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 h as been demonstrated to be sufficient for superoxide production. In contras t, nNOS was reported not to be involved in superoxide formation, because su ch formation occurred with a mixture of the boiled enzyme and redox-active cofactors. We aimed to resolve these controversial issues by examining supe roxide generation, without the addition of redox-active cofactors, by recom binant wild-type nNOS and by C415A-nNOS, which has a mutation in the haem p roximal site. In a superoxide-sensitive adrenochrome assay, the initial lag period of C415A-nNOS was increased 2-fold compared with that of native nNO S. 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, wherea s an enzyme preparation boiled for 5 min did not produce superoxide. Higher concentrations of NaCN (10 mM) decreased superoxide formation by 63%. Alth ough the activity of the reductase domain was intact, superoxide generation from C415A-nNOS was decreased markedly, to only 10 of that of the wild-typ e enzyme. These results demonstrate that nNOS truly catalyses superoxide fo rmation, that this involves the oxygenase domain, and that full-length nNOS hinders the reductase domain from producing superoxide.