ROLE OF SER457 OF NADPH-CYTOCHROME P450 OXIDOREDUCTASE IN CATALYSIS AND CONTROL OF FAD OXIDATION-REDUCTION POTENTIAL

Site-directed mutagenesis of Ser457 of NADPH-cytochrome P450 oxidoredu ctase demonstrates that this residue plays a major role in both hydrid e transfer from NADPH to FAD and modulation of FAD redox potential. Su bstitution of Ser457 with alanine or cysteine decreases the rates of r eduction of the substrates cytochrome c and potassium ferricyanide app roximately 100-fold, while substitution with threonine produces a 20-f old decrease in activity. No changes are observed in K-m(NADPH), K-i(N ADP+), Or flavin content, indicating that these substitutions have no effect on cofactor binding but affect catalysis only. K-m(cyt c) value s are decreased in parallel with the observed decreases in the rates o f the reductive half-reaction. Stopped-flow studies with the S457A mut ant show a 100-fold decrease in the rate of flavin reduction. The prim ary deuterium isotope effect on k(cat) for cytochrome c reduction incr eases from 2.7 for the wild-type enzyme to 9.0 for the S457A mutant, c onsistent with a change in the rate-determining step from NADP(+) rele ase in the wild-type enzyme to hydride transfer in the S457A mutant. T he primary deuterium isotope effect on k(1) for flavin reduction at hi gh ionic strength (I = 535 mM) increases from 12.2 for the wild-type e nzyme to >20 for the S457A mutant, consistent again with an increase i n the relative rate limitation of hydride transfer. Furthermore, anaer obic titration of S457A indicates that the redox potential of the FAD semiquinone has been decreased. Data presented in this study support t he hypothesis that Ser457 is involved in hydrogen bonding interactions which stabilize both the transition state for hydride transfer and th e reduced FAD.