No· NO from NO Synthase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 25; pp. 14492 - 14497
• Main Authors: Harald H. H. W. Schmidt, Hofmann, Heinrich, Schindler, Ursula, Shutenko, Zhanna S., Cunningham, David D., Feelisch, Martin
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 10.12.1996; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences of the USA
• Subjects: Biochemistry; Biological Sciences; Electrodes; Electron Transport; Enzymes; NADP - metabolism; Nitric Oxide - metabolism; Nitric Oxide Synthase - metabolism; Nitrogen; Oxidation; Oxides; Salts; Stoichiometry; Superoxides; Thiols
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.25.14492

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (· NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify· NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable · NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal · NO standards were only partially recovered and the dismutation of superoxide (O$_{2}^{\overline{\cdot}}$), which otherwise scavenges · NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO-resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing 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 H4Bip. Here, recovery of internal · NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to · NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from · NO decomposition, was consistent with formation of an · NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that · NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.