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.(ABSTRACT TRUNCATED AT 250 WORDS)