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 ni tric oxide (NO) inhibits NO synthase. Previous studies revealed that N O inhibits unpurified preparations of NO synthase. In the present stud y, the mechanism by which NO inhibits purified neuronal NO synthase fr om rat cerebellum was examined. The rate of L-citrulline formation fro m L-arginine was non-linear despite the presence of excess substrate a dn cofactors and was further inhibited by 30% by 200 units/ml superoxi de dismutase. In contrast, 30 mu M oxyhemoglobin increased NO synthase activity by 2-fold and made the reaction rate linear. These observati ons were consistent with the hypothesis that enzymatically generated N O inhibits NO synthase activity. Exogenous NO (0.1-10 mu M) (but not N O2, nitrite, or nitrate) also inhibited NO synthase, and enzyme inhibi tion 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 mu M) or a tetrahydrobiopterin-regenerating system, and the latter mad e the reaction rate linear. In contrast, NO synthase inhibition by NO was markedly enhanced by heme oxidants (10 mu M methylene blue; 3 mu M ferricynanide), and these oxidants directly inhibited NO synthase act ivity. In support of this view, NO inhibited enzyme activity in the ab sence of turnover, when the heme iron is in the ferric state, and this inhibition was reversed by tetrahydrobiopterin. Therefore, the oxidat ion state of heme iron appears to be one important determinant for the inhibitory action of NO, and tetrahydrobiopterin may increase NO synt hase activity by diminishing the inhibitory action of NO.