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

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 Mn₄CaO₅ cluster that cycles through five oxidation states S_i (i = 0–4). The S₃ state is the last metastable state before the O₂ evolution. Its electronic structure and nature of the S₂ → S₃ transition are key topics of persisting controversy. Most spectroscopic studies suggest that the S₃ state consists of four Mn­(IV) ions, compared to the Mn­(III)­Mn­(IV)₃ of the S₂ 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 S₃ 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 S₃ 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 S₃ 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.