Apport d'un nouvel inhibiteur, la fuscine, dans l'étude des chaînes respiratoires de mitochondries animales et de mitochondries de levures

Abstract

Fuscin, a quinoid compound elaborated by Oidiodendron fuscum, is a powerful inhibitor of mitochondrial respiration in animal and yeast mitochondria. It is not an uncoupler.In rat liver mitochondria fuscin inhibits the oxidation of glutamate, but is without effect on the oxidation of succinate. Bound fuscin cannot be removed by washing mitochondria. Inhibition of glutamate oxidation is of the same order of magnitude in state 3 and in state 4 and the efficiency of oxidative phosphorylation remains unaltered. Half‐inhibition is obtained with 14 nmoles of fuscin per mg of mitochondrial protein. The inhibition of electron transfer by fuscin is not released by 2,4‐dinitrophenol. Menadione does not induce a by‐pass of electron transfer around the site inhibited by fuscin, as is the case for rotenone inhibition.In rat liver mitochondria, fuscin prevents the reduction of cytochrome b by glutamate and the succinate‐linked reduction of mitochondrial NAD. In beef heart mitochondria, fuscin inhibits to the same extent the NADH‐oxidase and the NADH‐menadione reductase. The extent of reduction of the non‐heme iron protein associated with NADH‐dehydrogenase (electron paramagnetic resonance spectra) is not altered by the addition of fuscin. These data suggest that the site of action of fuscin in the NAD respiratory chain of animal mitochondria is located between the non‐heme iron component of the NADH‐dehydrogenase and cytochrome b.In mitochondria of Candida utilis, and by contrast to animal mitochondria, fuscin inhibits both succinate and pyruvate oxidations; it partially prevents the reduction of cytochrome b by succinate and the reoxidation of ubiquinone reduced by pyruvate. The amount of fuscin required for half inhibition of respiration is four times smaller in the case of succinate oxidation, (14 nmoles/mg of protein) than in the case of pyruvate oxidation (46–56 nmoles/mg of protein). No uncoupling effect is observed. Contrary to rotenone which is some 500 times less active on Candida utilis mitochondria than on animal mitochondria, fuscin exerts its inhibitory effect on animal and on yeast mitochondria in the same range of concentrations.Respiration of mitochondria of Saccharomyces cerevisiae Which are rotenone‐insensitive and mitochondria of Candida utilis which are rotenone‐sensitive is inhibited by fuscin to the same extent whether succinate or pyruvate is used as substrate. Moreover, mitochondria obtained from iron‐limited Candida utilis, which have lost their sensitivity to rotenone, are still sensitive to fuscin. These data clearly point to different sites of action of fuscin and rotenone in the respiratory chain of yeast mitochondria.Fuscin inhibits the reduction by succinate of the non‐heme iron protein associated with succinate dehydrogenase in Candida utilis mitochondria, but it does not alter the reduction of the non‐heme iron protein of NADH dehydrogenase. Electron paramagnetic resonance studies point to a site of action of fuscin, in yeast mitochondria, on the succinate side of the non‐heme iron protein linked to succinate dehydrogenase. This site of action could also account for NADH oxidase inhibition if, in yeast mitochondria the two non‐heme iron proteins linked to the flavoproteins FPS and FPD are closely connected.The different patterns of inhibitory effects of fuscin in animal mitochondrial and in yeast mitochondria are discussed in terms of organization of the respiratory chain according to the source of mitochondria.