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

Abstract 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 the51V-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.