Kα X-ray Emission Spectroscopy on the Photosynthetic Oxygen-Evolving Complex Supports Manganese Oxidation and Water Binding in the S3 State

The unique manganese calcium-catalyst in photosystem II (PSII) is the natural paragon for efficient light driven water oxidation to yield O-2. The oxygen-evolving complex (OEC) in the dark-stable state (S-1) comprises a Mn4CaO4 core with five metal-bound water species. Binding and modification of the water molecules that are substrates of the water-oxidation reaction is mechanistically crucial but controversially debated. Two recent crystal structures of the OEC in its highest oxidation state (S-3) show either a vacant Mn coordination site or a bound peroxide species. For purified PSII at room temperature, we collected Mn K alpha X-ray emission spectra of the S-0, S-1, S-2, and S-3 intermediates in the OEC cycle, which were analyzed by comparison to synthetic Mn compounds, spectral simulations, and OEC models from density functional theory. Our results contrast both crystallographic structures. They indicate Mn oxidation in three S-transitions and suggest additional water binding at a previously open Mn coordination site. These findings exclude Mn reduction and render peroxide formation in S-3 unlikely.