Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6 - Lack of an allosteric role of nadph-cytochrome P450 reductase in catalytic regioselectivity

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human de brisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regiosel ectivity of oxidation products formed in systems containing NADPH-P450 redu ctase/NADPH and the model oxidant cumene hydroperoxide have been proposed b y others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., W olf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We ex amined the differences in the formation of oxidation products of N-methyl-4 -phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reduc tase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalyt ic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influ ences. The presence of the reductase had little effect on the affinity of P 450 2D6 for any of these three substrates. The addition of the reaction rem nants of the model oxidants (cumyl alcohol and iodobenzene) to the reductas e-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from (H2 O)-O-18 was quantitatively incorporated into 1'-hydroxybufuralol in the iod osylbenzene- but not in the reductase- or cumene hydroperoxide-supported re actions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distri butions, not an allosteric influence of the reductase.