The reaction between ubiquinol and peroxynitrite

SCHÖPFER F, RIOBÓ N, LISDERO C, VALDEZ A, BOVERIS A, PODEROSO JJ

Estocolmo, Suecia

Congreso; 6th International Meeting, Biology of Nitric Oxide; 1999

NO Society

Previously, we have reported a reaction of nitric oxide(·NO) with ubiquinol to produce nitroxyl anion and ubisemiquinone and, in the presence of O2, superoxide anion (O2-); the simultaneous production of ·NO and O2- leads to intramitochondrial fomation of peroxynitrite (ONOO-). The aim of this study was to demonstrate that ONOO- oxidizes ubiquinol to form ubisemiquinone, and to evaluate a protective effect of this reaction. In this way, spectrophotometrical analysis showed ubiquinol oxidation to ubiquinone by ONOO-. Also, flow EPR allowed us to detect the formation of the intermediary ubisemiquinone. Oxidation of different ubiquinol concentrations by ONOO- in the presence of NADH as competitive ONOO- scavenger (k=233±27 M-1 s-1) was assessed and the data was analyzed using a simple competition kinetic model calculating k=485±54 M-1 s-1. In addition, we observed that mitochondrial proteins tyrosine nitration as well as albumin nitration induced by 0.5 mM ONOO- were decreased by supplementation with 100-400 mM ubiquinol as assessed by Western blot with an antinitrotyrosine antibody. The same protective effect at 0.3-0.5 mM ONOO- was observed after the supplementation of 4 mg/ml submitochondrial particles (SMP) with 0-100 mM UQ-2 and, to achieve enzymatic UQ-2 reduction, with 6 mM succinate and 6 mM myxothiazol. The pattern of SMP nitration by 30 mM ONOO- was identical in myxothiazol or cyanide treated SMP, showing that ubiquinol is more protective than cytochome c. These data demonstrate that peroxynitrite reacts with ubiquinol in mitochondria. The protective effect of this reaction on mitochondrial protein oxidative damage suggests that it follows the interplay of oxygen free radicals and ·NO.