Studies on the Mechanism of Oxidative Phosphorylation. 8. Purification of the Reduced Cytochrome C Coupling Factor.

It seems likely that the mechanism of mitochondrial oxidative phosphorylation will be understood only after the components of the system have been separated and identified, and the mechanism of their action examined in detail. Experiments in this laboratory, therefore, have been directed toward the resolution of the oxidative phosphorylation system of beef heart mitochondria into its component parts (1). A major step in this resolution has been the isolation by Hansen and Smith (2) of a particle from mitochondria which has the same phosphorylative efficiency (P:O ratio of 3 with reduced diphosphopyridine nucleotide as substrate and 2 with succinate) as has often been found in intact mitochondria. The oxidative phosphorylation system of beef heart mitochondria can be further separated into two major fractions: a submitochondrial particle having a phosphorylative efficiency considerably lower than that of intact mitochondria, and a soluble protein fraction which partially restores the phosphorylative efficiency of the particle (3-5). Evidence has been reported for the occurrence in the soluble protein fraction of at least three distinct proteins, each of which will restore a portion of the phosphorylative ability of the particles (1, 6-9). This communication describes the purification and certain properties of the soluble protein from beef heart mitochondria which specifically restores the phosphorylation coupled to the oxidation of reduced cytochrome c. This protein has been tentatively designated the reduced cytochrome c coupling factor (1).