Properties and Reconstitution of a Cytochrome Oxidase Deficient in Subunit III

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Abstract
Three different preparations of beer heart cytochrome oxidase were reconstituted into the membranes of artificial liposomes, and the electrical change/electron ratios were determined for charge translocation coupled to enzymic activity. A previously characterized subunit-III-deficient preparation, which apparently lacks H+ translocation capacity has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of K in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of 3 minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparatios, but with a small shift to the blue of the .alpha.-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.