Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation

Abstract 1. An ATP-dependent oxidation of cytochrome b was observed in submitochondrial particles from beef heart in the presence of succinate, malonate, sulfide, and antimycin A. The reaction was inhibited by rotenone, rutamycin, and uncoupling agents. Under the same conditions an ATP-dependent H2O2 production took place. 2. Spectrophotometric measurements also revealed an ATP-dependent reduction of flavin. In contrast to the oxidation of cytochrome b, the reduction of flavin required addition of catalytic amounts of NAD to the depleted submitochondrial particles. 3. An ATP-dependent, rotenone-sensitive, reduction of ferricyanide by succinate was observed which did not require the addition of NAD. 4. It was concluded that in beef heart submitochondrial particles the reversed electron transfer from cytochrome b driven by ATP takes place via a slowly autoxidizable electron carrier. In the absence of NAD, ferricyanide or oxygen can accept electrons from this carrier, and in the presence of NAD, the electrons can be channeled to a flavoprotein and to NAD. 5. These findings strongly suggest that in the reversal of electron transfer the energy coupling site is located between cytochrome b and the flavin of NADH dehydrogenase.