Metal ligation by Walker homology B aspartate betaD262 at site 3 of the latent but not activated form of the chloroplast F(1)-ATPase from Chlamydomonas reinhardtii.

Site-directed mutations D262C, D262H, D262N, and D262T were made to the beta subunit Walker Homology B aspartate of chloroplast F(1)-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3-14% of wild type as were ATPase activities of purified chloroplast F(1) indicating that betaD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F(1) as VO(2+)-ATP gave rise to two EPR species designated B and C in wild type and mutants. (51)V-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 VO(2+), 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 (51)V-hyperfine parameters of species B, the predominant conformation of VO(2+)-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.