The participation of metals in the mechanism of the F(1)-ATPase.

The Mg(2+) cofactor of the F(1)F(0) ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (V(IV)=O)(2+) is a functional surrogate for Mg(2+) in the F(1)-ATPase. The (51)V-hyperfine parameters derived from EPR spectra of VO(2+) bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the (51)V-hyperfine parameters of the bound VO(2+), thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to beta(E) in mitochondrial F(1), activation of the chloroplast F(1)-ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the gamma subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the beta(DP) site of mitochondrial F(1), the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from betaE197 in Chlamydomonas CF(1). Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.