title: Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica).
abstract: Recombinant house fly (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromatography. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD, as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c had no effect on the enzyme activity, while NADP+ and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and noncompetitively with respect to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher than to NADP+ (Kd of 0.31 and 3.3 microM, respectively). Such an affinity change during catalysis could account for a +30 mV shift of the redox potential of FAD. Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP+ decreased 9-fold compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinamide binding site of the active, full-size enzyme in solution.
mesh_terms: D000818:Animals; D003545:Cysteine / Q000235:genetics; D003574:Cytochrome c Group / Q000378:metabolism; D004583:Electrons; D006793:Houseflies / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics