On the Composition and Structural Organization of Complex III of the Mitochondrial Electron Transfer Chain

Abstract

Abstract The sulfhydryl groups of Complex III of the electron transport chain were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) under a variety of conditions. In the absence of added detergents, a total of about eight sulfhydryl groups per complex was readily titratable by DTNB, whether the complex was oxidized, reduced, or inhibited with antimycin A. Titration of eight sulfhydryl groups with DTNB or with mersalyl did not affect enzymic activity. The enzyme was, however, reversibly inactivated by 12 eq of mersalyl. In the presence of high levels of taurocholate or guanidine hydrochloride, 23 sulfhydryl groups were titrated by DTNB per mole of oxidized complex. When the complex was reduced prior to prolonged incubation with DTNB in concentrated detergent, 14 to 15 sulfhydryl groups were titrated. In the presence of antimycin A, only 12 groups were titrated in the oxidized complex. The decreased titer in these latter two cases probably reflected an increased conformational stability of the complex attendant upon reduction or treatment with antimycin A. However, the decreased titration in the case of the reduced complex was due only indirectly to this stabilizing effect. Enzymatic activity was lost upon digestion of the complex with phospholipase A but was partially restored by treatment with phospholipid. The complex was irreversibly inactivated by incubation with trypsin or chymotrypsin, the rate of this inactivation being greater when the complex was in the reduced state. The non-heme iron protein of the complex was the component most sensitive to tryptic digestion. Electrophoresis of the complex on polyacrylamide gel revealed six major, staining species. Fractionation procedures were used to assign identities to these species. In order of increasing mobility, these were a new major protein component (core protein); polymeric cytochrome c1; non-heme iron protein; monomeric cytochrome c1; cytochrome b; and a new, as yet uncharacterized, component. A new technique was used to separate the components of the complex, based on the solubility of certain species in methanol or chloroform-methanol following precipitation by acid. The over-all organization of the complex is discussed in the light of these findings.