Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H(2)S) donor Na(2)S with IC(50) values of ∼2.4·10(−5) and ∼7.9·10(−5) m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H(2)S does not interfere with substrate or cofactor binding. The IC(50) decreased to ∼1.5·10(−5) m at pH 6.0 and increased to ∼8.3·10(−5) m at pH 8.0. Preincubation of concentrated nNOS with H(2)S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H(2)S and NO. Likewise, NADPH oxidation was inhibited with an IC(50) of ∼1.9·10(−5) m in the presence of Arg and BH4 but exhibited much higher IC(50) values (∼1.0–6.1·10(−4) m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na(2)S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC(50) ∼ 1.3–2.0·10(−5) m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H(2)S/NO at modest concentrations of H(2)S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H(2)S formation.