Properties and reconstitution of a cytochrome oxidase deficient in subunit III.
Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three 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 preparations, 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.
D000818:Animals; D002417:Cattle; D004563:Electrochemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008567:Membranes, Artificial; D008929:Mitochondria, Heart / Q000201:enzymology; D010446:Peptide Fragments; D011522:Protons
D008081:Liposomes; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011522:Protons; D003576:Electron Transport Complex IV