Studies on the Transmembrane Orientation of Cytochrome c Oxidase in Phospholipid Vesicles

We report investigations into the direction of orientation of cytochrome c oxidase in reconstituted vesicles and the factors determining this. Measurement of the enzyme orientation employed two independent techniques: monitoring of the level of haem reduction by membrane-permeant and membrane-impermeant reagents and a kinetic analysis of the reduction of a spin label covalently bound to the oxidase surface. The method of preparation of the oxidase vesicles had a pronounced effect on the enzyme orientation and the two measurement techniques agreed in indicating that the proportion of mitochondrially oriented enzyme was approximately 85% and 50% for vesicles prepared by cholate dialysis and sonication respectively. Our results show that the membrane orientation of the oxidase is determined by interactions between the phospholipid bilayer and the portion of the enzyme embedded therein, as opposed to gross physical constraints. In particular, we demonstrate that the orientation of the oxidase is affected by the fluidity and surface charge of the membrane.