REGULATION OF CYTOCHROME-C-OXIDASE BY INTERACTION OF ATP AT 2 BINDING-SITES, ONE ON SUBUNIT-VIA

Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit VIa was higher than that of the wild-type enzyme. Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain. These results indicate an interaction of ATP with subunit VIa in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme. The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme. Quantitative titrations with the fluorescent adenine nucleotide analogues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit VIa. Labeling of both tissue specific isoforms of bovine cytochrome c oxidase in subunit VIa indicated that the ATP binding site is conserved in the subunit from different species as well as in different isoforms. Since the C-terminal part of subunit VIa, which is located in the intermembrane space, is much more strongly conserved than the N-terminal part of the polypeptide, the labeling results suggest a common ATP binding site located at the C-terminal part of the polypeptide. Taken together, these observations support a regulatory role for subunit VIa of cytochrome c oxidase in which this subunit monitors the concentration of ATP in the intermembrane space and inhibits the enzyme activity at physiological concentrations of ATP.