Vanadyl as a probe of the function of the F1-ATPase-Mg2+ cofactor.

The Mg2+ dependent asymmetry of the F(1)-ATPase catalytic sites leads to the differences in affinity for nucleotides and is an essential component of the binding-change mechanism. Changes in metal ligands during the catalytic cycle responsible for this asymmetry were characterized by vanadyl (V(IV) + O)2+, a functional surrogate for Mg2+. The (51)V-hyperfine parameters derived from EPR spectra of VO2+ bound to specific sites on F(1) provide a direct probe of the metal ligands. Site-directed mutations of metal ligand residues cause measurable changes in the (51)V-hyperfine parameters of the bound VO2+, thereby providing a means to identification. Initial binding of the metal-nucleotide to the low-affinity catalytic site conformation results in metal coordination by hydroxyl groups from the P-loop threonine and catch-loop threonine. Upon conversion to the high-affinity conformation, carboxyl groups from the Walker homology B aspartate and MF(1)betaE197 become ligands in lieu of the hydroxyl groups.