Substitution of Mg²⁺ cofactor with Ca²⁺ disrupts positive cooperativity in F₁F_O-ATP(hydrol)ase catalysis

The mitochondrial F₁F_O-ATPase is a dual-function enzyme that synthesizes ATP using the proton motive force and hydrolyzes ATP to reenergize the membrane. Mg²⁺ is the physiological cofactor of F₁F_O-ATPase, enabling both ATP synthesis and hydrolysis, while Ca²⁺ supports only ATP hydrolysis. Mg²⁺-dependent F₁F_O-ATPase exhibits positive cooperativity in ATP hydrolysis Hill coefficient (nHi) of 2.01 ± 0.21, whereas Ca²⁺-dependent activity shows Michaelian kinetics, nHi 1.41 ± 0.06. Ca²⁺ acts as an uncompetitive inhibitor on Mg²⁺-dependent ATP hydrolysis, suggesting distinct binding sites and conformational effects. The differential kinetic behavior underlies the enzyme's multifunctionality of F₁F_O-ATPase in physio-pathological conditions, depending on the cofactor.