THE NUMBER OF NUCLEOTIDE-BINDING SITES IN CYTOCHROME-C-OXIDASE

The binding of 2'(3')-O-(3,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), [S-35]ATP alpha S and 8-azido-[gamma-P-32]ATP to isolated cytochrome c oxidase of bovine heart and liver and to the two-subunit enzyme of Paracoccus denitrificans was studied by measuring the fluorescence change or bound radioactivity, respectively. With TNP-ATP three binding sites were determined at cytochrome c oxidase from bovine heart and liver, both with two dissociation constants K-d of about 0.2 and 0.9 mu M. Trypsin treatment of the enzyme from bovine heart, resulted in one binding site with a K-d of 0.3 mu M The two-subunit enzyme of Paracoccus denitrificans had only one binding site with a K-d of 3.6 mu M. The binding of [S-35]ATP alpha S to cytochrome c oxidase was studied by equilibrium dialysis. With the enzyme of bovine heart seven and the enzyme of liver six high-affinity binding sites with apparent K-d's of 7.5 and 12 mu M, respectively, were obtained. The two-subunit enzyme of Paracoccus denitrificans had one binding site with a K-d of 20 mu M. The large number of binding sites at cytochrome c oxidase from bovine heart, mainly at nuclear coded subunits, was verified by photoaffinity labelling with 8-azido-[gamma-(32)]ATP. (C) 1995 academic Press, Inc.