Nature of S-States in the Oxygen-Evolving Complex Resolved by High-Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy

[Image: see text] Photosystem II, the water splitting enzyme of photosynthesis, utilizes the energy of sunlight to drive the four-electron oxidation of water to dioxygen at the oxygen-evolving complex (OEC). The OEC harbors a Mn(4)CaO(5) cluster that cycles through five oxidation states S(i) (i = 0–4). The S(3) state is the last metastable state before the O(2) evolution. Its electronic structure and nature of the S(2) → S(3) transition are key topics of persisting controversy. Most spectroscopic studies suggest that the S(3) state consists of four Mn(IV) ions, compared to the Mn(III)Mn(IV)(3) of the S(2) state. However, recent crystallographic data have received conflicting interpretations, suggesting either metal- or ligand-based oxidation, the latter leading to an oxyl radical or a peroxo moiety in the S(3) state. Herein, we utilize high-energy resolution fluorescence detected (HERFD) X-ray absorption spectroscopy to obtain a highly resolved description of the Mn K pre-edge region for all S-states, paying special attention to use chemically unperturbed S(3) state samples. In combination with quantum chemical calculations, we achieve assignment of specific spectroscopic features to geometric and electronic structures for all S-states. These data are used to confidently discriminate between the various suggestions concerning the electronic structure and the nature of oxidation events in all observable catalytic intermediates of the OEC. Our results do not support the presence of either peroxo or oxyl in the active configuration of the S(3) state. This establishes Mn-centered storage of oxidative equivalents in all observable catalytic transitions and constrains the onset of the O–O bond formation until after the final light-driven oxidation event.