Metal ligation by Walker Homology B aspartate βD262 at Site 3 of the latent but not activated form of the chloroplast F₁-ATPase from Chlamydomonas reinhardtii

Site-directed mutations D262C, D262H, D262N, and D262T were made to the β subunit Walker Homology B aspartate of chloroplast F₁-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3- 14% of wild type as were ATPase activities of purified chloroplast F₁ indicating that βD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F₁ as VO²⁺-ATP gave rise to two EPR species designated B and C in wild type and mutants. ⁵¹V- hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO²⁺, nor is it hydrogen- bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the ⁵¹V-hyperfine parameters of species B, the predominant conformation of VO²⁺-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal- nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation.