Neuronal nitric oxide synthase generates superoxide from the oxygenase domain

When l-arginine is depleted, neuronal nitric oxide synthase (nNOS) has been reported to generate superoxide. A flavoprotein module construct of nNOS has been demonstrated to be sufficient for superoxide production. In contrast, nNOS was reported not to be involved in superoxide formation, because such formation occurred with a mixture of the boiled enzyme and redox-active cofactors. We aimed to resolve these controversial issues by examining superoxide generation, without the addition of redox-active cofactors, by recombinant wild-type nNOS and by C415A-nNOS, which has a mutation in the haem proximal site. In a superoxide-sensitive adrenochrome assay, the initial lag period of C415A-nNOS was increased 2-fold compared with that of native nNOS. With ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, prominent signals of the superoxide adduct were obtained with wild-type nNOS, whereas an enzyme preparation boiled for 5min did not produce superoxide. Higher concentrations of NaCN (10mM) decreased superoxide formation by 63%. Although the activity of the reductase domain was intact, superoxide generation from C415A-nNOS was decreased markedly, to only 10% of that of the wild-type enzyme. These results demonstrate that nNOS truly catalyses superoxide formation, that this involves the oxygenase domain, and that full-length nNOS hinders the reductase domain from producing superoxide.