Mechanism of superoxide generation by neuronal nitric-oxide synthase

Abstract

Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O2/·-) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO·). Yet the competition between O2 and L-arginine for electrons, and by implication formation of O2/·-, has until recently remained undefined. Herein, we investigated this relationship, observing O2/·- generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor N(G)-monomethyl L-arginine enhanced O2/·- production in the presence of L-arginine because this antagonist attenuated NO· formation. Whereas diphenyliodonium chloride inhibited O2/·-, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O2/·- at concentrations that inhibited NO· formation from L-arginine. Taken together these data demonstrate that NOS I generates O2/·- and the formation of this free radical occurs at the heme domain.