Seeing O: Mn Kβ2,5 and Kβ′′ X‐ray emission spectra arise from transitions from the ligand 2s and 2p shells of the metal complexes to the metal 1s levels. In biological systems, it is difficult to specifically probe the O and N ligands. This spectroscopic technique is used to study the O ligands of the Mn4Ca cluster that catalyzes photosynthetic water splitting and allows direct detection for the first time of the bridging oxo groups of Mn.

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 Ca2+-depleted S1 (S1′) and S2 (S2′) states, the S2′YZ• state, at which point the catalytic cycle of water oxidation is inhibited, and the Ca2+-reconstituted S1 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, 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 tetranuclear Mn cluster, which indicates that the Ca2+ ion in the OEC is not critical for structural maintenance of the cluster, at least in the S1 and S2 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 YZ• (D1-Tyr161).

Ligands play critical roles during the catalytic reactions in inorganic systems and in metalloproteins through bond formation/breaking, protonation/deprotonation, and electron/ spin delocalization. There are well-defined element-specific spectroscopic handles, such as X-ray spectroscopy and EPR, to follow the chemistry of metal catalytic sites. However, directly probing particular ligand atoms like C, N, and O, especially in a large protein matrix, is challenging due to their abundance in the protein. FTIR/Raman and ligandsensitive EPR techniques such as ENDOR and ESEEM have been applied to study metalligand interactions. X-ray absorption spectroscopy (XAS) can also probe the ligand environment; its element-specificity allows us to focus only on the catalytic metal site, and EXAFS and XANES provide metal-ligand distances, coordination numbers, and symmetry of ligand environments. However, the information is limited, because one cannot distinguish among ligand elements with similar atomic number (i.e. C, N. and O).