REDOX CONTROL OF THE CATALYTIC CYCLE OF FLAVOCYTOCHROME P-450 BM3

Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polype ptlde whose heme and diflavln domains are fused to produce a catalytic ally self-sufficient fatty acid monooxygenase. Redox potentiometry stu dies have been performed with intact flavocytochrome P-450 BM3 and wit h its component heme, diflavin, FAD, and FMN domains, Results indicate that elctron flow occurs from the NADPH donor through FAD, then FMN a nd on to the heme center where fatty acid substrate is bound and monoo xygenation occurs. Prevention of futile cycling of electrons is avoide d 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 val ues in the larger constructs, providing further evidence for the discr ete domain organization of this flavocytochrome. The reduction potenti als of the dr-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquin one 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 accumul ate under turnover conditions. In contrast, the FMN hydroquinone is th ermodynamically more favorable than the semiquinone.