Removal of Ca2+ from the Oxygen-Evolving Complex in Photosystem II Has Minimal Effect on the Mn4O5 Core Structure: A Polarized Mn X-ray Absorption Spectroscopy Study

Ca2+-depleted and Ca2+-reconstituted spinach photosystem II was studied using polarized X-ray absorption spectroscopy of oriented PS II preparations to investigate the structural and functional role of the Ca2+ ion in the Mn4O5Ca cluster of the oxygen-evolving complex (OEC). Samples were prepared by low pH/citrate treatment as one-dimensionally ordered membrane layers and poised in the Ca-2-depleted S-1 (S-1') and S-2 (S-2') states, the S-2'Y-z(center dot) state, at which point the catalytic cycle of water oxidation is inhibited, and the Ca2+-reconstituted S-1 state. Polarized Mn K-edge XANES and EXAFS spectra exhibit pronounced dichroism. Polarized EXAFS data of all states of Ca2+ depleted PS II investigated show only minor changes in distances and orientations of the Mn Mn vectors compared to the Ca2+-containing OEC, 3 which may be attributed to some loss of rigidity of the core structure. Thus, removal of the Ca2+ ion does not lead to fundamental distortion or rearrangement of the tetranudear Mn cluster, which indicates that the Ca2+ ion in the OEC is not critical for structural maintenance of the cluster, at least in the S-1 and S-2 states, but fulfills a crucial catalytic function in the mechanism of the water oxidation reaction. On the basis of this structural information, reasons for the inhibitory effect of Ca2+ removal are discussed, attributing to the Ca2+ ion a fundamental role in organizing the surrounding (substrate) water framework and in proton-coupled electron transfer to Y-z(center dot) (D1-Tyr161).