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Alejandro K Samhanarias - One of the best experts on this subject based on the ideXlab platform.
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human erythrocytes exposure to juglone leads to an increase of superoxide anion production associated with Cytochrome B5 reductase uncoupling
Biochimica et Biophysica Acta, 2020Co-Authors: Gabriel N Valerio, Carlos Gutierrezmerino, Fatima Nogueira, Alejandro K Samhanarias, Isabel Moura, José J. G. MouraAbstract:Abstract Cytochrome B5 reductase is an enzyme with the ability to generate superoxide anion at the expenses of NADH consumption. Although this activity can be stimulated by Cytochrome c and could participate in the bioenergetic failure accounting in apoptosis, very little is known about other molecules that may uncouple the function of the Cytochrome B5 reductase. Naphthoquinones are redox active molecules with the ability to interact with electron transfer chains. In this work, we made an inhibitor screening against recombinant human Cytochrome B5 reductase based on naphthoquinone properties. We found that 5-hydroxy-1,4-naphthoquinone (known as juglone), a natural naphthoquinone extracted from walnut trees and used historically in traditional medicine with ambiguous health and toxic outcomes, had the ability to uncouple the electron transfer from the reductase to Cytochrome B5 and ferricyanide. Upon complex formation with Cytochrome B5 reductase, juglone is able to act as an electron acceptor leading to a NADH consumption stimulation and an increase of superoxide anion production by the reductase. Our results suggest that Cytochrome B5 reductase could contribute to the measured energetic failure in the erythrocyte apoptosis induced by juglone, that is concomitant with the reactive oxygen species produced by Cytochrome B5 reductase.
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topography of human Cytochrome B5 Cytochrome B5 reductase interacting domain and redox alterations upon complex formation
Biochimica et Biophysica Acta, 2018Co-Authors: Alejandro K Samhanarias, Carlos Gutierrezmerino, Rui M Almeida, Susana Ramos, Cristina M Cordas, Isabel Moura, José J. G. MouraAbstract:Abstract Cytochrome B5 is the main electron acceptor of Cytochrome B5 reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of Cytochrome B5 reductase flavin autofluorescence that was dependent upon the presence of Cytochrome B5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of Cytochrome B5 reductase redox potential in presence of Cytochrome B5 was also measured. These experiments suggest that the FAD group of Cytochrome B5 reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.
Steven L. Kelly - One of the best experts on this subject based on the ideXlab platform.
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biodiversity of the p450 catalytic cycle yeast Cytochrome B5 nadh Cytochrome B5 reductase complex efficiently drives the entire sterol 14 demethylation cyp51 reaction
FEBS Letters, 1999Co-Authors: David C Lamb, Nigel J. Manning, Diane E. Kelly, Mustak A Kaderbhai, Steven L. KellyAbstract:The widely accepted catalytic cycle of Cytochromes P450 (CYP) involves the electron transfer from NADPH Cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal Cytochrome B5/NADH Cytochrome B5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cyto- chrome P450 reductase, Cytochrome B5 and NADH Cytochrome B5 reductase, we show here that fungal CYP51 mediated sterol 14K-demethylation can be wholly and efficiently supported by the Cytochrome B5/NADH Cytochrome B5 reductase electron trans- port system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH Cytochrome B5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disrup- tion of the gene encoding CPR. z 1999 Federation of European Biochemical Societies.
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Biodiversity of the P450 catalytic cycle: yeast Cytochrome B5/NADH Cytochrome B5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction.
FEBS Letters, 1999Co-Authors: David C Lamb, Nigel J. Manning, Diane E. Kelly, Mustak A Kaderbhai, Steven L. KellyAbstract:The widely accepted catalytic cycle of Cytochromes P450 (CYP) involves the electron transfer from NADPH Cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal Cytochrome B5/NADH Cytochrome B5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cyto- chrome P450 reductase, Cytochrome B5 and NADH Cytochrome B5 reductase, we show here that fungal CYP51 mediated sterol 14K-demethylation can be wholly and efficiently supported by the Cytochrome B5/NADH Cytochrome B5 reductase electron trans- port system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH Cytochrome B5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disrup- tion of the gene encoding CPR. z 1999 Federation of European Biochemical Societies.
Sergey A. Usanov - One of the best experts on this subject based on the ideXlab platform.
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mechanism of electron transfer in fusion protein Cytochrome B5 nadh Cytochrome B5 reductase
Biochemistry, 2008Co-Authors: A V Yantsevich, Andrei A. Gilep, Sergey A. UsanovAbstract:In the present work we summarize results on construction of expression plasmid, heterologous expression in Escherichia coli, isolation and purification, as well as physicochemical characterization of chimeric protein consisting of hydrophilic domain of Cytochrome B5 and truncated from the N-terminal sequence (Δ23) form of NADH-Cytochrome B5 reductase. The kinetics and mechanism of electron transfer between NADH-Cytochrome B5 reductase and Cytochrome B5 in the frames of fusion protein consisting of Cytochrome B5 (94 amino acids) and truncated form of NADH-Cytochrome B5 reductase (277 amino acids) have been studied. It is shown that electron transfer takes place between redox partners belonging to two different molecules of the chimeric protein. Using computer modeling, we built the model of the tertiary structure of the fusion protein, which is in agreement with experimental data. By using Marcus theory of electron transfer in polar media, we demonstrate the inability of the hypothesis of electrostatic repulsions to explain the increase of electron transfer rate on increase of ion concentration in media due to elimination of the repulsion of similar charges. The real reason for the increase of the first order rate constant in some oxidation-reduction reactions between proteins, as shown in the present work, is a decrease of the media reorganization energy resulting in decrease of activation energy for oxidation-reduction reactions.
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mechanism of electron transfer in fusion protein Cytochrome B5 nadh Cytochrome B5 reductase
Biochemistry, 2008Co-Authors: A V Yantsevich, Andrei A. Gilep, Sergey A. UsanovAbstract:In the present work we summarize results on construction of expression plasmid, heterologous expression in Escherichia coli, isolation and purification, as well as physicochemical characterization of chimeric protein consisting of hydrophilic domain of Cytochrome B5 and truncated from the N-terminal sequence (Δ23) form of NADH-Cytochrome B5 reductase. The kinetics and mechanism of electron transfer between NADH-Cytochrome B5 reductase and Cytochrome B5 in the frames of fusion protein consisting of Cytochrome B5 (94 amino acids) and truncated form of NADH-Cytochrome B5 reductase (277 amino acids) have been studied. It is shown that electron transfer takes place between redox partners belonging to two different molecules of the chimeric protein. Using computer modeling, we built the model of the tertiary structure of the fusion protein, which is in agreement with experimental data. By using Marcus theory of electron transfer in polar media, we demonstrate the inability of the hypothesis of electrostatic repulsions to explain the increase of electron transfer rate on increase of ion concentration in media due to elimination of the repulsion of similar charges. The real reason for the increase of the first order rate constant in some oxidation-reduction reactions between proteins, as shown in the present work, is a decrease of the media reorganization energy resulting in decrease of activation energy for oxidation-reduction reactions.
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Expression of outer mitochondrial membrane Cytochrome B5 in Escherichia coli. purification of the recombinant protein and studies of its interaction with electron-transfer partners.
Biochemistry. Biokhimiia, 2006Co-Authors: G. V. Sergeev, Andrei A. Gilep, Ronald W. Estabrook, Sergey A. UsanovAbstract:In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length Cytochrome B5 from outer mitochondrial membrane. Optimization of expression conditions for Cytochrome B5 from outer mitochondrial membrane allowed reaching expression level up to 104 nmol of the hemeprotein per liter of culture. Recombinant Cytochrome B5 from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of Cytochrome B5 from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial Cytochrome B5 was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:Cytochrome P450 reductase with both forms of Cytochrome B5 an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:Cytochrome P450 reductase is different for isoforms of Cytochrome B5.
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Site-directed mutagenesis of Cytochrome B5 for studies of its interaction with Cytochrome P450.
Biochemistry. Biokhimiia, 2001Co-Authors: M V Chudaev, Andrei A. Gilep, Sergey A. UsanovAbstract:We have shown earlier that microsomal Cytochrome B5 can form a specific complex with mitochondrial Cytochrome P450 (Cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized Cytochrome B5, and by measuring the cholesterol side-chain cleavage activity of Cytochrome P450scc in a reconstituted system in the presence of Cytochrome B5. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of Cytochrome B5, which are located at the site responsible for interaction with electron transfer partners, we used site-directed mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of Cytochrome B5 were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these Cytochrome B5 mutants were purified. Addition of the truncated forms of Cytochrome B5 (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing Cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of Cytochrome B5 in its interaction with Cytochrome P450scc. However, full-length Cytochrome B5 and the full-length Glu48Lys and Asp65Ala mutant forms of Cytochrome B5 stimulated the cholesterol side-chain cleavage reaction catalyzed by Cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of Cytochrome B5 are not directly involved in its interaction with Cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of Cytochrome B5 about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of Cytochrome P450scc, indicating that residue Glu42 of Cytochrome B5 is involved in electrostatic interactions with Cytochrome P450scc. Residues Glu42 and Glu48 of Cytochrome B5 appear to participate in electrostatic interaction with microsomal type Cytochrome P450.
Seyed Abbas Mirzaei - One of the best experts on this subject based on the ideXlab platform.
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human Cytochrome B5 reductase structure function and potential applications
Critical Reviews in Biotechnology, 2014Co-Authors: Fatemeh Elahian, Bahareh Moghimi, Zargham Sepehrizadeh, Seyed Abbas MirzaeiAbstract:AbstractCytochrome B5 reductase is a flavoprotein that is produced as two different isoforms that have different localizations. The amphipathic microsomal isoform, found in all cell types with the exception of erythrocytes, consists of one hydrophobic membrane-anchoring domain and a larger hydrophilic flavin catalytic domain. The soluble Cytochrome B5 reductase isoform, found in human erythrocytes, is a truncated protein that is encoded by an alternative transcript and consists of the larger domain only. Cytochrome B5 reductase is involved in the transfer of reducing equivalents from the physiological electron donor, NADH, via an FAD domain to the small molecules of Cytochrome B5. This protein has received much attention from researchers due to its involvement in many oxidation and reduction reactions, such as the reduction of methemoglobin to hemoglobin. Autosomal Cytochrome B5 reductase gene deficiency manifests with the accumulation of oxidized Fe+3 and recessive congenital methemoglobinemia in humans....
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human Cytochrome B5 reductase structure function and potential applications
Critical Reviews in Biotechnology, 2014Co-Authors: Fatemeh Elahian, Bahareh Moghimi, Zargham Sepehrizadeh, Seyed Abbas MirzaeiAbstract:Cytochrome B5 reductase is a flavoprotein that is produced as two different isoforms that have different localizations. The amphipathic microsomal isoform, found in all cell types with the exception of erythrocytes, consists of one hydrophobic membrane-anchoring domain and a larger hydrophilic flavin catalytic domain. The soluble Cytochrome B5 reductase isoform, found in human erythrocytes, is a truncated protein that is encoded by an alternative transcript and consists of the larger domain only. Cytochrome B5 reductase is involved in the transfer of reducing equivalents from the physiological electron donor, NADH, via an FAD domain to the small molecules of Cytochrome B5. This protein has received much attention from researchers due to its involvement in many oxidation and reduction reactions, such as the reduction of methemoglobin to hemoglobin. Autosomal Cytochrome B5 reductase gene deficiency manifests with the accumulation of oxidized Fe+3 and recessive congenital methemoglobinemia in humans. In this article, we provide a comprehensive overview of the structure and function of Cytochrome B5 reductase from different eukaryotic sources and its potential use in the food industry, biosensor, and diagnostic areas.
Lauren A Trepanier - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome B5 and nadh Cytochrome B5 reductase genotype phenotype correlations for hydroxylamine reduction
Pharmacogenetics and Genomics, 2010Co-Authors: James C Sacco, Lauren A TrepanierAbstract:Objectives NADH Cytochrome B5 reductase (B5R) and Cytochrome B5 (B5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in B5 and B5R cDNA.
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unusual dehydroxylation of antimicrobial amidoxime prodrugs by Cytochrome B5 and nadh Cytochrome B5 reductase
Drug Metabolism and Disposition, 2005Co-Authors: Janelle Y Saulter, Lauren A Trepanier, Joseph R Kurian, Richard R Tidwell, Arlene S Bridges, David W Boykin, Chad E Stephens, Mariappan Anbazhagan, James Edwin HallAbstract:Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by Cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble Cytochrome B5 and NAD Cytochrome B5 reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to Cytochrome B5 and B5 reductase and by chemical inhibitors of B5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain Cytochrome B5/B5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed Cytochrome B5/B5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. Km values were similar to those for microsomes, and Vmax values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with Cytochrome B5 or B5 reductase alone or with Cytochrome P450 reductase alone or with Cytochrome B5. These results indicate that Cytochrome B5 and B5 reductase play a direct role in metabolic activation of DB289 to furamidine.
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unusual dehydroxylation of antimicrobial amidoxime prodrugs by Cytochrome B5 and nadh Cytochrome B5 reductase
Drug Metabolism and Disposition, 2005Co-Authors: Janelle Y Saulter, Lauren A Trepanier, Joseph R Kurian, Richard R Tidwell, Arlene S Bridges, David W Boykin, Chad E Stephens, Mariappan Anbazhagan, James Edwin HallAbstract:Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by Cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble Cytochrome B5 and NAD Cytochrome B5 reductase, expressed in Escherichia coli .K inetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to Cytochrome B5 and B5 reductase and by chemical inhibitors of B5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain Cytochrome B5/B5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed Cytochrome B5/B5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. Km values were similar to those for microsomes, and Vmax values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with Cytochrome B5 or B5 reductase alone or with Cytochrome P450 reductase alone or with Cytochrome B5. These results indicate that Cytochrome B5 and B5 reductase play a direct role in metabolic activation of DB289 to furamidine. 2,5-Bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289) is an antimicrobial prodrug developed for the treatment of a variety of microbial infections. DB289 has completed phase II clinical trials for African trypanosomiasis in Angola and the Democratic Republic of Congo and is currently enrolled in phase II trials for malaria in Thailand and for Pneumocystis pneumonia in Peru. In the phase II clinical trials involving patients with primary-stage African trypanosomiasis, treatment with DB289 achieved cure rates of approximately 95%. Moreover, DB289 was found to be well tolerated, with no significant side effects (J. Allen, unpublished). The study of drug metabolism is a key component in the drug discovery process. A compound’s metabolic pathway can provide valuable information, including the identification of metabolites, the rate and extent of metabolism, the enzymes responsible for catalyzing metabolism, and potentially dangerous drug-drug interactions. More importantly for DB289 is the role of drug metabolism for activation of this inactive prodrug. The phase I metabolic pathway for DB289 conversion to the active dicationic compound 2,5-bis(4-amidinophenyl)furan (furamidine; DB75) has been determined in vitro using freshly isolated rat hepatocytes (Zhou, 2001). DB289 uptake and metabolism by rat liver hepatocytes was rapid, with furamidine detectable inside the cells within 30 min. Further investigations resulted in the detection of four other intermediate phase I metabolites as shown in Fig. 1. The metabolic conversion of DB289 to furamidine is complex with two different metabolic routes that converge on M4. Efficient transformation and enzymatic activation through this metabolic pathway are required for sufficient quantities of furamidine to reach its target. Therefore, characterizing the enzymes involved in the metabolic conversion is important for evaluating the metabolic disposition of these compounds in vivo. Preliminary results using rat hepatocytes with 1-aminobenzotria
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UNIT 4.16 Evaluation of the Cytochrome B5/Cytochrome B5 Reductase Pathway
Current protocols in immunology, 2005Co-Authors: Lauren A Trepanier, Sunil U Bajad, Joseph R KurianAbstract:NADH Cytochrome B5 reductase (B5R; EC 1.6.2.2; Diaphorase I; NADH: ferriCytochrome B5 oxidoreductase) is an FAD-containing protein, which, along with the hemoprotein Cytochrome B5 (cyt B5), mediates electron transfer from NADH to fatty acid desaturases, P450 oxidases, methemoglobin, and ascorbyl free radical. In addition, B5R and cyt B5 can directly catalyze the reduction of hydroxylamine and amidoxime metabolites. This unit provides protocols for measuring the activity and mRNA expression of the Cytochrome B5/Cytochrome B5 reductase pathway, and for obtaining heterologous expression and purification of the soluble forms of each protein. Keywords: Cytochrome B5 reductase; Cytochrome B5; hydroxylamine; ferrihemoglobin reduction; qRT-PCR; protein purification
