Activation of peroxynitrite by inducible nitric-oxide synthase: a direct source of nitrative stress.
In mammals, nitric oxide (NO) is an essential biological mediator that is exclusively synthesized by nitric-oxide synthases (NOSs). However, NOSs are also directly or indirectly responsible for the production of peroxynitrite, a well known cytotoxic agent involved in numerous pathophysiological processes. Peroxynitrite reactivity is extremely intricate and highly depends on activators such as hemoproteins. NOSs present, therefore, the unique ability to both produce and activate peroxynitrite, which confers upon them a major role in the control of peroxynitrite bioactivity. We report here the first kinetic analysis of the interaction between peroxynitrite and the oxygenase domain of inducible NOS (iNOSoxy). iNOSoxy binds peroxynitrite and accelerates its decomposition with a second order rate constant of 22 x 10(4) m(-1)s(-1) at pH 7.4. This reaction is pH-dependent and is abolished by the binding of substrate or product. Peroxynitrite activation is correlated with the observation of a new iNOS heme intermediate with specific absorption at 445 nm. iNOSoxy modifies peroxynitrite reactivity and directs it toward one-electron processes such as nitration or one-electron oxidation. Taken together our results suggest that, upon binding to iNOSoxy, peroxynitrite undergoes homolytic cleavage with build-up of an oxo-ferryl intermediate and concomitant release of a NO(2)(.) radical. Successive cycles of peroxynitrite activation were shown to lead to iNOSoxy autocatalytic nitration and inhibition. The balance between peroxynitrite activation and self-inhibition of iNOSoxy may determine the contribution of NOSs to cellular oxidative stress.
Maréchal
Amandine
A
Laboratoire de Stress Oxydant et Détoxication, iBiTec-S, Commissariat à l'Energie Atomique, Saclay, Gif-sur-Yvette Cedex, France.
Mattioli
Tony A
TA
Stuehr
Dennis J
DJ
Santolini
Jérôme
J
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000494:pharmacology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D010648:Phenylacetates / Q000378:metabolism
D010648:Phenylacetates; D002245:Carbon Dioxide; D030421:Peroxynitrous Acid; D002956:Citrulline; D009569:Nitric Oxide; C008070:4-hydroxyphenylacetic acid; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D010100:Oxygen