Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise
The sites and rates of mitochondrial production of superoxide and H 2 O 2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity ( e.g. the outer quinol site in complex III (site III Qo ) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site I F ) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H 2 O 2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site I Q ) and the flavin site in complex II (site II F ) each account for about a quarter of the total measured rate of H 2 O 2 production. Site I F , site III Qo , and perhaps site E F in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site I F dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H 2 O 2 in vivo .Ten mitochondrial sites of superoxide/H 2 O 2 generation are known, but their contributions in vivo are undefined. Results We assessed their rates ex vivo in conditions mimicking rest and exercise. Conclusion Sites I Q and II F generated half the signal at rest. During exercise, rates were lower, and site I F dominated. Significance Contributing sites ex vivo probably reflect those in vivo .