Chloroplast F 1 -ATPase from Chlamydomonas Reveals Changes in Metal Ligation Resulting from Mutations to the Phosphate-binding Loop Threonine (bT168)*

Site-directed mutations were made to the phosphatebinding loop threonine in the b-subunit of the chloroplast F1-ATPase in Chlamydomonas (bT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from bT168D, bT168C, and bT168L mutants had <10% of the wild type rates, as did rates of Mg 21 -ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO 21 -ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51 V hyperfine parameters that depend on the equatorial VO 21 ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphatebinding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg 21 ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg 21 -ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.