Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

Abstract

The binding of TNP-ATP (2′ or 3′-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 μM) and one with lower affinity (K_d = 0.9 μM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 μM). The binding of [³⁵S]ATPαS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 μM) and six at the liver enzyme (K_d =12 μM). The Paracoccus enzyme had only one binding site (K_d =16 μM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)