Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress

We have determined the underlying sites of H2O2 generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H2O2 production was determined using the fluorogenic Amplex red and peroxidase system. H2O2 production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H2O2 production stimulated by all respiratory substrates. H2O2 production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H2O2 generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H2O2 production but stimulated H2O2 production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H2O2 production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H2O2 production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca2+ overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H2O2 production by complex I and an increase in succinate-supported H2O2 production by complex III, (ii) increased glutamate/malate-induced H2O2 generation by complex I and (iii) increased alpha-glycerophosphate-supported H2O2 generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H2O2 generation and that, in both normal and stressed states, the level and location of H2O2 production depend on the predominant energetic substrate.