Localization of the site of oxygen radical generation inside the complex Iof heart and nonsynaptic brain mammalian mitochondria

Mitochondrial production of oxygen radicals seems to be involved in many di seases and aging. Recent studies clearly showed that a substantial part of the free radical generation of rodent mitochondria comes from complex I. It is thus important to further localize the free radical generator site with in this respiratory complex. In this study, superoxide production by heart and nonsynaptic brain submitochondrial particles from up to seven mammalian species, showing different longevities, were studied under different condi tions. The results, taking together, show that rotenone stimulates NADH-sup ported superoxide generation, confirming that complex I is a source of oxyg en radicals in mammals, in general. The rotenone-stimulated NADH-supported superoxide production of the heart and nonsynaptic brain mammalian submitoc hondrial particles was inhibited both by p-chloromercuribenzoate and by eth oxyformic anhydride. These results localize the complex I oxygen radical ge nerator between the ferricyanide and the ubiquinone reduction site, making iron-sulfur centers possible candidates: although unstable semiquinones can not be discarded. The results also indicate that the previously described inverse correlation between rates of mitochondrial oxygen radical generatio n and mammalian longevity operates through mechanisms dependent on the pres ence of intact functional mitochondria.