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 III_Qo) and the flavin site of electron transfer flavoprotein (site E_F). We found no evidence of ROS production from complex I ubiquinone-binding site (site I_Q) by reverse electron transport during oxidation of palmitoylcarnitine. Pyruvate oxidation also drove H₂O₂ emission primarily from sites III_Qo; however, the flavin sites of pyruvate dehydrogenase (site P_F) and complex II (site II_F) contributed substantially. The effect of Cu depended on substrate and redox site, with effects at sites O_F and III_Qo 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.