Native rates of superoxide production from multiple sites in isolated mitochondria measured using endogenous reporters

Abstract

Individual sites of superoxide production in the mitochondrial respiratory chain have previously been defined and partially characterized using specific inhibitors, but the native contribution of each site to total superoxide production in the absence of inhibitors is unknown. We estimated rates of superoxide production (measured as H2O2) at various sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site IF) was calibrated to the reduction state of endogenous NAD(P)H. Similarly, the rate of superoxide production by the complex III site of quinol oxidation (site IIIQo) was calibrated to the reduction state of endogenous cytochrome b566. We then measured the endogenous reporters in mitochondria oxidizing NADH-generating substrates, without added respiratory inhibitors, with and without ATP synthesis. We used the calibrated reporters to calculate the rates of superoxide production from sites IF and IIIQo. The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site IF was the dominant superoxide producer. Under nonphosphorylating conditions, overall rates were higher, and sites IF and IIIQo and unidentified sites (perhaps the complex I site of quinone reduction, site IQ) all made substantial contributions to measured H2O2 production.