Identification of the P-Loop Lysine as a Metal Ligand in the Absence of Nucleotide at Catalytic Site 3 of Chloroplast F_1-ATPase from Chlamydomonas reinhardtii

摘要

Site-directed mutations were made to the phosphate-binding loop lysine in the fJ-subunit of le chloroplast F1-ATPase in Chlamydomonas reinhardtii (j3K167) to investigate the participation of this ~sidue in the binding of metal to catalytic site 3 in the absence of nucleotide. The cw-EPR spectra of ro2+ bound to site 3 of CF1-ATPase from wild type and mutants revealed changes in metal ligation ~sulting from mutations to fJK167. The three-pulse ESEEM spectrum of the wild-type CF 1-A TPase with TO~(2+) bound to site 3 shows an equatorially coordinating 14N from an amine. The ESEEM spectra of the iJ.utants do not show evidence of an equatorially coordinating amine group. The results presented here how that, in the absence of nucleotide, fJK167 is a ligand to the metal bound at catalytic site 3, suggesting regulatory role for the P-loop lysine in addition to its known role in catalysis.