Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O₂^{{radical dot} -} generation in mitochondria respiring on the complex I substrates pyruvate + malate, an effect fully inhibited by rotenone. Antimycin A increased O₂^{{radical dot} -} production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O₂^{{radical dot} -} production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O₂^{{radical dot} -} formation driven with the complex II substrate succinate. At 0.6 μM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O₂^{{radical dot} -} formation; however, at 40 μM myxothiazol (which completely inhibits both complexes I and III) eliminated O₂^{{radical dot} -} production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O₂^{{radical dot} -} from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.