Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart.

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.