Substitution of Mg2+ cofactor with Ca2+ disrupts positive cooperativity in F1FO-ATP(hydrol)ase catalysis

The mitochondrial F1FO-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 F1FO-ATPase, enabling both ATP synthesis and hydrolysis, while Ca2+ supports only ATP hydrolysis. Mg2+-dependent F1FO-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 F1FO-ATPase in physio-pathological conditions, depending on the cofactor.