Novel purification of cytochrome c1 from mitochondrial Complex III. Reconstitution of antimycin-insensitive electron transfer with the iron-sulfur protein and cytochrome c1.

Abstract

Complex III of beef heart mitochondria was separated into the iron-sulfur protein and the complex devoid of it as described previously (Shimomura, Y., Nishikimi, M., and Ozawa, T. (1984) J. Biol. Chem. 259, 14059-14063). From the latter preparation, cytochrome c1 was subsequently purified by detergent-exchange chromatography on a phenyl-Sepharose column and DEAE-Sepharose column chromatography. In the former chromatography, the resolution of the iron-sulfur protein-depleted complex was achieved by changes of detergents on the surface of the complex; nearly homogeneous cytochrome c1 was eluted from the column with dodecyl octaethylene glycol mono-ether after dissociation of core proteins and subunit VI with guanidine and cholate. The purified cytochrome c1 consists of a single polypeptide as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 39 nmol of heme/mg of protein. The isolated iron-sulfur protein catalyzes reduction of cytochrome c by ubiquinol, which is insensitive to antimycin, at a rate of 0.03 mumol of cytochrome c reduced/min/nmol of protein, while the purified cytochrome c1 has no such catalytic activity. When cytochrome c1 and the iron-sulfur protein form a complex, the rate of cytochrome c reduction increases to 0.12 mumol/min/nmol of the iron-sulfur protein. In this reaction, cytochrome c1 mediates antimycin-insensitive electron transfer from the iron-sulfur protein to cytochrome c, thereby constituting a pathway of electrons: ubiquinol----iron-sulfur protein----cytochrome c1----cytochrome c. The complex formation between the iron-sulfur protein and cytochrome c1 was verified by binding of cytochrome c1 to a column of protein A-Sepharose to which the iron-sulfur protein was linked with immobilized anti-iron-sulfur protein antibody. The electron-transfer activity of the mixture is at a comparable level to that of antimycin-inhibited Complex III, and both activities are partially sensitive to superoxide dismutase. Thus, the above-described coupling of the iron-sulfur protein and cytochrome c1 is considered as reconstitution of the antimycin-insensitive pathway of electrons in Complex III.