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

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

Research output: Contribution to journalArticlepeer-review

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
Publication statusPublished - 11 Nov 1997

Keywords

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

Cite this