be single-value dependent on LET alone, but also on particle species, due to the different dose deposition...... profiles on microscopic scale. Hence LET is not sufficient as a predictor of RBE, and this is one of the motivations for development of radiobiological models which explicitly depend on the detailed particle energy spectrum of the applied radiation field. The aim of the present study is to summarize...... the available data in the literature regarding the dependency of RBE on LET for different particles. Method. As RBE is highly dependent on cell type and endpoint, we discriminated the RBE-LET relationship for the three investigated cell lines and at the same endpoint (10% survival in colony formation). Data...

Electrochemistry, surface plasmon resonance, and quartz crystal microbalance: an associative study on cytochrome c adsorption on pyridine tail-group monolayers on gold.

Science.gov (United States)

Paulo, Tércio de F; de Sousa, Ticyano P; de Abreu, Dieric S; Felício, Nathalie H; Bernhardt, Paul V; Lopes, Luiz G de F; Sousa, Eduardo H S; Diógenes, Izaura C N

2013-07-25

Quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and electrochemistry techniques were used to study the electron-transfer (ET) reaction of cytochrome c (Cyt c) on gold surfaces modified with thionicotinamide, thioisonicotinamide, 4-mercaptopyridine, 5-(4-pyridyl)-1,3,4-oxadiazole-2-thiol, 5-phenyl-1,3,4-oxadiazole-2-thiol, 4,4'-bipyridine, and 4,4'-dithiopyridine. The electrochemical results showed that the ET process is complex, being chiefly diffusional with steps depending on the orientation of the pyridine or phenyl tail group of the modifiers. The correlation between the electrochemical results and those acquired by SPR and QCM indicated the presence of an adlayer of Cyt c adsorbed on the thiolate SAMs. This adlayer, although being not electroactive, is essential to assess the ET reaction of Cyt c in solution. The results presented in this work are consistent with the statement (Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Electroanal. Chem. 1995, 394, 149-154) that the ET reaction of Cyt c can be explained in terms of the through-bond tunneling mechanism.

Upper and lower limits of the proton stoichiometry of cytochrome c oxidation in rat liver mitoplasts.

Science.gov (United States)

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

1986-06-25

The stoichiometry of vectorial H+ translocation coupled to oxidation of added ferrocytochrome c by O2 via cytochrome-c oxidase of rat liver mitoplasts was determined employing a fast-responding O2 electrode. Electron flow was initiated by addition of either ferrocytochrome c or O2. When the rates were extrapolated to level flow, the H+/O ratios in both cases were less than but closely approached 4; the directly observed H+/O ratios significantly exceeded 3.0. The mechanistic H+/O ratio was then more closely fixed by a kinetic approach that eliminates the necessity for measuring energy leaks and is independent of any particular model of the mechanism of energy transduction. From two sets of kinetic measurements, an overestimate and an underestimate and thus the upper and lower limits of the mechanistic H+/O ratio could be obtained. In the first set, the utilization of respiratory energy was systematically varied through changes in the concentrations of valinomycin or K+. From the slope of a plot of the initial rates of H+ ejection (JH) and O2 uptake (JO) obtained in such experiments, the upper limit of the H+/O ratio was in the range 4.12-4.19. In the second set of measurements, the rate of respiratory energy production was varied by inhibiting electron transport. From the slope of a plot of JH versus JO, the lower limit of the H+/O ratio, equivalent to that at level flow, was in the range 3.83-3.96. These data fix the mechanistic H+/O ratio for the cytochrome oxidase reaction of mitoplasts at 4.0, thus confirming our earlier measurements (Reynafarje, B., Alexandre, A., Davies, P., and Lehninger, A. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7218-7222). Possible reasons for discrepancies in published reports on the H+/O ratio of cytochrome oxidase in various mitochondrial and reconstituted systems are discussed.

HmsC Controls Yersinia pestis Biofilm Formation in Response to Redox Environment

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Gai-Xian Ren

2017-08-01

Full Text Available Yersinia pestis biofilm formation, controlled by intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP, is important for blockage-dependent plague transmission from fleas to mammals. HmsCDE is a tripartite signaling system that modulates intracellular c-di-GMP levels to regulate biofilm formation in Y. pestis. Previously, we found that Y. pestis biofilm formation is stimulated in reducing environments in an hmsCDE-dependent manner. However, the mechanism by which HmsCDE senses the redox state remains elusive. Using a dsbA mutant and the addition of Cu2+ to simulate reducing and oxidizing periplasmic environments, we found that HmsC protein levels are decreased and the HmsC-HmsD protein-protein interaction is weakened in a reducing environment. In addition, we revealed that intraprotein disulphide bonds are critical for HmsC since breakage lowers protein stability and diminishes the interaction with HmsD. Our results suggest that HmsC might play a major role in sensing the environmental changes.

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...

The cytochrome b p.278Y>C mutation causative of a multisystem disorder enhances superoxide production and alters supramolecular interactions of respiratory chain complexes

DEFF Research Database (Denmark)

Ghelli, Anna; Tropeano, Concetta V; Calvaruso, Maria Antonietta

2013-01-01

, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2......IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.......Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe...

Boron-Based Catalysts for C-C Bond-Formation Reactions.

Science.gov (United States)

Rao, Bin; Kinjo, Rei

2018-05-02

Because the construction of the C-C bond is one of the most significant reactions in organic chemistry, the development of an efficient strategy has attracted much attention throughout the synthetic community. Among various protocols to form C-C bonds, organoboron compounds are not just limited to stoichiometric reagents, but have also made great achievements as catalysts because of the easy modification of the electronic and steric impacts on the boron center. This review presents recent developments of boron-based catalysts applied in the field of C-C bond-formation reactions, which are classified into four kinds on the basis of the type of boron catalyst: 1) highly Lewis acidic borane, B(C 6 F 5 ) 3 ; 2) organoboron acids, RB(OH) 2 , and their ester derivatives; 3) borenium ions, (R 2 BL)X; and 4) other miscellaneous kinds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

Science.gov (United States)

Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

2013-12-01

Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations

Czech Academy of Sciences Publication Activity Database

Kovářová, Nikola; Vrbacká-Čížková, Alena; Pecina, Petr; Stránecký, V.; Pronicka, E.; Kmoch, S.; Houštěk, Josef

2012-01-01

Roč. 1822, č. 7 (2012), s. 1114-1124 ISSN 0925-4439 R&D Projects: GA MZd(CZ) NS9759; GA MZd(CZ) NT12370; GA ČR(CZ) GD305/08/H037 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : mitochondrial disorder * SURF1 gene * Leigh syndrome * gene expression * oxidative phosphorylation * cytochrome c oxidase Subject RIV: FG - Pediatrics Impact factor: 4.910, year: 2012

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.

The moderating role of shopping trip type in store satisfaction formation

NARCIS (Netherlands)

Hunneman, Auke; Verhoef, Pieter; Sloot, Laurentius

Consumers may weigh store attributes differently depending on the type of shopping trip. For example, fill-in shoppers likely value convenience, due to the ad-hoc nature and urgency of such trips. However, no study has yet explored the effects of shopping trip types on satisfaction formation. This

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis[W][OA

Science.gov (United States)

Christ, Bastien; Süssenbacher, Iris; Moser, Simone; Bichsel, Nicole; Egert, Aurelie; Müller, Thomas; Hörtensteiner, Stefan

2013-01-01

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis. PMID:23723324

Metabolism of styrene to styrene oxide and vinylphenols in cytochrome P450 2F2- and P450 2E1-knockout mouse liver and lung microsomes.

Science.gov (United States)

Shen, Shuijie; Li, Lei; Ding, Xinxin; Zheng, Jiang

2014-01-21

Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e., styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. A dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes relative to that in the wild-type mouse lung microsomes; however, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knockout and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed a susceptibility to lung toxicity of styrene similar to that of the wild-type animals; however, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene.

Auto Poisoning of the Respiratory Chain by a Quorum Sensing Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance

Science.gov (United States)

Hazan, Ronen; Que, Yok Ai; Maura, Damien; Strobel, Benjamin; Majcherczyk, Paul Anthony; Hopper, Laura Rose; Wilbur, David J.; Hreha, Teri N.; Barquera, Blanca; Rahme, Laurence G.

2015-01-01

Summary Bacterial programmed cell death and quorum sensing are direct examples of prokaryote group behaviors, wherein cells coordinate their actions to function cooperatively like one organism for the benefit of the whole culture. We demonstrate here that 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a Pseudomonas aeruginosa quorum sensing -regulated low-molecular-weight excreted molecule, and triggers autolysis by self-perturbing the electron transfer reactions of the cytochrome bc1 complex. HQNO induces specific self-poisoning by disrupting the flow of electrons through the respiratory chain at the cytochrome bc1 complex, causing a leak of reducing equivalents to O2 whereby electrons that would normally be passed to cytochrome c are donated directly to O2. The subsequent mass production of reactive oxygen species (ROS) reduces membrane potential and disrupts membrane integrity, causing bacterial cell autolysis and DNA release. DNA subsequently promotes biofilm formation and increases antibiotic tolerance to beta-lactams, suggesting that HQNO-dependent cell autolysis is advantageous to the bacterial populations. These data both identify a new programmed cell death system, and a novel role for HQNO as a critical-inducer of biofilm formation and antibiotic tolerance. This newly identified pathway suggests intriguing mechanistic similarities with the initial mitochondrial-mediated steps of eukaryotic apoptosis. PMID:26776731

Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect

Czech Academy of Sciences Publication Activity Database

Kovářová, Nikola; Pecina, Petr; Nůsková, Hana; Vrbacký, Marek; Zeviani, M.; Mráček, Tomáš; Viscomi, C.; Houštěk, Josef

2016-01-01

Roč. 7, June 01 (2016), s. 1004-1009 ISSN 2352-3409 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MZd(CZ) NT12370 Institutional support: RVO:67985823 Keywords : cytochrome c oxidase * respiratory chain * SURF1 * knockout * doxycycline Subject RIV: EB - Genetics ; Molecular Biology

Guarded dependent type theory with coinductive types

DEFF Research Database (Denmark)

Bizjak, Aleš; Grathwohl, Hans Bugge; Clouston, Ranald

2016-01-01

We present guarded dependent type theory, gDTT, an extensional dependent type theory with a later' modality and clock quantifiers for programming and proving with guarded recursive and coinductive types. The later modality is used to ensure the productivity of recursive definitions in a modular......, type based, way. Clock quantifiers are used for controlled elimination of the later modality and for encoding coinductive types using guarded recursive types. Key to the development of gDTT are novel type and term formers involving what we call delayed substitutions’. These generalise the applicative...... functor rules for the later modality considered in earlier work, and are crucial for programming and proving with dependent types. We show soundness of the type theory with respect to a denotational model....

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

Dicty_cDB: Contig-U04737-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available se I (COI) gen... 44 5.8 1 ( AY225873 ) Lasius austriacus isolate Laus5COI cytoch...rome c o... 44 5.8 1 ( AY225872 ) Lasius austriacus isolate Laus4COI cytochrome c o... 44 5.8 1 ( AY225871 ) Lasius austria...cus isolate Laus3COI cytochrome c o... 44 5.8 1 ( AY225870 ) Lasius austriacus isolate Laus2C...OI cytochrome c o... 44 5.8 1 ( AY225869 ) Lasius austriacus isolate Laus1COI cytochrome c o... 44 5.8 1 ( A...9 ) Prenolepis imparis mitochondrial COI gene for cyt... 44 5.8 1 ( AB371009 ) Lasius austriacus mitochondri

Silencing, positive selection and parallel evolution: busy history of primate cytochromes C.

Science.gov (United States)

Pierron, Denis; Opazo, Juan C; Heiske, Margit; Papper, Zack; Uddin, Monica; Chand, Gopi; Wildman, Derek E; Romero, Roberto; Goodman, Morris; Grossman, Lawrence I

2011-01-01

Cytochrome c (cyt c) participates in two crucial cellular processes, energy production and apoptosis, and unsurprisingly is a highly conserved protein. However, previous studies have reported for the primate lineage (i) loss of the paralogous testis isoform, (ii) an acceleration and then a deceleration of the amino acid replacement rate of the cyt c somatic isoform, and (iii) atypical biochemical behavior of human cyt c. To gain insight into the cause of these major evolutionary events, we have retraced the history of cyt c loci among primates. For testis cyt c, all primate sequences examined carry the same nonsense mutation, which suggests that silencing occurred before the primates diversified. For somatic cyt c, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses yielded the same tree topology. The evolutionary analyses show that a fast accumulation of non-synonymous mutations (suggesting positive selection) occurred specifically on the anthropoid lineage root and then continued in parallel on the early catarrhini and platyrrhini stems. Analysis of evolutionary changes using the 3D structure suggests they are focused on the respiratory chain rather than on apoptosis or other cyt c functions. In agreement with previous biochemical studies, our results suggest that silencing of the cyt c testis isoform could be linked with the decrease of primate reproduction rate. Finally, the evolution of cyt c in the two sister anthropoid groups leads us to propose that somatic cyt c evolution may be related both to COX evolution and to the convergent brain and body mass enlargement in these two anthropoid clades.

Silencing, positive selection and parallel evolution: busy history of primate cytochromes C.

Directory of Open Access Journals (Sweden)

Denis Pierron

Full Text Available Cytochrome c (cyt c participates in two crucial cellular processes, energy production and apoptosis, and unsurprisingly is a highly conserved protein. However, previous studies have reported for the primate lineage (i loss of the paralogous testis isoform, (ii an acceleration and then a deceleration of the amino acid replacement rate of the cyt c somatic isoform, and (iii atypical biochemical behavior of human cyt c. To gain insight into the cause of these major evolutionary events, we have retraced the history of cyt c loci among primates. For testis cyt c, all primate sequences examined carry the same nonsense mutation, which suggests that silencing occurred before the primates diversified. For somatic cyt c, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses yielded the same tree topology. The evolutionary analyses show that a fast accumulation of non-synonymous mutations (suggesting positive selection occurred specifically on the anthropoid lineage root and then continued in parallel on the early catarrhini and platyrrhini stems. Analysis of evolutionary changes using the 3D structure suggests they are focused on the respiratory chain rather than on apoptosis or other cyt c functions. In agreement with previous biochemical studies, our results suggest that silencing of the cyt c testis isoform could be linked with the decrease of primate reproduction rate. Finally, the evolution of cyt c in the two sister anthropoid groups leads us to propose that somatic cyt c evolution may be related both to COX evolution and to the convergent brain and body mass enlargement in these two anthropoid clades.

Electron transfer kinetics of cytochrome c immobilized on a phenolic terminated thiol self assembled monolayer determined by scanning electrochemical microscopy

International Nuclear Information System (INIS)

Alizadeh, Vali; Mousavi, Mir Fazlollah; Mehrgardi, Masoud Ayatollahi; Kazemi, Sayed Habib; Sharghi, Hashem

2011-01-01

Highlights: → Preparing a thiolated phenolic self-assembled monolayer surface (SAM). → Application of this SAM to immobilize cytochrome C. → Scanning electrochemical microscopy used for these studies. → Determination of both tunneling electron transfer and bimolecular rate constants between the immobilized protein-substrate and probe. - Abstract: In the present manuscript, the electrochemical behavior of cytochrome c (cyt-c) immobilized onto a phenolic terminated self assembled monolayer (SAM) on a gold electrode is investigated using cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM). The tunneling electron transfer (ET) rate constant between the immobilized protein and the underlying electrode surface, and also the bimolecular ET rate constant between the immobilized protein and a probe has been obtained using approach curves that were obtained by SECM. The approach curves were recorded at different substrate overpotentials in the presence of various concentrations of ferrocyanide as a probe and various surface concentrations of cyt-c; then the standard tunneling ET and bimolecular rate constants are obtained as 3.4 ± 0.3 s -1 and (2.0 ± 0.5) x 10 7 cm 3 mol -1 s -1 , respectively.

Electron transfer kinetics of cytochrome c immobilized on a phenolic terminated thiol self assembled monolayer determined by scanning electrochemical microscopy

Energy Technology Data Exchange (ETDEWEB)

Alizadeh, Vali [Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran (Iran, Islamic Republic of); Mousavi, Mir Fazlollah, E-mail: mousavim@modares.ac.ir [Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran (Iran, Islamic Republic of); Mehrgardi, Masoud Ayatollahi [Department of Chemistry, University of Isfahan, Isfahan (Iran, Islamic Republic of); Kazemi, Sayed Habib [Department of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan (Iran, Islamic Republic of); Sharghi, Hashem [Department of Chemistry, Shiraz University, Shiraz (Iran, Islamic Republic of)

2011-07-01

Highlights: > Preparing a thiolated phenolic self-assembled monolayer surface (SAM). > Application of this SAM to immobilize cytochrome C. > Scanning electrochemical microscopy used for these studies. > Determination of both tunneling electron transfer and bimolecular rate constants between the immobilized protein-substrate and probe. - Abstract: In the present manuscript, the electrochemical behavior of cytochrome c (cyt-c) immobilized onto a phenolic terminated self assembled monolayer (SAM) on a gold electrode is investigated using cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM). The tunneling electron transfer (ET) rate constant between the immobilized protein and the underlying electrode surface, and also the bimolecular ET rate constant between the immobilized protein and a probe has been obtained using approach curves that were obtained by SECM. The approach curves were recorded at different substrate overpotentials in the presence of various concentrations of ferrocyanide as a probe and various surface concentrations of cyt-c; then the standard tunneling ET and bimolecular rate constants are obtained as 3.4 {+-} 0.3 s{sup -1} and (2.0 {+-} 0.5) x 10{sup 7} cm{sup 3} mol{sup -1} s{sup -1}, respectively.

Atomistic simulations of the formation of <c>-component dislocation loops in α-zirconium

Energy Technology Data Exchange (ETDEWEB)

Dai, Cong, E-mail: dai.cong@queensu.ca; Balogh, Levente; Yao, Zhongwen; Daymond, Mark R., E-mail: mark.daymond@queensu.ca

2016-09-15

The formation of <c>-component dislocation loops in α-Zr is believed to be responsible for the breakaway irradiation growth experimentally observed under high irradiation fluences. However, while <c>-loop growth is well described by existing models, the atomic mechanisms responsible for the nucleation of <c>-component dislocation loops are still not clear. In the present work, both interstitial and vacancy -type dislocation loops are initially equilibrated at different temperatures. Cascades simulations in the vicinity of the -type loops are then performed by selecting an atom as the primary knock-on atoms (PKAs) with different kinetic energies, using molecular dynamics simulations. No <c>-component dislocation loop was formed in cascades simulations with a 10 keV PKA, but <c>-component interstitial loops were observed after the interaction between discontinuous 50 keV PKAs and pre-existing -type interstitial loops. The comparisons of cascades simulations in volumes having pre-existing -type interstitial and vacancy loops suggest that the reaction between the PKAs and -type interstitial loops is responsible for the formation of <c>-component interstitial loops.

Irradiation temperature dependence of defect formation of nitrides (A1N and c-BN) during neutron irradiations

International Nuclear Information System (INIS)

Atobe, Kozo.; Okada, Moritami; Nakagawa, Masuo

2000-01-01

The nitrogen vacancy concentration in the more refractory nitrides (A1N and c-BN) is determined as a function of reactor fluence up to 5.2x10 17 thermal neutrons/cm 2 and a function of the irradiation temperature at 25, 50, 100, 150, 200, 250 K. It is found that there is no remarkable dependence of the defect formation in nitrides on the irradiation temperature. The production of damage in the nitrides is considerably different from that in oxides. From the irradiation experiments using thermal neutron irradiation field, it is suggested in reactor irradiation that the atomic displacements in the nitrides occur predominately from energetic particles of the nuclear reactions with thermal neutrons in addition to the elastic collisions by fast neutron

Direct observation of vibrational energy flow in cytochrome c.

Science.gov (United States)

Fujii, Naoki; Mizuno, Misao; Mizutani, Yasuhisa

2011-11-10

Vibrational energy flow in ferric cytochrome c has been examined by picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) measurements. By taking advantage of the extremely short nonradiative excited state lifetime of heme in the protein (energy of 20000-25000 cm(-1) was optically deposited selectively at the heme site. Subsequent energy relaxation in the protein moiety was investigated by monitoring the anti-Stokes UVRR intensities of the Trp59 residue, which is a single tryptophan residue involved in the protein that is located close to the heme group. It was found from temporal changes of the anti-Stokes UVRR intensities that the energy flow from the heme to Trp59 and the energy release from Trp59 took place with the time constants of 1-3 and ~8 ps, respectively. These data are consistent with the time constants for the vibrational relaxation of the heme and heating of water reported for hemeproteins. The kinetics of the energy flow were not affected by the amount of excess energy deposited at the heme group. These results demonstrate that the present technique is a powerful tool for studying the vibrational energy flow in proteins.

Type II cGMP‑dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

Science.gov (United States)

Wu, Min; Wu, Yan; Qian, Hai; Tao, Yan; Pang, Ji; Wang, Ying; Chen, Yongchang

2017-10-01

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

12(R)-hydroxyicosatetraenoic acid: a cytochrome P450-dependent arachidonate metabolite that inhibits Na/sup +/, K/sup +/-ATPase in the cornea

Energy Technology Data Exchange (ETDEWEB)

Schwartzman, M.L.; Balazy, M.; Masferrer, J.; Abraham, N.G.; McGiff, J.C.; Murphy, R.C.

1987-11-01

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na/sup +/, K/sup +/-ATPase from the corneal epithelium in a dose-dependent manner. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive- and negative-ionization modes was carried out on derivatized compound C. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from (/sup 2/H/sub 8/)arachidonate were retained in the structure. Compound C was characterized as a 12-hydroxyicosatetraenoic acid. However, only 12(R) isomer was found to be an inhibitor of the Na/sup +/, K/sup +/-ATPase from the corneal epithelium, suggesting that the biologically active compound C was 12(R)-hydroxyy-5,8,10,14-icosatetraenoic acid. Such an inhibitor of Na/sup +/, K/sup +/-ATPase synthesized in the cornea may have an important role in regulating ocular transparency and aqueous human secretion.

Iron hexacyanide/cytochrome-C - intramolecular electron transfer and binding constants - (pulse radiolytic study). Progress report

International Nuclear Information System (INIS)

Ilan, Y.; Shafferman, A.

Internal oxidation and reduction rates of horse cytochrome-c in the complexes, CII.Fe/sup III/(CN) -3 6 and CIII.Fe/sup II/(CN) -4 6 , are 4.6.10 4 s -1 and 3.3.10 2 s -1 , respectively. The binding sites of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87.10 3 to 2.10 3 M -1 . The present pulse radiolytic kinetic data are compared with that from N.M.R, T-jump and equilibrium dialysis studies

Molecular Computational Investigation of Electron Transfer Kinetics across Cytochrome-Iron Oxide Interfaces

International Nuclear Information System (INIS)

Kerisit, Sebastien N.; Rosso, Kevin M.; Dupuis, Michel; Valiev, Marat

2007-01-01

The interface between electron transfer proteins such as cytochromes and solid phase mineral oxides is central to the activity of dissimilatory-metal reducing bacteria. A combination of potential-based molecular dynamics simulations and ab initio electronic structure calculations are used in the framework of Marcus' electron transfer theory to compute elementary electron transfer rates from a well-defined cytochrome model, namely the small tetraheme cytochrome (STC) from Shewanella oneidensis, to surfaces of the iron oxide mineral hematite (a-Fe2O3). Room temperature molecular dynamics simulations show that an isolated STC molecule favors surface attachment via direct contact of hemes I and IV at the poles of the elongated axis, with electron transfer distances as small as 9 Angstroms. The cytochrome remains attached to the mineral surface in the presence of water and shows limited surface diffusion at the interface. Ab initio electronic coupling matrix element (VAB) calculations of configurations excised from the molecular dynamics simulations reveal VAB values ranging from 1 to 20 cm-1, consistent with nonadiabaticity. Using these results, together with experimental data on the redox potential of hematite and hemes in relevant cytochromes and calculations of the reorganization energy from cluster models, we estimate the rate of electron transfer across this model interface to range from 1 to 1000 s-1 for the most exothermic driving force considered in this work, and from 0.01 to 20 s-1 for the most endothermic. This fairly large range of electron transfer rates highlights the sensitivity of the rate upon the electronic coupling matrix element, which is in turn dependent on the fluctuations of the heme configuration at the interface. We characterize this dependence using an idealized bis-imidazole heme to compute from first principles the VAB variation due to porphyrin ring orientation, electron transfer distance, and mineral surface termination. The electronic

Higher Collagen VI Formation Is Associated With All-Cause Mortality in Patients With Type 2 Diabetes and Microalbuminuria

DEFF Research Database (Denmark)

Rasmussen, Daniel G K; Hansen, Tine W; von Scholten, Bernt J

2018-01-01

OBJECTIVE: Type 2 diabetes is a common risk factor for the development of chronic kidney disease (CKD). Enhanced de novo collagen type VI (COL VI) formation has been associated with renal fibrosis and CKD. We investigated the hypothesis that PRO-C6, a product specifically generated during COL VI...... formation, is prognostic for adverse outcomes in patients with type 2 diabetes and microalbuminuria. RESEARCH DESIGN AND METHODS: In a prospective, observational study, we measured PRO-C6 in serum (S-PRO-C6) and urine (U-PRO-C6) of 198 patients with type 2 diabetes and microalbuminuria without symptoms...... = 0.004). U-PRO-C6 was not significantly associated with any of the outcomes. CONCLUSIONS: S-PRO-C6 generated during COL VI formation predicts cardiovascular events, all-cause mortality, and disease progression in patients with type 2 diabetes and microalbuminuria....

IDENTIFICATION OF PARAMECIUM BURSARIA SYNGENS THROUGH MOLECULAR MARKERS – COMPARATIVE ANALYSIS OF MITOCHONDRIAL CYTOCHROME C OXIDASE SUBUNIT I (COI

Directory of Open Access Journals (Sweden)

Patrycja Zagata

2014-08-01

Full Text Available The aim of this study is an identification of Paramecium bursaria syngens originating from different geographical locations and proving the correlation between distributions and belonging to any of five syngens. Ten strains of Paramecium bursaria belonging to five different syngens and strain of Paramecium multimicronucleatum were investigated using molecular marker — mitochondrial cytochrome c oxidase subunit I (COI. According to results, obtained in this study, using phylogenetic methods like Neighbor Joining (NJ and Maximum Likelihood (ML, relationship between analyzing strains through their clustering in clusters and correlation between strains belonging to any syngen and syngen’s distribution was confirmed. Phylograms constructed using NJ and ML methods revealed strains’ grouping in five clusters. Results which were obtained revealed usefulness of COI as a biomarker, which is important in identification of Paramecium bursaria syngens. This reports to a great potential of COI as a molecular marker and obtaining dependable results through combination of molecular methods with classical ones.

Roles of calcium/calmodulin-dependent kinase II in long-term memory formation in crickets.

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Makoto Mizunami

Full Text Available Ca(2+/calmodulin (CaM-dependent protein kinase II (CaMKII is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca(2+/CaM and cAMP signaling participates in long-term memory (LTM formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM. Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca(2+ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca(2+ signaling and cAMP signaling for LTM formation, a new role of Ca

Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli

International Nuclear Information System (INIS)

Sargentini, N.J.; Smith, K.C.

1989-01-01

γ-radiation mutagenesis Escherichia coli K-12. Mutagenesis (argE3(OC) A rg + ) was blocked in a δ(recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the γ-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but non all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC T and AT GC transitions were essentially umuC independent, while the yields of (AT or GC) TA transversions were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to γ-radiation mutagenesis. (author). 48 refs.; 1 tab.; 6 refs

Hydrazine and hydroxylamine as probes for O2-reduction site of mitochondrial cytochrome c oxidase.

Science.gov (United States)

Kubota, T; Yoshikawa, S

1993-01-01

Reactions of hydrazine and hydroxylamine with bovine heart cytochrome c oxidase in the fully reduced state were investigated under anaerobic conditions following the visible-Soret spectral change. Hydrazine gave a sharp band at 575 nm with 20% decrease in the alpha band at 603 nm, and hydroxylamine induced a 2 nm blue-shift for the alpha band without any clear splitting. The Soret band at 443 nm was decreased significantly in intensity, with the concomitant appearance of a shoulder with hydrazine or a peak with hydroxylamine, both near 430 nm. The dependence on pH of the affinity of these reagents for the enzyme indicates that only the deprotonated forms of these reagents bind to the enzyme, suggesting a highly hydrophobic environment of the haem ligand-biding site. These spectral changes were largely removed by addition of cyanide or CO. However, detailed analysis of these spectral changes indicates that hydrazine perturbs the shape of the spectral change induced by cyanide and hydroxylamine perturbs that induced by CO. These results suggest that these aldehyde reagents bind to haem a3 iron as well as to a second site which is most likely to be the formyl group on the haem periphery, and that these two sites bind these reagents anti-cooperatively with each other. PMID:8389138

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.

The IpaC carboxyterminal effector domain mediates Src-dependent actin polymerization during Shigella invasion of epithelial cells.

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Joëlle Mounier

2009-01-01

Full Text Available Shigella, the causative agent of bacillary dysentery, invades epithelial cells by locally reorganizing the actin cytoskeleton. Shigella invasion requires actin polymerization dependent on the Src tyrosine kinase and a functional bacterial type III secretion (T3S apparatus. Using dynamic as well as immunofluorescence microscopy, we show that the T3S translocon component IpaC allows the recruitment of the Src kinase required for actin polymerization at bacterial entry sites during the initial stages of Shigella entry. Src recruitment occurred at bacterial-cell contact sites independent of actin polymerization at the onset of the invasive process and was still observed in Shigella strains mutated for translocated T3S effectors of invasion. A Shigella strain with a polar mutation that expressed low levels of the translocator components IpaB and IpaC was fully proficient for Src recruitment and bacterial invasion. In contrast, a Shigella strain mutated in the IpaC carboxyterminal effector domain that was proficient for T3S effector translocation did not induce Src recruitment. Consistent with a direct role for IpaC in Src activation, cell incubation with the IpaC last 72 carboxyterminal residues fused to the Iota toxin Ia (IaC component that translocates into the cell cytosol upon binding to the Ib component led to Src-dependent ruffle formation. Strikingly, IaC also induced actin structures resembling bacterial entry foci that were enriched in activated Src and were inhibited by the Src inhibitor PP2. These results indicate that the IpaC effector domain determines Src-dependent actin polymerization and ruffle formation during bacterial invasion.

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...

Radiation-induced DNA damage in tumors and normal tissues. III. Oxygen dependence of the formation of strand breaks and DNA-protein crosslinks

International Nuclear Information System (INIS)

Zhang, H.; Wallen, C.A.; Wheeler, K.T.; Joch, C.J.

1995-01-01

Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicated on both published and nonpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-produced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37 degrees C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the K m value for radiation-induced strand breaks and DPCs were similar when 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4 degrees C and nonideal gas-liquid equilibrium conditions at 37 degrees C. At 4 degrees C under ideal gas-liquid equilibrium conditions, the K m for the formation of strand breaks was approximately 0.0045 mM, and Km for radiation sensitivity was approximately 0.005mM. A similar comparison for the formation of DPCs at 4 degrees C could not be made, because the efficiency of the formation of DPC was much lower at 4 degrees C than at 37 degrees C. 30 refs., 3 figs

Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

Science.gov (United States)

Koopmann, Edda; Hahlbrock, Klaus

1997-01-01

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. PMID:9405720

Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

International Nuclear Information System (INIS)

Koopmann, E.; Hahlbrock, K.

1997-01-01

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H

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.

Cytochrome P450 1B1 and 2C9 genotypes and risk of ischemic vascular disease, cancer, and chronic obstructive pulmonary disease

DEFF Research Database (Denmark)

Kaur-Knudsen, Diljit; Bojesen, Stig E; Nordestgaard, Børge G

2012-01-01

The aim of this review is to summarize present knowledge of genetic variation in cytochrome P450 1B1 (CYP1B1) and 2C9 (CYP2C9) genes and risk of tobacco-related cancer, female cancer, chronic obstructive pulmonary disease and ischemic vascular disease. The CYP1B1 and CYP2C9 enzymes metabolize pol...

A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart.

OpenAIRE

Li, Y; Naqui, A; Frey, T G; Chance, B

1987-01-01

A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (max...

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.

Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

Science.gov (United States)

Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

2012-12-15

Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. Copyright © 2012 Elsevier Inc. All rights reserved.

Thermodynamics of interactions between mammalian cytochromes P450 and b5.

Science.gov (United States)

Yablokov, Evgeny; Florinskaya, Anna; Medvedev, Alexei; Sergeev, Gennady; Strushkevich, Natallia; Luschik, Alexander; Shkel, Tatsiana; Haidukevich, Irina; Gilep, Andrei; Usanov, Sergey; Ivanov, Alexis

2017-04-01

Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes.

Science.gov (United States)

Zebothsen, Inga; Kunze, Thomas; Clement, Bernd

2006-07-01

Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.