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

The mitochondrial F 1 F O -ATPase is a dual-function enzyme that synthesizes ATP using the proton motive force and hydrolyzes ATP to reenergize the membrane. Mg 2+ is the physiological cofactor of F 1 F O -ATPase, enabling both ATP synthesis and hydrolysis, while Ca 2+ supports only ATP hydrolysis. Mg 2+ -dependent F 1 F O -ATPase exhibits positive cooperativity in ATP hydrolysis Hill coefficient (nHi) of 2.01 ± 0.21, whereas Ca 2+ -dependent activity shows Michaelian kinetics, nHi 1.41 ± 0.06. Ca 2+ acts as an uncompetitive inhibitor on Mg 2+ -dependent ATP hydrolysis, suggesting distinct binding sites and conformational effects. The differential kinetic behavior underlies the enzyme's multifunctionality of F 1 F O -ATPase in physio-pathological conditions, depending on the cofactor. • Mg 2+ enables cooperative ATP hydrolysis; Ca 2+ shifts kinetics toward Michaelian. • Ca 2+ supports ATP hydrolysis only and inhibits uncompetitively Mg 2+ -dependent F 1 F O -ATPase. • Distinct Mg 2+ /Ca 2+ effects tune F 1 F O -ATPase function(s) in health and disease.