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 muta nt in which the gene for subunit VIa had been disrupted were used to s tudy the interaction of adenine nucleotides with the enzyme complex. A t low ionic strength (25 mM potassium phosphate), in the absence of nu cleotides, the cytochrome c oxidase activity of the mutant enzyme lack ing subunit VIa was higher than that of the wild-type enzyme. Increasi ng concentrations of ATP, in the physiological range, enhanced the cyt ochrome c oxidase activity of the mutant much more than the activity o f 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 su bunit 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 enzym e. Quantitative titrations with the fluorescent adenine nucleotide ana logues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TN P-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucl eotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cyt ochrome c oxidase with radioactive 2-azido-ATP further confirmed the p resence of an ATP binding site on subunit VIa. Labeling of both tissue specific isoforms of bovine cytochrome c oxidase in subunit VIa indic ated that the ATP binding site is conserved in the subunit from differ ent species as well as in different isoforms. Since the C-terminal par t 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 intermembr ane space and inhibits the enzyme activity at physiological concentrat ions of ATP.