The participation of metals in the mechanism of the F₁-ATPase

The Mg²⁺ cofactor of the F₁F₀ 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 Mg²⁺ in the F₁-ATPase. The ⁵¹V-hyperfine parameters derived from EPR spectra of VO²⁺ 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 ⁵¹V-hyperfine parameters of the bound VO²⁺, 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 β(E) in mitochondrial F₁, activation of the chloroplast F₁-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 γ 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 β(DP) site of mitochondrial F₁, the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from βE197 in Chlamydomonas CF₁. Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity. Copyright (C) 2000 Elsevier Science B.V.