Calcium is an indispensable cofactor for photosynthetic oxygen evolution. We have studied structural relevance of Ca2+ 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 Ca2+-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 Ca2+ 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 N-15-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 Ca2+ depletion, it was concluded that (i) this carboxylate serves as a bridging ligand between the redox-active Mn and the Ca2+ ions, (ii) upon S-2 formation, the coordination bond of this carboxylate to Ca2+ is selectively broken, and (iii) upon depletion of Ca2+, 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 Ca2+ depletion. This indicates that some perturbations on the protein conformations around the Mn-cluster induced by the S-2 formation require the presence of Ca2+ in OEC. Possible roles of Ca2+ in the oxygen-evolving reactions are discussed based on these findings.
