Peptides from complex II active in reconstitution of succinate-ubiquinone reductase.

A preparation has been made from Complex I1 of beef heart mitochondria which contains in purified form two peptides, designated CItw3 and Ctt-4, with molecular weights of 13,500 and 7,000, respectively. Recombination of soluble succinate dehydrogenase with the peptides elicits ubiquinone reductase activity and, with Complex 111, antimycin-sensitive cytochrome c reductase activity, while the “low K,”-ferricyanide reductase activity (Vinogradov, A. D., Gavrikova, E. V., and Goloveshkina, V. G. (1975) Biochem Biophys. Res. Commun. 65, 1264-1269) is lost. A ratio of 6 to 7 mol of the peptides/mol of succinate dehydrogenase is required for maximal ubiquinone reductase activity in the reconstituted system. The characteristics of the recombined mixture are those of Complex 11, rather than ETP, in respect to the turnover number in the phenazine methosulfate reductase assay (38°C) and the pattern of inhibition by thenoyltrifluoroacetone. Direct evidence for physical association of the enzyme and peptides during reconstitution has been obtained by immunoprecipitation of the mixture with antibody specific for the 70,000-dalton subunit of succinate dehydrogenase. Chymotryptic digestion causes destruction f Peptide CI I -~ and loss of ability to restore ubiquinone reductase activity, without apparent effect on Peptide c11-4. Thus an essential role of Ctt-3 in ubiquinone reductase activity may be inferred. Peptide c11-4 after this treatment is still able to combine with succinate dehydrogenase, as shown by immunoprecipitation. It is suggested that may serve to bind the enzyme in the membrane or to bind and orient C I I ~ for function in ubiquinone reduction. An intermediate stage where activity is retained is detected in the chymotryptic breakdown of CII.3 a t > 7000 daltons. Similarly, chymotryptic digestion of intact Complex I1 results in breakdown only of CtI+ and stops at an intermediate stage of 9000 daltons, with no loss of activity. The rest of the molecule of CII.3, as well as CII-~, thus appears to be protected from chymotryptic attack in Complex 11.