Search

A soluble enzymically active cytochrome b.c1 complex has been purified from baker's yeast mitochondria by a procedure involving solubilization in cholate, differential fractionation with ammonium sulfate, and ultracentrifugation. The resulting particle is free of both cytochrome c oxidase and succinate dehydrogenase activities. The complex contains cytochromes b and c1 in a ratio of 2:1 and quinone and iron-sulfur protein in amounts roughly stoichiometric with cytochrome c1. EPR spectroscopy has shown the iron-sulfur protein to be present mainly as the Rieske protein. EPR spectroscopy also shows a heterogeneity in the cytochrome b population with resonances appearing at g = 3.60 (cytochrome bK) and g = 3.76 (cytochrome bT). A third EPR resonance appearing in the region associated with low spin ferric hemes (g = 3.49) is assigned to cytochrome c1. Anaerobic titration of the complex with dithionite confirmed the heterogeneity in the cytochrome b population and demonstrated that the oxidation-reduction potential of the iron-sulfur protein is approximately 30 mV more positive than cytochrome c1. An intense EPR signal assigned to the coenzyme Q free radical appeared midway in the reductive titration; this signal disappeared toward the end of the titration. A conformational change in the iron-sulfur protein attendant on reduction of a low potential species was noted.