Mechanism of azoreduction of dimethylaminoazobenzene by rat liver NADPH-cytochrome P-450 reductase and partially purified cytochrome P-450. Oxygen and carbon monoxide sensitivity and stimulation by FAD and FMN.

We have reported that the hepatocarcinogen dimethylaminoazobenzene (DAB) is reduced by rat liver microsomes in an oxygen- and carbon monoxide-insensitive manner and that activity is induced by clofibrate but no other recognized inducers of cytochrome P-450 activity. In the present study we have shown that the reaction proceeds in a partially purified reconstituted cytochrome P-450 system as well as with purified NADPH-cytochrome P-450 reductase alone. In the latter system, activity is totally inhibited in air whereas the former system is active in air as well as in a carbon monoxide atmosphere. Although clofibrate induces both DAB azoreductase and laurate hydroxylase activities, the suicide substrate 10-undecynoic acid blocks the latter but not the former, implying catalysis by distinct enzymes. FAD and FMN stimulate DAB azoreduction 40-50-fold by both NADPH-cytochrome P-450 reductase alone and by the reconstituted cytochrome P-450 system. However, it was shown that these flavins facilitate electron flow to DAB only from reductase and not from cytochrome P-450. The fact that the reconstituted system, which contains NADPH-cytochrome P-450 reductase, is oxygen insensitive suggests that there is an obligatory electron flow through cytochrome P-450 to DAB, bypassing the oxygen-sensitive step.