Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation

Abstract 1. Cytochrome oxidase was combined with a hydrophobic protein fraction derived from bovine heart mitochondria in the presence of cytochrome c, phosphatidylcholine, phosphatidylethanolamine, and sodium cholate. After removal of cholate by dialysis and of external cytochrome c by washing, the vesicles were combined with coupling factors. The reconstituted vesicles catalyzed oxidative phosphorylation in the presence of ascorbate plus N-methylphenazinium methyl sulfate with a P:O ratio between 0.25 and 0.5. 2. For optimal activity the ratios of the protein and of the phospholipid components were critical. Excess cytochrome oxidase or cytochrome c yielded vesicles with a low P:O ratio. The ratio of cholate to phospholipids and the proportions of individual phospholipids also influenced the effectiveness of reconstitution. With purified phosphatidylcholine and phosphatidylethanolamine a ratio of 1:4 was optimal. At a ratio of 1:1 the rate of phosphorylation was low but was markedly increased when cardiolipin was added to the phospholipid mixture. 3. Synthetic phospholipids replaced the natural phospholipids even when they contained unnatural side chains. With synthetic phosphatidylcholine and phosphatidylethanolamine the P:O ratio was lower and optimal conditions were different from those used with natural phospholipids. 4. The reconstituted vesicles oxidized both external and internal ferrocytochrome c. Removal of external cytochrome c was essential for effective coupled phosphorylation. In the presence of external ferrocytochrome c the P:O ratio as well as the net rate of phosphorylation was depressed. This uncoupling effect by external cytochrome c was not observed with submitochondrial particles. It appears therefore that in contrast to the membrane of mitochondria and of submitochondrial particles, which are asymmetrical, the reconstituted particles exhibit symmetrical features of assembly. Asymmetry, required for phosphorylation, was only induced by removal of external cytochrome c.