Protein-lipid interactions within purified and reconstituted cytochrome C reductase and oxidase.

Analysis of the rotational mobility of spin-labeled bovine heart cytochrome c oxidase by saturation transfer electron paramagnetic resonance(ST-EPR) has recently demonstrated that this enzymic complex can be obtained as an aggregate of individual protein molecules which display little or no rotational mobility on the submillisecond time scale (1). Aggregation appears to occur as a result of protein-protein interactions, as rotationally mobile (r2 =1 X 10-7S) enzymes can be isolated by further purification of the complex to remove contaminating polypeptides, followed by gel filtration to separate aggregated from individual enzyme molecules. This tendency of cytochrome oxidase to exist as protein "patches" may reflect the situation within the mitochondrial inner membrane (2). Correlation of information gained on the rotational mobility of cytochrome oxidase to the fluidity of lipid adjacent to the protein indicates that an immobilized or "boundary" lipid population is only detectable, using EPR, when the protein moiety of the complex is immobilized on the submillisecond time scale (1). This presumably is due to the formation of enzyme aggregates resulting in the entrapment of spin-labeled lipid between cytochrome oxidase molecules. Steady-state activity displayed by cytochrome oxidase is unaffected by the rotational mobility, and therefore the aggregation state, of either the purified or reconstituted complex. Furthermore, electron transport rates are not influenced by the fluidity of the hydrophobic environment adjacent to the protein. In the present study, the rotational mobility of another mitochondrial electron transport complex, ubiquinol:cytochrome c oxidoreductase(1.10.2.2), has been studied by ST-EPR and correlated with lipid fluidity adjacent to the purified and reconstituted protein. Because both cytochrome c reductase and oxidase interact with cytochrome c, the only water-soluble protein component of the electron transport chain, it was of interest to investigate whether cytochrome c reductase also possessed the type of aggregation behavior displayed by oxidase, and whether the