The beef heart mitochondrial ATPase (F₁) exhibits a single binding site for inorganic phosphate (P_i). The interaction with P_i is reversible and partially dependent on the presence of divalent metal ions. The dissociation constant at pH 7.5 is 80 μM. Various substances that influence oxidative phosphorylation or the activity of F₁ also affect P_i binding. Aurovertin, an inhibitor of oxidative phosphorylation, enhances P_i binding by increasing the enzyme's affinity for P_i (K_D = 20 μM) without altering binding stoichiometry. Anions such as sulfate, chromate, and 2,4-dinitrophenolate also enhance P_i binding. Inhibitors of ATPase activity, such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy, inhibit P_i binding. Adenine nucleotides (ADP, ATP, and adenylyl imidodiphosphate) and the P_i analog arsenate also inhibit P_i binding. These observations suggest that the P_i binding site is located in or near an adenine nucleotide binding site on the molecule. The cold-labile ATPase of oxidative phosphorylation was first described about 15 years ago. It was proposed that the ATPase activity of the soluble beef heart enzyme was an artifact of isolation and that when the enzyme was bound to the mitochondrial membrane, it served as a phosphate transfer catalyst in the terminal transphosphorylation step of oxidative phosphorylation. Over the years, it has been suggested that F₁ functions as a component of a system that synthesizes ATP via the reversal of the hydrolytic reaction and that energy-dependent conformational changes altering the affinity of adenine nucleotide binding sites on F₁ are critical features of ATP synthesis in oxidative phosphorylation. A detailed understanding of the mechanism of action of F₁ in oxidative phosphorylation at the molecular level is important for elucidating the overall reaction. Investigations have revealed that the soluble beef heart enzyme, along with other energy-transducing ATPases, is complex, consisting of five chemically distinct subunits and up to 10 subunits/oligomer. The enzyme can be isolated with 5 mol of nucleotide tightly bound per mol of protein, and five adenine nucleotide binding sites per oligomer of molecular weight 347,000 have been demonstrated. Three of the five sites on beef heart F₁ are occupied by tightly bound adenine nucleotides, and two sites are engaged in readily reversible binding of adenine nucleotides. The function of all sites remains to be elucidated, but one or more may be control sites, and at least one site is the hydrolytic site. The role of adenine nucleotide binding sites in the mechanism of action of F₁ has been studied, but little attention has been given to the second major chemical componentThe beef heart mitochondrial ATPase (F₁) exhibits a single binding site for inorganic phosphate (P_i). The interaction with P_i is reversible and partially dependent on the presence of divalent metal ions. The dissociation constant at pH 7.5 is 80 μM. Various substances that influence oxidative phosphorylation or the activity of F₁ also affect P_i binding. Aurovertin, an inhibitor of oxidative phosphorylation, enhances P_i binding by increasing the enzyme's affinity for P_i (K_D = 20 μM) without altering binding stoichiometry. Anions such as sulfate, chromate, and 2,4-dinitrophenolate also enhance P_i binding. Inhibitors of ATPase activity, such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy, inhibit P_i binding. Adenine nucleotides (ADP, ATP, and adenylyl imidodiphosphate) and the P_i analog arsenate also inhibit P_i binding. These observations suggest that the P_i binding site is located in or near an adenine nucleotide binding site on the molecule. The cold-labile ATPase of oxidative phosphorylation was first described about 15 years ago. It was proposed that the ATPase activity of the soluble beef heart enzyme was an artifact of isolation and that when the enzyme was bound to the mitochondrial membrane, it served as a phosphate transfer catalyst in the terminal transphosphorylation step of oxidative phosphorylation. Over the years, it has been suggested that F₁ functions as a component of a system that synthesizes ATP via the reversal of the hydrolytic reaction and that energy-dependent conformational changes altering the affinity of adenine nucleotide binding sites on F₁ are critical features of ATP synthesis in oxidative phosphorylation. A detailed understanding of the mechanism of action of F₁ in oxidative phosphorylation at the molecular level is important for elucidating the overall reaction. Investigations have revealed that the soluble beef heart enzyme, along with other energy-transducing ATPases, is complex, consisting of five chemically distinct subunits and up to 10 subunits/oligomer. The enzyme can be isolated with 5 mol of nucleotide tightly bound per mol of protein, and five adenine nucleotide binding sites per oligomer of molecular weight 347,000 have been demonstrated. Three of the five sites on beef heart F₁ are occupied by tightly bound adenine nucleotides, and two sites are engaged in readily reversible binding of adenine nucleotides. The function of all sites remains to be elucidated, but one or more may be control sites, and at least one site is the hydrolytic site. The role of adenine nucleotide binding sites in the mechanism of action of F₁ has been studied, but little attention has been given to the second major chemical component