Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria

Abstract

The objective of this study was to explore the possible cause(s) underlying the previously observed, age-related increase in the rate of mitochondrial H 2O 2 release in the housefly. The hypothesis that an imbalance between different respiratory complexes may be a causal factor was tested. Cytochrome c oxidase activity was found to sharply decline in the latter part of the life span of the flies. Effects of different substrates and respiratory inhibitors were determined in order to ascertain if a decrease in cytochrome c oxidase activity could be responsible for the increased H 2O 2 release. H 2O 2 was measured spectrofluorometrically using horseradish peroxidase and p-hydrophenylacetate as an indicator. Neither NADH-linked substrates nor succinate caused a stimulation of H 2O 2 production. H 2O 2 release by mitochondria, inhibited with rotenone and antimycin A, was greatly increased upon supplementation with α-glycerophosphate; however, the further addition of KCN or myxothiazol, to such preparations, caused a depression of H 2O 2 generation. In contrast, relatively low concentrations of KCN or myxothiazol were found to stimulate H 2O 2 release in insect mitochondria supplemented with α-glycerophosphate and exposed to rotenone, but not antimycin A. Results are interpreted to suggest that partial inhibition of cytochrome c oxidase activity can lead to the stimulation of mictochondrial H 2O 2 production in the housefly at site(s) other than NADH dehydrogenase and ubisemiquinone/ cytochrome b region; a possible source may be glycerophosphate dehydrogenase.