Investigation of the high-spin S2 states in photosystem II using Mn X-ray absorption spectroscopy with insights from XFEL crystallography.

Water oxidation in photosynthesis occurs through a four-electron redox reaction that is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II), that consists of the Mn4CaO5 cluster. Among the four stable intermediates in the water oxidation reaction, it has been reported that the paramagnetic S2 state with the formal oxidation of Mn(III)Mn(IV)3 has multiple spin states, that include high spin (HS, 5/2, g≥4.1) and low spin (LS, 1/2, g = 2) forms. Previous computational studies proposed that these two forms differ in their structure and the high spin form may represent an intermediate form of the OEC during the S2 to S3 transition, the step in which the first substrate water binds to the last open coordination site of the cluster. In the current study, we examined the pH-induced HS-S2 state of T. vestitus PS II with MnK edge extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge spectroscopy (XANES). Small, but notable differences were observed in the EXAFS and XANES between the HS and LS S2 states. EXAFS data and the fits indicate that the basic geometry of the cluster remains the same between these two states. Therefore, we hypothesize that the difference between the HS and LS S2 state may be due to chemical differences such as different protonation states of the oxo and/or water ligands, or its surroundings. We also examined the detailed oxygen positions of the S2 states in the XFEL-based room temperature crystal structures, and confirmed that at neutral pH at room temperature, the structure is consistent with that of the LS-S2 model with open cubane-like geometry.