Calcium is an indispensable cofactor for photosynthetic oxygen evolution. We have studied structural relevance of Ca 2 + to the oxygen-evolving center (OEC) of Photosystem II (PS II) by means of Fourier transform infrared (FTIR) spectroscopy. The single-pulse induced FTIR difference spectra of PS II membranes reflecting solely the structural changes of OEC between in the S 1 and S 2 states were measured by controlling the redox potential and pH of the buffer. Comparison between the two S 2 S 1 difference spectra using untreated and Ca 2 + -depleted PS II membranes showed that the negative bands at 1560 and 1403 cm - 1 (belonging to S 1 ) and the positive bands at 1587 and 1364 cm - 1 (belonging to S 2 ) were lost upon Ca 2 + depletion. These bands were assigned to the asymmetric (higher frequency bands) and symmetric (lower frequency bands) COO - stretching modes of a certain carboxylate group in Asp, Glu or the C-termini, based on the infrared data of 20 amino acids and the S 2 S 1 spectra of 1 5 N-labeled PS II membranes. The frequency differences of the asymmetric and symmetric COO - bands, i.e., 157 cm - 1 for S 1 and 223 cm - 1 for S 2 , indicated that this carboxylate group possesses the structure of bridging bidentate coordination in the S 1 state and that of unidentate coordination in the S 2 state. Taking together the observation of disappearance of these bands upon Ca 2 + depletion, it was concluded that (i) this carboxylate serves as a bridging ligand between the redox-active Mn and the Ca 2 + ions, (ii) upon S 2 formation, the coordination bond of this carboxylate to Ca 2 + is selectively broken, and (iii) upon depletion of Ca 2 + , this carboxylate ligand is liberated even from the Mn ion. Along with the changes of COO - bands, several intense bands in 1680-1630 cm - 1 , which were assigned to the amide I modes of backbone amide groups, were lost upon Ca 2 + depletion. This indicates that some perturbations on the protein conformations around the Mn-cluster induced by the S 2 formation require the presence of Ca 2 + in OEC. Possible roles of Ca 2 + in the oxygen-evolving reactions are discussed based on these findings.