Characterization of the metal binding environment of catalytic site 1 of chloroplast F(1)-ATPase from Chlamydomonas

Metal ligands of the VO²⁺-adenosine diphosphate (ADP) complex bound to high-affinity catalytic site 1 of chloroplast F₁ adenosine triphosphatase (CF₁ ATPase) were characterized by electron paramagnetic resonance (EPR) spectroscopy. This EPR spectrum contains two EPR species designated E and F not observed when VO²⁺-nucleotide is bound to site 3 of CF₁. Site-directed mutations βE197C, βE197D, and βE197S in Chlamydomonas CF₁ impair ATP synthase and ATPase activity catalyzed by CF₁F₀ and soluble CF₁, respectively, indicating that this residue is important for enzyme function. These mutations caused large changes in the ⁵¹V hyperfine tensors of VO²⁺-nucleotide bound to site 1 but not to site 3. Mutations to the Walker homology B aspartate βD262C, βD262H, and βD262T of Chlamydomonas CF₁ caused similar effects on the EPR spectrum of VO²⁺-ADP bound to site 1. These results indicate that the conversion of the low-affinity site 3 conformation to high-affinity site 1 involves the incorporation βE197 and βD262 as metal ligands.