Spin-label study of the relation between enzymatic activity and lipid–protein organization in reconstituted cytochrome c oxidase

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Abstract
Lipid-depleted [beef heart] cytochrome c oxidase (EC 1.9.3.1) containing < 20 .mu.g lipids/mg protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and(or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipids such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology and a high specific activity, was examined by ESR spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid-containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, apparently is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.