The study investigates the reversible binding of Pi to beef heart mitochondrial ATPase (F1). The interaction is characterized by a dissociation constant of 80 μM at pH 7.5 and is influenced by divalent metal ions and various substances known to affect oxidative phosphorylation or ATPase activity. Aurovertin, an inhibitor of oxidative phosphorylation, increases the affinity of F1 for Pi (Kd = 20 μM) without altering binding stoichiometry. Anions such as SO42-, SO32-, chromate, and 2,4-dinitrophenolate, which stimulate ATPase activity, also enhance Pi binding. Inhibitors of ATPase activity, such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy, inhibit Pi binding. Adenine nucleotides (ADP, ATP) and the ATP analog adenylyl imidodiphosphate, as well as the Pi analog arsenate, inhibit Pi binding. These findings suggest that the Pi binding site is located in or near an adenine nucleotide binding site on the molecule. The study also examines the effects of pH, metal ions, and other substances on Pi binding, providing insights into the mechanism of oxidative phosphorylation and the role of F1 in ATP synthesis.The study investigates the reversible binding of Pi to beef heart mitochondrial ATPase (F1). The interaction is characterized by a dissociation constant of 80 μM at pH 7.5 and is influenced by divalent metal ions and various substances known to affect oxidative phosphorylation or ATPase activity. Aurovertin, an inhibitor of oxidative phosphorylation, increases the affinity of F1 for Pi (Kd = 20 μM) without altering binding stoichiometry. Anions such as SO42-, SO32-, chromate, and 2,4-dinitrophenolate, which stimulate ATPase activity, also enhance Pi binding. Inhibitors of ATPase activity, such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy, inhibit Pi binding. Adenine nucleotides (ADP, ATP) and the ATP analog adenylyl imidodiphosphate, as well as the Pi analog arsenate, inhibit Pi binding. These findings suggest that the Pi binding site is located in or near an adenine nucleotide binding site on the molecule. The study also examines the effects of pH, metal ions, and other substances on Pi binding, providing insights into the mechanism of oxidative phosphorylation and the role of F1 in ATP synthesis.