COOPERATIVITY IN OXIDATIONS CATALYZED BY CYTOCHROME-P450 3A4

Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidize s a diversity of substrates, including various drugs, steroids, carcin ogens, and macrolide natural products. In some reactions, positive coo perativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone. alpha NF), have been shown to stimulate some reactions but not others. In systems containing purifie d recombinant bacterial P450 3A4, positive cooperativity was seen in o xidations of several substrates, including testosterone, 17 beta-estra diol, amitriptyline, and most notably aflatoxin (AF) B-1. With these a nd other reactions, alpha NF typically reduced cooperativity (i.e., th e n value in a Hill plot) while either stimulating or inhibiting react ions. With the substrate AFB(1), alpha NF both stimulated 8,9-epoxidat ion and inhibited 3 alpha-hydroxylation. The same patterns were seen w ith AFB(1) in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins . The patterns of AFB(1) oxidation to the two products were modified c onsiderably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroper oxide. AFB(2), which differs from AFB(1) only in the presence of a sat urated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB(1) oxidation. These and other results are considered in the context of s everal possible models. The results support a model in which an allost eric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of vet. In order to expla in the differential effects of alpha NF and reduction systems on the t wo oxidations of AFB(1), a model is presented in which binding of subs trate in a particular conformation can facilitate oxygen activation to enhance catalysis.