High-affinity binding of ADP and of ADP analogues to mitochondrial F1-ATPase.

Nucleotide-depleted F1-ATPase was prepared from beef heart mitochondria. By use of fluorescence techniques and isotope binding analyses, we investigated the occupation of the high-affinity binding sites on F1 by ADP and the ADP analogues 3'-O-(1-naphthoyl)adenosine diphosphate (N-ADP) and 3'-O-[1-(5-dimethylamino)-naphthoyl]adenosine diphosphate (DMAN-ADP). F1-ATPase was found to exhibit three binding sites for ADP (Kd = 50 nM for one site; Kd = 3 microM for the remaining two sites), two binding sites for N-ADP (Kd = 20 - 50 nM for both of the sites), and three binding sites for DMAN-ADP (Kd = 50 nM for all of the sites). Since the adenine nucleotides under consideration are bound to the same class of sites, the binding data can be explained best on the basis of the hypothesis that the binding process is anticooperative with ADP, whereas the analogues are able to overcome anticooperativity partially (N-ADP) or completely (DMAN-ADP). This binding model is consistent with the view that the exchangeable tight sites are involved directly in the catalytical process of ATP-synthesis in oxidative phosphorylation.