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 H₂O₂in 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/H₂O₂ 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 H₂O₂ 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/H₂O₂in vivo.