Site-directed mutations were made to the phosphate-binding loop lysine in the β-subunit of the chloroplast F1-ATPase in Chlamydomonas reinhardtii (βK167) to investigate the participation of this residue in the binding of metal to catalytic site 3 in the absence of nucleotide. The cw-EPR spectra of VO2+ bound to site 3 of CF1-ATPase from wild type and mutants revealed changes in metal ligation resulting from mutations to βK167. The three-pulse ESEEM spectrum of the wild-type CF1-ATPase with VO2+ bound to site 3 shows an equatorially coordinating 14N from an amine. The ESEEM spectra of the mutants do not show evidence of an equatorially coordinating amine group. The results presented here show that, in the absence of nucleotide, βK167 is a ligand to the metal bound at catalytic site 3, suggesting a regulatory role for the P-loop lysine in addition to its known role in catalysis.
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