Composition of complex V of the mitochondrial oxidative phosphorylation system.

Complex V isolated from bovine heart mitochondria (Stiggall, D. L., Galante, Y. M., and Hatefi, Y. (1978) J. Biol. Chem. 253, 956-964) was further purified to remove minor respiratory chain contaminants. The purified preparation exhibited an oligomycin-sensitive ATPase activity of 15 to 20 pmol/min/mg of protein, and an uncouplerand oligomycin-sensitive ATP-33Pi exchange activity of about 190 nmol/min/mg of protein, both at 30°C. Upon electrophoresis on three different gel systems in the presence of sodium dodecyl sulfate, purified Complex V exhibits 12 to 13 polypeptide bands. The only known impurity is 2 to 3% of an iron-sulfur flavoprotein with a composition and M, characteristic of ETF dehydrogenase. Using purified coupling factors for comparison of M, values and coelectrophoresis with Complex V, photoaffinity labeling with the uncoupler [3H]2-azido-4-nitrophenol, labeling with [‘4C]dicyclohexylcarbodiimide, and direct isolation from Complex V, the following components have been identified in the purified complex: the five subunits of F1-ATPase, oligomycin sensitivity-conferring protein, uncoupler-binding protein, dicyclohexylcarbodiimide-binding protein, a membrane sector protein corresponding in M, to subunit 6 of the yeast oligomytin-sensitive ATPase complex, and coupling factor F+ In addition, there is a polypeptide band with M, (-11,000) similar to those of the ATPase inhibitor protein and purified coupling factor B (You, K. S., and Hatefi, Y. (19’76) Biochim. Biophys. Acta 423, 398-412). Small amounts of the ATPase inhibitor protein are present in Complex V.