Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica)

Recombinant house Ay (Musca domestica) cytochrome P450 reductase has been p urified by anion exchange and affinity chromatography. Steady-state kinetic s of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as c atalytic intermediate. NADP(H) binding is essential for fast hydride ion tr ansfer to FAD, as well as for electron transfer from FMN to cytochrome c. R educed cytochrome c had no effect on the enzyme activity, while NADP(+) and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and no ncompetitively with respect to cytochrome c. The affinity of the P450 reduc tase to NADPH is 10 times higher than to NADP(+) (K-d of 0.31 and 3.3 mu M, 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 ra te constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP(+) decreased 9-f old compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinami de binding site of the active, full-size enzyme in solution. (C) 1999 Elsev ier Science Ltd. All rights reserved.