Effects of nucleotides on the protein ligands to metals at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase.

Abstract

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 CF1 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 (VIV = 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 (alpha T176), 2 H2O, and 1 RCOO- (alpha D269 or alpha 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 (alpha 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.(ABSTRACT TRUNCATED AT 250 WORDS)