Characterization of Ligands of a High-Affinity Metal-Binding Site in the Latent Chloroplast F₁-ATPase by EPR Spectroscopy of Bound VO²⁺

Vanadyl, (V=O)²⁺, is able to substitute for Mg²⁺ as a cofactor for ATPase activity catalyzed by the chloroplast F₁-ATPase (CF₁). Mg²⁺-dependent ATPase activity was also observed with CFi that contained VO²⁺-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg²⁺-ATPase activity induced by VO²⁺ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF₁ is depleted of nucleotides from all but the N1 site, a single Mg²⁺ remains bound at a site designated Ml. Addition of VO²⁺ to the depleted protein gives rise to an EPR spectrum characteristic of a CF₁-bound VO²⁺ species. The binding curve of the VO²⁺ complex to latent, nucleotide-depleted CFi was determined by the integrated intensities of the —5/2∥ peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO²⁺ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF₁-VO²⁺ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated ¹⁴N nucleus. Analysis of the bound VO²⁺ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO²⁺ bound to CFi under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.