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 oxidoreductase demonstrates that this residue plays a major role in both hydride transfer from NADPH to FAD and modulation of FAD redox potential. Substitution of Ser457 with alanine or cysteine decreases the rates of reduction of the substrates cytochrome c and potassium ferricyanide approximately 100-fold, while substitution with threonine produces a 20-fold decrease in activity. No changes are observed in k(m)NADPH, KiNADP+, or flavin content, indicating that these substitutions have no effect on cofactor binding but affect catalysis only. k(m)cyt c values are decreased in parallel with the observed decreases in the rates of the reductive half-reaction. Stopped-flow studies with the S457A mutant show a 100-fold decrease in the rate of flavin reduction. The primary deuterium isotope effect on Kcat for cytochrome c reduction increases from 2.7 for the wild-type enzyme to 9.0 for the S457A mutant, consistent with a change in the rate-determining step from NADP+ release in the wild-type enzyme to hydride transfer in the S457A mutant. The primary deuterium isotope effect on K1 for flavin reduction at high ionic strength (I = 535 mM) increases from 12.2 for the wild-type enzyme to > 20 for the S457A mutant, consistent again with an increase in the relative rate limitation of hydride transfer. Furthermore, anaerobic titration of S457A indicates that the redox potential of the FAD semiquinone has been decreased. Data presented in this study support the hypothesis that Ser457 is involved in hydrogen bonding interactions which stabilize both the transition state for hydride transfer and the reduced FAD.
Shen
A L
AL
McArdle Laboratory for Cancer Research, Madison, Wisconsin 53706, USA.
Kasper
C B
CB
D001483:Base Sequence; D003574:Cytochrome c Group / Q000378:metabolism; D017931:DNA Primers; D004227:Dithionite / Q000494:pharmacology; D004579:Electron Transport; D005292:Ferricyanides / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry
D003574:Cytochrome c Group; D017931:DNA Primers; D005292:Ferricyanides; D009243:NAD; C007931:hexacyanoferrate III; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D012694:Serine; D009249:NADP; C005941:3-acetylpyridine adenine dinucleotide; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase