Mitochondria are the major site for the generation of ATP at the expense of molecular oxygen. Significant fractions (approximately 2%) of oxygen are converted to the superoxide radical and its reactive metabolites (ROS) in and around mitochondria. Although ROS have been known to impair a wide variety of biological molecules including lipids, proteins and DNA, thereby causing various diseases, they also play critical roles in the maintenance of aerobic life. Because mitochondria are the major site of free radical generation, they are highly enriched with antioxidants including GSH and enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase, on both sides of their membranes to minimize oxidative stress in and around this organelle. The present work reviews the sites and mechanism of ROS generation by mitochondria, mitochondrial localization of Mn-SOD and Cu,Zn-SOD which has been postulated for a long time to be a cytosolic enzyme. The present work also describes that a cross-talk of molecular oxygen, nitric oxide (NO) and superoxide radicals regulates the circulation, energy metabolism, apoptosis, and functions as a major defense system against pathogens. Pathophysiological significance of ROS generation by mitochondria in the etiology of aging, cancer and degenerative neuronal diseases is also described.

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Although oxygen is required for the energy metabolism in aerobic organisms, it generates reactive oxygen and nitrogen species that impair a wide variety of biological molecules, including lipids, proteins, and DNA, thereby causing various diseases. Because mitochondria are the major site of free radical generation, they are highly enriched with enzymes, such as Mn-type superoxide dismutase in matrix, and antioxidants including GSH on both sides of inner membranes, thus minimizing oxidative stress in and around this organelle. We recently showed that a cross talk of nitric oxide and oxygen radicals regulates the circulation, energy metabolism, reproduction, and remodeling of cells during embryonic development, and functions as a major defense system against pathogens. The present work shows that Cu/Zn-type superoxide dismutase, which has been postulated for a long time to be a cytosolic enzyme, also localizes bound to inner membranes of mitochondria, thereby minimizing oxidative stress in and around this organelle, while mitochondrial association decreases markedly with the variant types of the enzyme found in patients with familial amyotrophic lateral sclerosis. We also report that a cross talk of nitric oxide, superoxide, and molecular oxygen cooperatively regulates the fates of pathogens and their hosts and that oxidative stress in and around mitochondria also determines cell death in the development of animals and tissue injury caused by anticancer agents by some carnitine-inhibitable mechanism.

The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe3+. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10(-5)M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10(-5)-10(-6) M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2,6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2,4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears likely that the inhibition of oxygen consumption caused by CV-2619 is related to the effect on non-respiratory systems such as lipid peroxidation which also consumes oxygen.