Reactive oxygen species production by brain mitochondria depends linearly on oxygen level

Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism involved in several cellular signaling pathways and pathophysiological processes, including brain ischemia-reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is a lack of agreement on how ROS release depends on oxygen concentration. We determined how the rate of H2O2 release by intact mouse brain mitochondria oxidizing different substrates depends on oxygen concentrations. The highest rate of H2O2 release occurs in conditions of reverse electron transfer, with complex I flavin as a major source of ROS generation. Our data indicate that the rate of H2O2 release by respiring mitochondria is linearly dependent on oxygen concentration. We also found that complex II can significantly contribute to ROS generation when the quinone pool is reduced even in the absence of its substrate succinate. Our results underscore the critical importance of reverse electron transfer in brain, where a significant accumulation of succinate during ischemia favors a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that in brain tissue mitochondria ROS generation under hypoxic conditions is lower than in normoxia.