Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase

Iyanagi, Takashi; Fk, Anan; Imai, Yoh; Hs, Mason

doi:10.1021/bi00604a032

Cited by 78 publications

(38 citation statements)

References 21 publications

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“…We found no changes in the electron-spin relaxation time compared to the power dependence of the ESR saturation properties of the protease-treated and detergent-solubilized forms of the P-450 reductase. The same observations have been made by Iyanagi et al [42].…”

Section: Discussionsupporting

confidence: 84%

A ³¹P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP⁺ reductase complex

Bonants

Müller

Vervoort

et al. 1990

European Journal of Biochemistry

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'P-nuclear-magnetic-resonance spectroscopy has been employed to probe the structure of the detergentsolubilized form of liver microsomal NADPH -cytochrome-P-450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2'-phosphate (2'-AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2'-AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220 -72281 for the protease-solubilized enzyme, the addition of Mn(I1) to the oxidized enzyme did not result in any observable line-broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2'-AMP was broadened by z 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2'-AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P-NMR spectrum as compared with the native enzyme. ferredoxin-NADP' reductase (EC 1.18.1.2). P-450. During this electron transfer, the FAD and FMN coenzymes function at both semiquinone and hydroquinone redox levels.31P-NMR spectroscopy is a powerful technique to investigate the environments of phosphorus groups in proteins and has been profitably used to study the flavin coenzyme and protein-bound phosphorus residues in Megasphaera elsdenii flavodoxin [18], Aspergillus niger glucose oxidase [19], bovine milk xanthine oxidase [20], and Azotobucter vinelandii flavodoxin [21, 221. Otvos et al. [23] have recently investigated the properties of the bound flavin phosphates in the proteolytically solubilized form of the P-450 reductase, by 31P NMR. The work reported here, on the detergent-solubilized form of the reductase, represents an extension of their published studies.In addition, it is of importance to compare the properties of the two forms in as much as the detergent-solubilized enzyme retains activity in the cytochrome P-450 assay while the proteolytically solubilized form does not [16, 171. As documented in this paper, the most significant difference in the 31P-NMR spectral properties of the two forms of the enzyme is that the FMN phosphorus resonance is extensively broadened on formation of the air-stable FMN

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Section: Discussionsupporting

confidence: 84%

A ³¹P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP⁺ reductase complex

Bonants

Müller

Vervoort

et al. 1990

European Journal of Biochemistry

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“…The A2771A456 ratio for the rabbit liver reductase is 7.4 as compared with 8.7 for the rat liver reductase (13) and 6.7 and 6.3 for the rat liver enzyme solubilized by treatment with bromelain (41) or trypsin (42), respectively. The minimal molecular weight of the rabbit liver microsomal reductase was estimated from the flavin content to be 77,000; this figure is in reasonable agreement with the value of 78,000 reported by Iyanagi et al (17) and 74,000 determined by calibrated SDS-polyacrylamide gel electrophoresis in the present study.…”

supporting

confidence: 93%

“…The availability of the highly purified reductase as well as two forms of cytochrome P-450 (14,15) from a single source will permit more meaningful studies on the interactions of these enzymes in the reconstituted system (16). While this paper was in preparation, Iyanagi et al (17) reported the extensive purification and some of the properties of the rabbit liver reductase. The reductase is unusual in containing both FMN and FAD, as first reported for trypsin-solubilized preparations (18) and confirmed for the de-tergent-solubilized enzyme purified from both rat (10)(11)(12)(13) and rabbit liver microsomes (9,17).…”

mentioning

confidence: 99%

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes

French

Coon

1979

Archives of Biochemistry and Biophysics

184

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NADPH-cytochrome P-450 reductase has been purified to electrophoretic homogeneity from rabbit liver microsomes by a procedure that may be used in conjunction with the isolation of the major forms of cytochrome P-450. The purified reductase is active in a reconstituted hydroxylation system containingp-45OLM, or P-~~OLM.,. The enzyme contains one molecule each of FMN and FAD per polypeptide chain having an apparent minimal molecular weight of 74,000. Immunological techniques provided evidence for only a single form of the reductase; lower molecular weight forms occasionally seen are believed to be due to degradation by contaminating microsomal or bacterial proteases. Upon anaerobic photochemical reduction, the rabbit liver reductase undergoes spectral changes highly similar to those previously described by Vermilion and Coon for the rat liver enzyme; the fully reduced rabbit liver enzyme is converted to the three-electron-reduced form by the addition of NADP and then to the stable one-electron-reduced form by exposure to oxygen. The CD spectra of the fully oxidized enzyme, one-electron-reduced form (air-stable semiquinone), three-electron-reduced form, and fully reduced form are presented. The results obtained provide evidence that the FMN and FAD are in highly different environments in the enzyme, as also indicated by the different redox potentials and oxygen reactivities of the flavins.

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“…The molecular weights of the human and rabbit proteins were 77 500 and 76 500, respectively. While the molecular weight of the human enzyme is 3500 higher than that reported by Guengerich et al (1981), the molecular weight of the rabbit reductase compares favourably with other values in the literature (Vermillion &Coon 1974;Iyanagi et al 1978;Black & Coon 1982). In this study the human reductase was purified in the presence of phenylmethylsulfonyl fluoride and the appearance of lower molecular weight fragments due to proteolysis was avoided (Guengerich et al 198 1).…”

Section: Discussionsupporting

confidence: 73%

Immunochemical and Catalytical Characterization of the Human Liver Nadph‐cytochrome P450 Reductase

McManus¹,

Huggett²,

Burgess³

et al. 1989

Clin Exp Pharma Physio

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1. The NADPH-cytochrome P450 reductases (EC 1.6.2.4) from human and rabbit liver have been purified to electrophoretic homogeneity. The human reductase had an apparent monomeric molecular weight of 77,500 and the rabbit enzyme of 76,500. 2. Both flavoproteins exhibited typical flavoprotein spectra and contained equimolar quantities of FAD and FMN. The two reductases were catalytically active in reducing cytochrome c, ferricyanide and dichlorophenolindophenol, and in supporting rabbit liver cytochrome P450 Form 4 metabolism of 2-acetylaminofluorene. 3. An antibody raised in the goat against the human enzyme formed a precipitin line with the human reductase in a double-diffusion assay, but did not react with the rabbit reductase. Similarly, an antibody raised in the goat against the rabbit reductase formed a precipitin line with the rabbit enzyme, but did not cross-react with the human reductase. 4. Both antibodies inhibited cytochrome c reduction by the two reductases suggesting some immunochemical recognition. 5. Immunochemical cross-reactivity was confirmed when both reductases were subjected to the more sensitive immunoblot technique using either anti-human or anti-rabbit reductase IgG. 6. The human and rabbit reductases are essentially similar in amino acid composition, except that the former has larger amounts of serine and glycine.

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Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase

Cited by 78 publications

References 21 publications

A ³¹P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP⁺ reductase complex

A ³¹P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP⁺ reductase complex

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes

Immunochemical and Catalytical Characterization of the Human Liver Nadph‐cytochrome P450 Reductase

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Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase

Cited by 78 publications

References 21 publications

A 31P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP+ reductase complex

A 31P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP+ reductase complex

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes

Immunochemical and Catalytical Characterization of the Human Liver Nadph‐cytochrome P450 Reductase

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A ³¹P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP⁺ reductase complex

A ³¹P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP⁺ reductase complex