Effects of Nucleotides on the Protein Ligands to Metals at the M2 and M3 Metal-Binding Sites of the Spinach Chloroplast F₁-ATPase

We have identified the most probable protein ligands at the catalytic M3 and noncatalytic M2 metal-binding sites in the spinach chloroplast F₁-ATPase (CF₁) and here propose possible residues in the protein sequence for these ligands in latent CFi in the absence of nucleotide. The changes in the metal ligands at these sites upon binding of nucleotide to the N2 and N3 sites and upon activation of latent CF₁ provide a possible molecular basis for inhibition of ATPase activity by free metal, for the lack of activity in the latent state, and for the gating mechanism of the ATPase H⁺ pump. To these ends, the Mg²⁺ analogue, vanadyl (V^IV=O)²⁺, was used as a paramagnetic probe at the M2 and M3 metal-binding sites. EPR and ESEEM spectra of VO²⁺ were obtained, and simulations of the full EPR spectra imply the ligand sets at the different metal-binding sites. When VO²⁺ is added to CF₁ in the absence of ATP, the most likely set of ligands at the M2 site are 1 ROH (±T176), 2 H₂O, and 1 RCOO^- (±D269 or ±D270), where the suggested amino acid designations of the residues are given in parentheses according to the mitochondrial sequence. Evidence suggests a possible axial nitrogen ligand at this site (±K175). When the M2 site is filled by addition of VO²⁺ and ATP, the metal binds as a second species in which N2-bound ATP and M2-bound VO²⁺ form a monodentate complex with concomitant exchange of the equatorial protein ligands by 3 H₂O. The M3 sites also exists in two forms: (i) in latent CF₁, the data are best fit with 1 ROH (βT163), 1 RCOO-(β256 or βE188), and 2 H₂O as ligands; and (ii) after the ATPase has been activated, the two phosphates of the ADP bound to the N3 site may coordinate to the metal bound to the M3 site. The best fit of the coordination sphere for this second form of M3-bound VO²⁺ is 2 ROH (β163 and aS344) and 2 phosphates (from ADP). Evidence suggests that there is an axial nitrogen ligand at this site (βK162) and/or the M2 site (αK175). The determination of metal ligands provided by this and earlier studies with vanadyl permits a correlation between the sites of the crystal structure and the order of filling: N1, N2, N3, etc.