NO CENTER-DOT-NO FROM NO SYNTHASE

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H(4)Bip)-dependent 5-electron oxid ation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O-2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (. NO). Analysis of NOS a ctivity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to direct ly quantify . NO formation from purified NOS. However, by two independ ent methods. NOS did not yield detectable . NO unless superoxide dismu tase (SOD; EC 1.15.1.1) was present. In the presence of H(4)Bip, inter nal . NO standards were only partially recovered and the dismutation o f superoxide (O-2(radical anion)), which otherwise scavenges . NO to y ield ONOO-, was a plausible mechanism of action of SOD. Under these co nditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:C it stoichiometry to 0.8-1.1, suggesting either that additional reducin g equivalents besides NADPH are required to explain Arg oxidation to . NO or that . NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H(4)Bip. Here, rec overy of internal . NO standards was unaffected. Thus, a second activi ty of SOD, the conversion of nitroxyl (NO-) to . NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N 2O) and hydroxylamine (NH2OH), which cannot arise from . NO decomposit ion, was consistent with formation of an . NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitroso glutathione was detected. Our results indicate that . NO is not the pr imary reaction product of NOS-catalyzed Arg turnover and an alternativ e reaction mechanism and stoichiometry have to be taken into account.