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NADPH-Cytochrome P-450 Oxidoreductase THE ROLE OF CYSTEINE 566 IN CATALYSIS AND COFACTOR BINDING*

Site-directed mutagenesis was employed to investigate the role of Cyssae in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 666 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase transhydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The K,,, for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The  conservative  substitution, Ser6“, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide  phosphate activity was unaffected.  The NADPH K,,, was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KC1 concentration was markedly  altered by the substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KC1 whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c K,, and flavin content were unaffected by these substitutions. These results demonstrate that Cyseee is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.