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Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex.

1984; Elsevier BV; Volume: 259; Issue: 22 Linguagem: Inglês

10.1016/s0021-9258(18)89855-6

1083-351X

Yoshiharu Shimomura, Morimitsu Nishikimi, Takayuki Ozawa,

RNA and protein synthesis mechanisms

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex 111) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl s-200 gel chromatography.Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography.The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein.When this preparation was incubated with an iron-sulfur proteindepleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex 111, whereas the recovery of the activity was marginal in the absence of the phospholipids.Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.Ubiquinol-ferricytochrome c oxidoreductase (Complex 111), which mediates electron transport from NADH-ubiquinone oxidoreductase and succinate-ubiquinone oxidoreductase to cytochrome c oxidase of the mitochondrial respiratory chain, contains cytochrome b, cytochrome cl, and an iron-sulfur protein as the main redox-active components (1).The ironsulfur protein was first isolated in a succinylated form from Complex I11 and shown to possess a 2Fe-2S cluster by Rieske et al. (2).Trumpower et al. (3, 4) purified the iron-sulfur protein from succinate-cytochrome c reductase complex and demonstrated that ubiquinol-cytochrome c reductase activity is restored by reconstituting the protein into the reductase complex which had been depleted of it.Their purification method consisted of many steps, and guanidine was used to isolate the iron-sulfur protein from the reductase complex.The reconstituted activity was not high compared to the activity of the parent reductase complex, probably due to the denaturing effect of guanidine.Recently, Engel et al. (5) have reported a relatively simple method for purification of the protein from Complex 111.Their method of dissociation of the complex was similar, in use of a chaotropic agent, to that of Trumpower and Edwards (3) in that urea was used in place