title: Redox control of the catalytic cycle of flavocytochrome P-450 BM3.
abstract: Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.
authors.affiliation: Department of Chemistry, University of Edinburgh, U.K.
authors.forename: S N
authors.initials: SN
authors.lastname: Daff
authors.forename: S K
authors.initials: SK
authors.lastname: Chapman
authors.forename: K L
authors.initials: KL
authors.lastname: Turner
authors.forename: R A
authors.initials: RA
authors.lastname: Holt
authors.forename: S
authors.initials: S
authors.lastname: Govindaraj
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
authors.forename: A W
authors.initials: AW
authors.lastname: Munro
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D011199:Potentiometry; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases