Mechanistic studies on the reductive half-reaction of NADPH-cytochrome P450 oxidoreductase

Site-directed mutagenesis has been employed to study the mechanism of hydri de transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specificall y, Ser(457), Asp(675), and Cys(630) have been selected because of their pro ximity to the isoalloxazine ring of FAD, Substitution of Asp(675) with aspa ragine or valine decreased cytochrome c reductase activities 17- and 677-fo ld, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytoc hrome c reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A, and Kasper, C, B, (1996) Biochemistry 35, 945 1-9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly mul tiplicative decreases in cytochrome c reductase activity (774- and 22000-fo ld, respectively) with corresponding decreases in the rates of flavin reduc tion. For each mutation, increases were observed in the magnitudes of the p rimary deuterium isotope effects with NADPD, consistent with decreased rate s of hydride transfer from NADPH to FAD and an increase in the relative rat e limitation of hydride transfer. Asp(675) substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shift ed the pK(a) of an ionizable group previously identified as necessary for c atalysis (Sem, D. S,, and Kasper, C, B. (1993) Biochemistry 32, 11539-11547 ) from 6.9 to 7.8, These results are consistent with a model in which Ser(4 57), Asp(675), and Cys(630) stabilize the transition state for hydride tran sfer. Ser(457) and Asp(675) interact to stabilize both the transition state and the FAD semiquinone, while Cys(630) interacts with the nicotinamide ri ng and the fully reduced FAD, functioning as a proton donor/acceptor to FAD .