title: 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.
abstract: 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.
authors.affiliation: Department of Chemistry, Arizona State University, Tempe, Arizona 85287-1601, USA.
authors.forename: C Y
authors.initials: CY
authors.lastname: Hu
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: W D
authors.initials: WD
authors.lastname: Frasch
mesh_terms: D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000254:growth & development; D002736:Chloroplasts / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010785:Photophosphorylation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020524:Thylakoids / Q000201:enzymology; D014638:Vanadates / Q000378:metabolism*
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014638:Vanadates; D006180:Proton-Translocating ATPases