Cytochrome oxidase vesicles with high oxidase activity and respiratory control ratio (greater than 3.5) were characterized by the freeze-etch technique for electron microscopy. By the use of this technique, cytochrome oxidase is shown to be an inner membrane particle. By locating cross-fractured vesicles in the same preparation, cytochrome oxidase particles are shown to extend across the phospholipid bilayer membranes. When cytochrome oxidase is added to preformed liposomes respiratory control is not observed, but high oxidase activity is maintained. In this preparation the cytochrome oxidase particles are located on the outer vesicle membrane surface. These observations provide direct evidence that cytochrome oxidase is found in a transmembranous position in closed, activecytochrome oxidase vesicles having respiratory control.

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