Copper modulates heart mitochondrial H2O2 emission differently during fatty acid and pyruvate oxidation

Although the preferred cardiac metabolic fuels are fatty acids, glucose metabolism also plays an important role. However, irrespective of substrate type, energy generation results in mitochondrial reactive oxygen species (ROS) formation. To determine if the preference of fat over carbohydrates predisposes cardiomyocytes to oxidant production, we measured total and site-specific H2O2 emission in heart mitochondria oxidizing palmitoylcarnitine or pyruvate during copper (Cu) exposure. H2O2 emission was higher during oxidation of palmitoylcarnitine compared with pyruvate. Moreover, the bulk of the H2O2 emitted during palmitoylcarnitine oxidation originated from the outer ubiquinone binding site of complex III (site IIIQo) and the flavin site of electron transfer flavoprotein (site E-F). We found no evidence of ROS production from complex I ubiquinonebinding site (site I-Q) by reverse electron transport during oxidation of palmitoylcarnitine. Pyruvate oxidation also drove H2O2 emission primarily from sites IIIQo; however, the flavin sites of pyruvate dehydrogenase (site P-F) and complex II (site IIF) contributed substantially. The effect of Cu depended on substrate and redox site, with effects at sites O-F and IIIQo being more pronounced in mitochondria oxidizing pyruvate compared with palmitoylcarnitine. Cu imposed a concentration-saturable effect at site P-F but concentration-dependently stimulated H2O2 emission at site E-F. The substrate-dependent differences in H2O2 emission and effects of Cu suggest that fuel type and points of entry of electrons into the mitochondrial electron transport system determine the mitochondrial ROS production rate. Importantly, knowledge of sites of mitochondrial ROS production is crucial to the understanding of cardiac dysfunction associated with impaired substrate metabolism.