High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates

Despite the considerable interest in superoxide as a potential cause of pathology, the mechanisms of its deleterious production by mitochondria remain poorly understood. Previous studies in purified mitochondria have found that the highest rates of superoxide production are observed with succinate-driven reverse-electron transfer through complex I, although the physiological importance of this pathway is disputed because it necessitates high concentrations of succinate and is thought not to occur when NAD is in the reduced state. However, very few studies have examined the rates of superoxide production with mitochondria respiring on both NADH-linked (e.g. glutamate) and complex II-linked substrates. In the present study, we find that the rates of superoxide production (measured indirectly as H₂O₂) with glutamate+succinate (∼1100 pmol of H₂O₂·min⁻¹·mg⁻¹) were unexpectedly much higher than with succinate (∼400 pmol of H₂O₂·min⁻¹·mg⁻¹) or glutamate (∼80 pmol of H₂O₂·min⁻¹·mg⁻¹) alone. Superoxide production with glutamate+succinate remained high even at low substrate concentrations (<1 mM), was decreased by rotenone and was completely eliminated by FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), indicating that it must in large part originate from reverse-electron transfer through complex I. Similar results were obtained when glutamate was replaced with pyruvate, α-ketoglutarate or palmitoyl carnitine. In contrast, superoxide production was consistently lowered by the addition of malate (malate+succinate ∼30 pmol of H₂O₂·min⁻¹·mg⁻¹). We propose that the inhibitory action of malate on superoxide production can be explained by oxaloacetate inhibition of complex II. In summary, the present results indicate that reverse-electron transfer-mediated superoxide production can occur under physiologically realistic substrate conditions and suggest that oxaloacetate inhibition of complex II may be an adaptive mechanism to minimize this.