Publications

• Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis.

  Marohnic C.C., Crowley L.J., Davis C.A., Smith E.T., Barber M.J.

  The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" mo ... >> More

  The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor. << Less

  Biochemistry 44:2449-2461(2005) [PubMed] [EuropePMC]

• Human cytochrome b5 reductase: structure, function, and potential applications.

  Elahian F., Sepehrizadeh Z., Moghimi B., Mirzaei S.A.

  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 fla ... >> More

  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. << Less

  Crit Rev Biotechnol 34:134-143(2014) [PubMed] [EuropePMC]

• Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.

  Zhu H., Qiu H., Yoon H.-W., Huang S., Bunn H.F.

  Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) cont ... >> More

  Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using BLAST searches against DBEST for FAD-, NAD(P)H-binding sequences followed by reverse transcription-PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor. << Less

  Proc. Natl. Acad. Sci. U.S.A. 96:14742-14747(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of soluble NADH-cytochrome b5 reductase of rabbit erythrocytes.

  Yubisui T., Takeshita M.

  Soluble NADH-cytochrome b5 reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4B column. The enzyme wa ... >> More

  Soluble NADH-cytochrome b5 reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4B column. The enzyme was purified about 12,000-fold from hemolysate in terms of NADH-cytochrome b5 reductase activity with a high yield of 40%. The purified enzyme has absorption maxima at 273, 390, and 462 nm, and shoulders at 370, 435, and 488 nm. The ratio of the absorbance at 273 nm to that at 462 nm of the purified enzyme was 5.6-5.8. The prosthetic group of the enzyme was found to be FAD, and the flavin content in the enzyme was 1 mol/mol of the enzyme. The molecular weight of the purified enzyme was estimated to be 33,000 and 32,000 by gel filtration on a Sephadex G-75 column, and by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, respectively. The NADH-cytochrome b5 reductase activity decreased strikingly as the buffer or salt concentration in the assay mixture was increased, and the optimal pH for the reduction of cytochrome b5 with NADH was determined to be 6.6 in Tris-maleate buffer of constant ionic strength. The maximum velocity of NADH-cytochrome b5 reductase activity of the purified enzyme was very high, 1,280 mumol/min/mg of protein in 10 mM phosphate buffer (pH 6.6). The Michaelis constants for NADH and cytochrome b5 were determined to be 2.5 and 4 microM, respectively. The reduction of cytochrome b5 with NADH by the enzyme was suggested to follow the ordered-type reaction mechanism based on the modes of product inhibition. From these results, and also from the estimated enzyme content in the erythrocytes (16-20 mg protein per liter of packed erythrocytes), the possible contribution of the enzyme to functions other than methemoglobin reduction in rabbit erythrocytes is discussed. << Less

  J Biochem 91:1467-1477(1982) [PubMed] [EuropePMC]

• Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential.

  Shirabe K., Yubisui T., Nishino T., Takeshita M.

  Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). ... >> More

  Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced. << Less

  J. Biol. Chem. 266:7531-7536(1991) [PubMed] [EuropePMC]

• One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase.

  Iyanagi T., Watanabe S., Anan K.F.

  The one-electron oxidation-reduction properties of flavin in hepatic NADH-cytochrome b5 reductase were investigated by optical absorption spectroscopy, electron paramagnetic resonance (EPR), and potentiometric titration. An intermediate with a peak at 375 nm previously described by Iyanagi (1977) ... >> More

  The one-electron oxidation-reduction properties of flavin in hepatic NADH-cytochrome b5 reductase were investigated by optical absorption spectroscopy, electron paramagnetic resonance (EPR), and potentiometric titration. An intermediate with a peak at 375 nm previously described by Iyanagi (1977) [ Iyanagi , T. (1977) Biochemistry 16, 2725-2730] was confirmed to be a red anionic semiquinone. The NAD+-bound reduced enzyme was oxidized by cytochrome b5 via the semiquinone intermediate. This indicates that electron transfer from flavin to cytochrome b5 proceeds in two successive one-electron steps. Autoxidation of the NAD+-bound reduced enzyme was slower than that of the NAD+-free reduced enzyme and was accompanied by the appearance of an EPR signal. Midpoint redox potentials of the consecutive one-electron-transfer steps in the presence of excess NAD+ were Em,1 = -88 mV and Em,2 = 147 mV at pH 7.0. This corresponds to a semiquinone formation constant of 8. The values of Em,1 and Em,2 were also studied as a function of pH. A mechanism for electron transfer from NADH to cytochrome b5 is discussed on the basis of the one-electron redox potentials of the enzyme and is compared with the electron-transfer mechanism of NADPH-cytochrome P-450 reductase. << Less

  Biochemistry 23:1418-1425(1984) [PubMed] [EuropePMC]