The F1F0-ATP synthase harnesses the energy obtained by oxidation of metabolites to provide energy for most all vital organ and tissue systems. Damage to the genes that code for the F1F0 ATP synthase as a result of aging or via free radicals are associated with neurologic muscle weakness, ataxia, and retinitis pigmentosa. Increased levels of damage have been found in patients with Parkinson's disease and cardiomyopathies. ATP can serve as the energy currency for a cell because the F1F0 ATP synthase maintains the ratio of ATP to ADP/phosphate away from equilibrium. Since the catalytic sites of this enzyme rapidly interconvert ATP with ADP/phosphate such that the equilibrium constant of the bound substrates and products is approximately unity, the mechanism whereby the enzyme selectively releases ATP to maintain the nonequilibrium condition remains a major unanswered question. Our studies with VO+2 to probe the metal binding sites of the F1-ATPase have opened a fertile new avenue of inquiry into the mechanism of this important enzyme. These studies indicated the ligands change during catalysis. These results led us to the view that, by following the sequence in which ligands are inserted and displaced from the catalytic metal center, we can unravel the mechanism that enables the enzyme to release ATP selectively over ADP, even against a concentration gradient. First, specific groups that serve as metal-ligands at the catalytic site will be identified by observing diagnostic changes in the CW-EPR and/or ESEEM spectra of the metal VO+2 bound to the enzyme that has been altered using site-directed mutagenesis. Changes observed by EPR in the mutant enzyme will be anticipated by direct comparison to VO+2-model complexes of known crystallographic structure that contain ligands comparable to either wild type or mutant enzyme. Second, the effects of the differences in the ability of the mutant enzymes to bind the metal-nucleotide complexes will be compared to their catalytic activity. Third, the ability of the mutant enzymes to interconvert between two forms of the catalytic site that contain the metal-nucleotide complex will allow us to determine the importance of each amino acid in the ability to make this switch. By relating the structural information from our EPR studies of the mutants to the crystal structure of the enzyme, we will measure the ability of the enzyme to insert and displace metal ligands, and we will thereby elucidate the relationship between changes in the metal ligation and the enzymatic mechanism.
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