EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F-1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (beta T168)

Site-directed mutations were made to the phosphate-binding loop threonine i n the P-subunit of the chloroplast F-1-ATPase in Chlamydomonas (beta T168), Rates of photophosphorylation and ATPase-driven proton translocation measu red in coupled thylakoids purified from beta T168D, beta T168C, and beta T1 68L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase ac tivity of purified chloroplast F-1-ATPase (CF1), The EPR spectra of VO2+-AT P bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF, from each mutant in both signal intensity and in V-51 hyperfine parameters that depend on t he equatorial VO2+ ligands. These data provide the first direct evidence th at 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 la tent enzyme. Thus, the phosphate-binding loop threonine is an equatorial me tal 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 Mg2+-ADP complex that becomes entrapped in a catalyt ic site in a manner that regulates enzymatic activity. The lack of catalyti c function of CF1 with entrapped Mg2+-ADP may be explained in part by the a bsence of the phosphate-binding loop threonine as a metal ligand.