Direct detection of a carboxylate bridge between Mn and Ca2+ in the photosynthetic oxygen-evolving center by means of Fourier transform infrared spectroscopy

摘要

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 S1 and S2 states were measured by controlling the redox potential and pH of the buffer. Comparison between the two S2/S1 difference spectra using untreated and Ca2+-depleted PS II membranes showed that the negative bands at 1560 and 1403 cm?1 (belonging to S1) and the positive bands at 1587 and 1364 cm?1 (belonging to S2) 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 S2/S1 spectra of 15N-labeled PS II membranes. The frequency differences of the asymmetric and symmetric COO? bands, i.e., 157 cm?1 for S1 and 223 cm?1 for S2, indicated that this carboxylate group possesses the structure of bridging bidentate coordination in the S1 state and that of unidentate coordination in the S2 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 S2 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 S2 formation require the presence of Ca2+ in OEC. Possible roles of Ca2+ in the oxygen-evolving reactions are discussed based on these findings.