Nitric oxide inhibits mitochondrial complex I by peroxynitrite formation.

RIOBÓ N; CLEMENTI E; MELANI M; MONCADA S; PODEROSO JJ

Florianólpolis, Brasil

Congreso; First Meeting of the South American Group for Free Radical Research.; 1999

South American Group for Free Radical Research.

Previous reports have shown that nitric oxide (NO) reversibly inhibits cytochrome oxidase in whole cells and isolated mitochondria. In addition, long term exposure of J774 mcrophages to NO, results in inhibition of mitochondrial complex I-driven respiration. Our goal was to confirm the inhibitory effect on isolated mitochondria and to analyze the mechanism of inhibition. To this purpose, we compared the effect of NO on rat heart, liver and brain mitochondria which have different ubiquinol content and, consequently, different oxygen radical production. Exposure of mitochondria and submitochondrial particles (SMP) to 1 µM steady-state NO or increasing pulses of 0.5 µM NO in the presence of respiratory substrate, lead to the inhibition of malate-dependent respiration but did not modify the oxygen uptake rate in the presence of succinate. When SMP, supplemented with succinate to achieve ubiquinol reduction, were exposed during 15 minutes to 1 µM steady-state NO, NADH-quinol (representing complex I activity) was significantly reduced (77±2 vs 19±5 µM QH2 min-1 mg prot-1). This effect was significantly prevented by coincubation with 20 µM SOD and 2 mM uric acid (47±3 µM QH2 min-1 mg prot-1), suggesting the involving of peroxynitrite (ONOO-). In contrast, succinate-quinol reductase activity was not affected. In addition, the effect of NO in the absence of substrate was significantly reduced and was not sensitive to SOD. Nitric oxide inhibition was directly related to the hydrogen peroxide production of the different tissues triggered by a single pulse of 1 µM NO (brain: 0.01, liver: 0.12 and heart: 0.17 nmol H2O2/min/mg prot). Furthermore, addition of increasing concentrations of ONOO- to brain submitochondrial particles, mimicked the effects of NO. Complex I was selectively inhibited by low ONOO- concentrations (IC50= 75 µM);by contrast, complex II proved to be more resistant (IC50= 500µM). As previously reported, neither complex III nor cytochrome oxidase activities were modified by ONOO-. In accord, addition of ONOO- to coupled brain mitochondria progressively decreased the respiratory control ratio with malate/glutamate (IC50= 50 µM) but not with succinate (IC50= 550µM). Interestingly, the effect of ONOO- was diminished when testing mitochondria with higher ubiquinol contents (liver complex I IC50= 200 µM and heart complex I IC50= 600 µM). The ubiquinol-related protection was further observed by measuring tyrosine nitration levels by western blot in SPM exposed to a single bolus of 25-50 µM ONOO-. Taken together, these results suggest that NO-induced mitochondrial complex I inhibition is mediated by the formation of intramitochondrial peroxynitrite.