Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase

Abstract

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 μM, limiting both purine oxidation and formation of superoxide (O2.-). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, β-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO 2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2.- production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.