Ligand Binding Studies of the F₁ Moiety of Rat Liver ATP Synthase: Implications about the Enzyme's Structure and Mechanism

F₁-ATPase of rat liver was examined for its capacity to interact with both metal ions and nucleotides and for the effect of covalent ATPase inhibitors on these interactions. As isolated, rat liver F₁ contains about 2 mol of Mg²⁺/mol of F₁, 1 mol of which can be removed or exchanged. The remaining mole of Mg²⁺ per mole of F₁ remains very tightly associated with F₁ and is recovered in the αγ fraction after cold denaturation. Rat liver F₁also contains as isolated a nearly equivalent amount of nucleotide (~ 1.7 mol/mol of F₁) which is readily removed by incubation at room temperature followed by column centrifugation. The “2 Mg²⁺enzyme” binds almost 3 mol of 5’-adenylyl imidodiphosphate (AMP-PNP)/mol of F₁in the presence or absence of added divalent cation. When divalent cation is present as Co²⁺, an equivalent activator to Mg²⁺ in the ATPase reaction, 1 mol of F₁ binds 3 mol of both AMP-PNP and Co²⁺. Under these conditions, the very tight Mg²⁺ site remains loaded, the exchangeable Mg²⁺ site is replaced with AMP-PNPCo, and two additional AMP-PNPCo sites are filled. At this point, ADP can be loaded onto the enzyme as a fourth nucleotide at a site separate and distinct from the AMP-PNP sites. Significantly, rat liver F₁ contains only a single readily detectable ADP binding site in the presence or absence of divalent cation. The covalent labeling agent 7-chloro-4-nitro-2, l, 3-benzoxadiazole prevents binding of all three AMP-PNP molecules and two of the three Co²⁺ molecules and reduces bound ADP by about 50%. Carboxylate labeling agents and a variety of other covalent labeling agents are without effect on Co²⁺ binding to F₁. These results are best interpreted within the framework of the asymmetric structure of the rat liver F₁complex which contains only a single copy of the smaller subunits (γδ∊) per three α(β pairs. It is suggested that the ADP site, the tight Mg²⁺site, and the exchangeable Mg²⁺ site (capable of forming an Mg-ATP complex) lie at the asymmetric center of F₁i.e., on an αβ pair “tagged” by one or more of the smaller subunits. In contrast, the two additional ATP metal binding sites are considered to lie on “naked” or “pure” αβ subunit pairs. Three current models for the function of F₁ are discussed with respect to their capacity to accommodate the ligand binding data obtained for the rat liver enzyme.