XFEL-Detected Peroxide Formation From the S₃ State Supports the Low Oxidation State over the High Oxidation State Paradigm in Photosystem II

The mechanism of O–O bond formation in PSII is still debated. Although it is generally accepted to occur during the S₃ → [S₄] → S₀ transition, some studies suggest it may already begin in the S₃ state. Notably, the 2017 XFEL study by Suga et al. supports this hypothesis by reporting structural features consistent with a peroxide intermediate formed from the S₃ state. Here, we present DFT calculations showing that, in the high oxidation state (HOS) model, O–O coupling starting from the S₃ state with the hydroxo–oxo configuration is kinetically feasible, forming a peroxide intermediate via a modest activation energy of 19.5 kcal/mol. However, in the HOS model, the resulting peroxide would be readily converted to O₂ with ΔG^‡ = 11 kcal mol⁻¹, inconsistent with experimental observations that O₂ release proceeds only after reaching the [S₄] state. In contrast, we show that if peroxide forms from the S₃ state, its persistence is more plausibly explained by the low oxidation state (LOS) model, where further conversion to superoxide or O₂ is energetically unfavorable. This distinction between these two models stems from the greater oxidizing power of Mn(IV) in the HOS paradigm relative to Mn(III) in the LOS paradigm in promoting peroxide-to-O₂ conversion.