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No?·NO from NO?synthase

Schmidt, Harald H.H.W., Hofmann, Heinrich, Schindler, Ursula, Shutenko, Zhanna S., Cunningham, David D. and Feelisch, Martin (1996) No?·NO from NO?synthase Proceedings of the National Academy of Sciences, 93, (25), pp. 14492-14497.

Record type: Article

Abstract

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 (O2-.), 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.

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More information

Published date: 10 December 1996
Keywords: NADPH, superoxide dismutase, hydroxylamine, nitroxyl, peroxynitrite
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 337890
URI: http://eprints.soton.ac.uk/id/eprint/337890
ISSN: 0027-8424
PURE UUID: 170f5288-8f28-4bda-899a-1d9fac064aa7
ORCID for Martin Feelisch: ORCID iD orcid.org/0000-0003-2320-1158

Catalogue record

Date deposited: 29 Jun 2012 13:51
Last modified: 18 Jul 2017 06:00

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Contributors

Author: Harald H.H.W. Schmidt
Author: Heinrich Hofmann
Author: Ursula Schindler
Author: Zhanna S. Shutenko
Author: David D. Cunningham
Author: Martin Feelisch ORCID iD

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