Publications

• Electron transfer in human cytochrome P450 reductase.

  Gutierrez A., Grunau A., Paine M., Munro A.W., Wolf C.R., Roberts G.C., Scrutton N.S.

  Cytochrome P450 reductase (CPR) is a diflavin enzyme responsible for electron donation to mammalian cytochrome P450 enzymes in the endoplasmic reticulum. Dissection of the enzyme into functional domains and studies by site-directed mutagenesis have enabled detailed characterization of the mechanis ... >> More

  Cytochrome P450 reductase (CPR) is a diflavin enzyme responsible for electron donation to mammalian cytochrome P450 enzymes in the endoplasmic reticulum. Dissection of the enzyme into functional domains and studies by site-directed mutagenesis have enabled detailed characterization of the mechanism of electron transfer using stopped-flow and equilibrium-perturbation methods, and redox potentiometry. These studies and the mechanism of electron transfer in CPR are reported herein. << Less

  Biochem Soc Trans 31:497-501(2003) [PubMed] [EuropePMC]

• Resolution of the cytochrome P-450-containing omega-hydroxylation system of liver microsomes into three components.

  Lu A.Y., Junk K.W., Coon M.J.

  J Biol Chem 244:3714-3721(1969) [PubMed] [EuropePMC]

• Determination of the redox properties of human NADPH-cytochrome P450 reductase.

  Munro A.W., Noble M.A., Robledo L., Daff S.N., Chapman S.K.

  Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in th ... >> More

  Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/-6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/-7 mV, E(2) = -280 +/-8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/-8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/-5 mV, E(2) [FAD] = -382 +/-8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes. << Less

  Biochemistry 40:1956-1963(2001) [PubMed] [EuropePMC]

• Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver.

  WILLIAMS C.H. Jr., KAMIN H.

  J Biol Chem 237:587-595(1962) [PubMed] [EuropePMC]

• STUDIES ON THE MECHANISM OF MICROSOMAL TRIPHOSPHOPYRIDINE NUCLEOTIDE-CYTOCHROME C REDUCTASE.

  MASTERS B.S., KAMIN H., GIBSON Q.H., WILLIAMS C.H. Jr.

  J Biol Chem 240:921-931(1965) [PubMed] [EuropePMC]

• Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes.

  Wang M., Roberts D.L., Paschke R., Shea T.M., Masters B.S.S., Kim J.-J.P.

  Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat ... >> More

  Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 A resolution. The molecule is composed of four structural domains: (from the N- to C-termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase. << Less

  Proc. Natl. Acad. Sci. U.S.A. 94:8411-8416(1997) [PubMed] [EuropePMC]

• The mechanism of 1- and 2-electron transfers catalyzed by reduced triphosphopyridine nucleotide-cytochrome c reductase.

  Masters B.S., Bilimoria M.H., Kamin H., Gibson Q.H.

  J Biol Chem 240:4081-4088(1965) [PubMed] [EuropePMC]

• NADPH-P-450 reductase: structural and functional comparisons of the eukaryotic and prokaryotic isoforms.

  Sevrioukova I.F., Peterson J.A.

  The comparison of the properties of microsomal NADPH-P-450 reductase and the flavoprotein domain of P-450BM-3 (BMR) has revealed a significant difference in the mechanism of reduction of the hemoprotein P-450 by these flavoproteins. Microsomal NADPH-P-450 reductase transfers electrons to the hemop ... >> More

  The comparison of the properties of microsomal NADPH-P-450 reductase and the flavoprotein domain of P-450BM-3 (BMR) has revealed a significant difference in the mechanism of reduction of the hemoprotein P-450 by these flavoproteins. Microsomal NADPH-P-450 reductase transfers electrons to the hemoprotein by shuttling between hydroquinone and semiquinone forms of the FMN delivering one electron per cycle. Since the microsomal NADPH-P450 reductase has evolved as a component of multi-enzyme system, this type of mechanism may permit regulation of the steps of the P-450 reaction via variation in the affinity of the reductase for different P-450s, interaction with cytochrome b5, etc. In contrast, in the soluble, bacterial flavocytochrome P-450BM-3, the reductase domain has evolved together with a single unique heme domain. This enzyme was found to utilize the fastest and simplest way to reduce the heme iron, with the FMN moiety of BMR shuttling between the semiquinone and oxidized states. This mechanism of reduction provides the highest turnover number of any P-450 and tight coupling of the monooxygenation reaction. While there are clear differences in the intermediates involved in the reduction of P-450s by these two enzymes, the domain structure and presumably the mode of interaction between the reductase and P-450s has been maintained over evolutionary time. << Less

  Biochimie 77:562-572(1995) [PubMed] [EuropePMC]