Mitochondrial Ca2+‐activated F1FO‐ATPase hydrolyzes ATP and promotes the permeability transition pore

The properties of the mitochondrial F1FO‐ATPase catalytic site, which can bind Mg2+, Mn2+, or Ca2+ and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca2+‐activated F1FO‐ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg2+ activates the F1FO‐ATPase in competition with Mn2+, Ca2+ is a noncompetitive inhibitor in the presence of Mg2+. Selective F1 inhibitors (Is‐F1), namely NBD‐Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca2+‐ or Mg2+‐activated F1FO‐ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg2+ is replaced by Ca2+. In a partially purified F1 domain preparation, Ca2+‐activated F1‐ATPase maintained Is‐F1 sensitivity, and enzyme inhibition was accompanied by the maintenance of the mitochondrial calcium retention capacity and membrane potential. The data strengthen the structural relationship between Ca2+‐activated F1FO‐ATPase and the PTP, and, in turn, on consequences, such as physiopathological cellular changes. By combining different biochemical strategies, we provide results obtained from experiments carried out in swine heart mitochondria and in partially purified F1 fractions that may contribute to either solving or rejecting the hypothesis that Ca2+‐ATP hydrolysis, supported by the F1FO‐ATPase, is required to open the permeability transition pore.