The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A.

Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism that are involved in several cellular signaling pathways as well as tissue injury and pathophysiological processes, including brain ischemia/reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS release depends on oxygen concentration. The rate of H2 O2 release by intact brain mitochondria was measured with an Amplex UltraRed assay using a high-resolution respirometer (Oroboros) equipped with a fluorescent optical module and a system of controlled gas flow for varying the oxygen concentration. Three types of substrates were used: malate and pyruvate, succinate and glutamate, succinate alone or glycerol 3-phosphate. For the first time we determined that, with any substrate used in the absence of inhibitors, H2 O2 release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H2 O2 release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate. H2 O2 production by complex III is significant only in the presence of antimycin A and, in this case, the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. We also demonstrated that complex II in brain mitochondria could contribute to ROS generation even in the absence of its substrate succinate when the quinone pool is reduced by glycerol 3-phosphate. Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia providing a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that ROS generation in brain mitochondria is lower under hypoxic conditions than in normoxia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.