Site-Site Interaction on Mitochondrial F₁-ATPase Functional Symmetry of the High-Affinity Nucleotide Binding Sites

Interactions between the high affinity binding sites on mitochondrial F 1 were analysed by combined use of the nucleotide analogues 3'-0-(l-naphthoyl)-ADP (N-ADP) and 2\3'-0-(2, 4, 6-trinitrophenyl)-ADP (TNP-ADP). The binding behaviour of F 1 with respect to these ligands was studied by measuring the fluorescence of F 1 and of TNP-ADP and the fluorescence anisotropy of N-ADP. A total of 3 high affinity binding sites can be occupied by TNP-ADP. By exchange experiments, it could be shown that binding of TNP-ADP to such a site considerably accelerates the dissociation of a ligand bound to a neighbouring site. These results support the notion that the functional behaviour of F 1 is symmetric: during the catalytic cycle any individual site can successively assume different affinity states as has been predicted by hypotheses such as the binding change model.