A New Protein Component of Complex III of the Mitochondrial Electron Transfer Chain

Abstract

Abstract Methods are described for the isolation of a new protein component, namely core protein, from Complex III of the electron transfer chain. One method involves prior precipitation of cytochrome b and non-heme iron protein from a taurocholate-ammonium sulfate dispersion of the unreduced intact complex, followed by precipitation of core protein by treating the supernatant solution containing cytochrome c1 with sodium mersalyl. A second method involves precipitation of non-heme iron protein alone from a taurocholate-ammonium sulfate dispersion of the dithionite-reduced complex, followed by precipitation of core protein by addition of sodium mersalyl to the supernatant solution containing both cytochromes b and c1. Core protein, as isolated, is homogeneous upon electrophoresis on polyacrylamide gel and accounts for about 50% of the protein of Complex III. It is essentially free of heme, phospholipids, and bile salts and contains no prosthetic group as yet identified. It has a molecular weight of 40,000 to 50,000; there are, therefore, 3 to 4 molecules of core protein per molecule of Complex III. Studies with a number of different sulfhydryl reagents provide evidence that cleavage of core protein from the other components of Complex III, followed by its precipitation, involves the blocking of a free sulfhydryl group (or groups), either those occurring on the core protein, or possibly the sulfhydryl groups of some other component of Complex III. It is shown that antimycin A decreases the rate of mersalyl-induced release of core protein from the supernatant solution containing, in addition, both cytochrome b and cytochrome c1, but not its rate of release from the supernatant solution containing only cytochrome c1. The rate of release of core protein from the supernatant solution containing both cytochromes b and c1 parallels the rate of destruction of the antimycin-binding site as measured by the release of antimycin A. This finding provides strong evidence that core protein is an integral and functional part of Complex III.