Characterization of superoxide production sites in isolated rat brain and skeletal muscle mitochondria

In this report, we have quantified the superoxide and H2O2 production rates of intact rat brain and skeletal muscle mitochondria under condition of oxygen saturation applying p-hydroxyphenylacetate as fluorescent probe for H2O2 generation and hydroethidine as probe for superoxide formation. The localisation of superoxide producing sites was determined by evaluating the effects of SOD addition. At comparable respiration rates and functional quality of mitochondria, we detected in brain mitochondria, a high reversed electron flow-dependent H2O2 generation while the bc1-complex-dependent H2O2 generation in the presence of succinate+antimycin was low. On the other hand, the reversed electron flow-dependent superoxide generation rate was small while the bc1-complex-dependent superoxide production was considerable. In contrast, isolated skeletal muscle mitochondria of comparable quality showed at almost comparable reversed electron flow-dependent H2O2 generation more than 10-fold higher bc1-complex-dependent H2O2 generation. Our data are compatible with the following suppositions: (i) The major ROS generation site in complex I visible during reversed electron flow (very likely the FMN moiety) is liberating superoxide predominantly to the mitochondrial matrix space. (ii) Similarly, the bc1-complex-dependent superoxide generation site (the semiquinone at center 'o') liberates superoxide with preference to the cytosolic space and (iii) Muscle mitochondria, most probably due to their higher endogenous CoQ content, generate at comparable maximal rates of respiration considerable larger amounts of superoxide at center 'o' of complex III.