Coordination of Nucleotides to Metals at the M2 and M3 Metal-Binding Sites of Spinach Chloroplast F₁-ATPase

Vanadyl (V^IV=O)²⁺ was used as a paramagnetic probe at the M2 and M3 metal-binding sites of the spinach chloroplast F_rATPase (CF₁ in order to detect interaction of the metals with nucleotides. The M2 site can exist in two forms in the presence of ATP. When ATP and VO²⁺ are added in a 1.5:1 ratio to CF₁, the VO²⁺ EPR spectrum is identical to that of CF₁-VO²⁺ in the absence of ATP. When the M2 site is filled by the addition of ATP and V0²⁺ in a 3:1 ratio, the VO²⁺ binds to M2 in a second form with equatorial coordination to a single phosphate. The treatments required to deplete CF₁ of the monodentate VO²⁺-nucleotide complex indicate that the VO²⁺ is coordinated to the ATP at the noncatalytic N2 site. The presence of uncomplexed nucleotide appears to induce formation of the second form, possibly via ATP binding to the N3 site. This change in coordination at the M2 noncatalytic site may regulate the ATPase activity of CF₁. The M3 site also exists in two forms: (i) in latent CF₁, no phosphate coordination is evident; and (ii) after the ATPase has been activated, the EPR line shape is consistent with the two phosphates from ADP at N3 coordinated to the VO²⁺ at M3. This work establishes a connection between the metal- and nucleotide-binding sites as M2-N2 and M3-N3.