Site-directed mutagenesis has been employed to study the mechanism of hydride transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specifically, Ser⁴⁵⁷, Asp⁶⁷⁵, and Cys⁶³⁰ have been selected because of their proximity to the isoalloxazine ring of FAD. Substitution of Asp⁶⁷⁵ with asparagine or valine decreased cytochromec reductase activities 17- and 677-fold, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytochromec reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A., and Kasper, C. B. (1996) Biochemistry 35, 9451–9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly multiplicative decreases in cytochrome c reductase activity (774- and 22000-fold, respectively) with corresponding decreases in the rates of flavin reduction. For each mutation, increases were observed in the magnitudes of the primary deuterium isotope effects with NADPD, consistent with decreased rates of hydride transfer from NADPH to FAD and an increase in the relative rate limitation of hydride transfer. Asp⁶⁷⁵substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shifted the pK _a of an ionizable group previously identified as necessary for catalysis (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 Ser⁴⁵⁷, Asp⁶⁷⁵, and Cys⁶³⁰ stabilize the transition state for hydride transfer. Ser⁴⁵⁷ and Asp⁶⁷⁵interact to stabilize both the transition state and the FAD semiquinone, while Cys⁶³⁰ interacts with the nicotinamide ring and the fully reduced FAD, functioning as a proton donor/acceptor to FAD.