Redox control of the catalytic cycle of flavocytochrome P-450 BM3

S N Daff, Stephen K Chapman, K L Turner, R A Holt, S Govindaraj, T L Poulos, A W Munro

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    Abstract

    Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptlde whose heme and diflavln 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 elctron 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 dr-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.

    Original languageEnglish
    Pages (from-to)13816-13823
    Number of pages8
    JournalBiochemistry
    Volume36
    Issue number45
    DOIs
    Publication statusPublished - 11 Nov 1997

    Keywords

    • ELECTRON-TRANSFER
    • NADPH
    • REDUCTASE
    • PURIFICATION
    • SUBSTRATE-BINDING
    • FLAVOPROTEIN DOMAIN
    • BACILLUS-MEGATERIUM
    • CYTOCHROME-P-450 BM3

    Cite this

    Daff, S. N., Chapman, S. K., Turner, K. L., Holt, R. A., Govindaraj, S., Poulos, T. L., & Munro, A. W. (1997). Redox control of the catalytic cycle of flavocytochrome P-450 BM3. Biochemistry, 36(45), 13816-13823. https://doi.org/10.1021/bi971085s