Purification of highly active cytochrome bc1 complexes from phylogenetically diverse species by a single chromatographic procedure.
A method has been developed for purification of highly active ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complexes from wild-type Rhodobacter sphaeroides, Rhodobacter capsulatus MT1131, bovine heart and yeast mitochondria. This is the first report of the isolation of cytochrome bc1 complex from a wild-type strain of Rb. sphaeroides and from any strain of Rb. capsulatus. The purification involves extraction of membranes with dodecyl maltoside and two successive DEAE column chromatography steps. All of the resulting bc1 complexes are free of succinate dehydrogenase and cytochrome c oxidase activities. The purified bc1 complexes from both photosynthetic bacteria contain four polypeptide subunits, although the molecular weights of some of their subunits differ. They are also free of reaction center and light-harvesting pigments and polypeptides. The turnover number of the Rb. sphaeroides complex is 128 s-1, and that of the Rb. capsulatus complex is 64 s-1. The bc1 complex from bovine heart contains eight polypeptides and has a turnover number of 1152 s-1, while the yeast complex contains nine polypeptides and has a turnover number of 219 s-1. The activities of these complexes are equal to or better than those commonly obtained by previously reported methods. This method of purification is relatively simple, reproducible, and yields cytochrome bc1 complexes which largely retain the turnover number of the starting material and are pure on the basis of optical spectra, enzymatic activities and polypeptide composition. The purification of cytochrome bc1 complexes from energy-transducing membranes which differ markedly in their lipid and protein composition makes it likely that with minor modifications this method could be applied to species other than those described here.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D014450:Electron Transport Complex III / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D010802:Phylogeny; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D012241:Rhodopseudomonas / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013045:Species Specificity
D014450:Electron Transport Complex III