Nitric oxide inhibits neuronal nitric oxide synthase by interacting with the heme prosthetic group. Role of tetrahydrobiopterin in modulating the inhibitory action of nitric oxide.

The objective of this study was to elucidate the mechanism by which nitric oxide (NO) inhibits NO synthase. Previous studies revealed that NO inhibits unpurified preparations of NO synthase. In the present study, the mechanism by which NO inhibits purified neuronal NO synthase from rat cerebellum was examined. The rate of L-citrulline formation from L-arginine was non-linear despite the presence of excess substrate and cofactors and was further inhibited by 30% by 200 units/ml superoxide dismutase. In contrast, 30 microM oxyhemoglobin increased NO synthase activity by 2-fold and made the reaction rate linear. These observations were consistent with the hypothesis that enzymatically generated NO inhibits NO synthase activity. Exogenous NO (0.1-10 microM) (but not NO2, nitrite, or nitrate) also inhibited NO synthase, and enzyme inhibition was not competitive with L-arginine. NO synthase inhibition by NO and other heme ligands supports the view that heme is involved in the catalytic activity of NO synthase. Oxyhemoglobin prevented but could not reverse enzyme inhibition by NO. NO synthase inhibition by NO was markedly diminished and reversed, however, by tetrahydrobiopterin (50 microM) or a tetrahydrobiopterin-regenerating system, and the latter made the reaction rate linear. In contrast, NO synthase inhibition by NO was markedly enhanced by heme oxidants (10 microM methylene blue; 3 microM ferricyanide), and these oxidants directly inhibited NO synthase activity. These observations suggest that NO interacts with enzyme-bound ferric heme to inhibit NO synthase activity. In support of this view, NO inhibited enzyme activity in the absence of turnover, when the heme iron is in the ferric state, and this inhibition was reversed by tetrahydrobiopterin. Therefore, the oxidation state of heme iron appears to be one important determinant for the inhibitory action of NO, and tetrahydrobiopterin may increase NO synthase activity by diminishing the inhibitory action of NO.