Reductive activation of mitomycin C by neuronal nitric oxide synthase.

Mitomycin C (MC) requires bioreduction prior to the generation of alkylating moieties. NADPH-cytochrome P450 reductase is predominant in metabolic activation of MC in hypoxic cancer cells. In this study, neuronal nitric oxide synthase (nNOS), whose reductase domain is structurally similar to that of NADPH-cytochrome P450 reductase, was assessed for its ability to activate MC. nNOS under anaerobic conditions catalyzed the reduction of MC, which was measured as the decrease in absorbance at 375 nm. Neither the heme blocker potassium cyanide (1 mM) nor the nNOS competitive inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM) affected the bioreduction of MC, whereas 0.1 mM diphenyleneiodonium chloride, which binds to the reductase domain of nNOS, inhibited MC reduction completely. The reduction of MC by nNOS was influenced by Ca(2+)/calmodulin. In the absence of Ca(2+)/calmodulin, the rate of MC reduction decreased by 28% at pH 6.6. The formation of an alkylated complex of 4-(p-nitrobenzyl)pyridine occurred in a manner analogous to that observed in MC metabolic experiments. The rate of MC reduction and the formation of the alkylated complex of 4-(p-nitrobenzyl)pyridine at pH 6.6 were increased by 43 and 54%, respectively, as compared with that at pH 7.6. nNOS-activated MC resulted in the consumption of oxygen in air. The rate of oxygen consumption decreased by 50% in the presence of 2000 U/mL of catalase. MC inhibited nNOS activity in a noncompetitive manner. These findings demonstrate that nNOS is capable of catalyzing the bioreduction of MC.