Two Distinct Oxygen-Radical Conformations in the X-ray Free Electron Laser Structures of Photosystem II

We report the existence of two distinct oxygen-radical-containing Mn₄CaO_5/6 conformations with short O···O bonds in the crystal structures of the oxygen-evolving enzyme photosystem II (PSII), obtained using an X-ray free electron laser (XFEL). A short O···O distance of <2.3 Å between the O4 site of the Mn₄CaO₅ complex and the adjacent water molecule (W539) in the proton-conducting O4-water chain was observed in the second flash-induced (2F) XFEL structure (2F-XFEL), which may correspond to S₃. By use of a quantum mechanical/molecular mechanical approach, the OH^• formation at W539 and the short O4···O_W539 distance (<2.3 Å) were reproduced in S₂ and S₃ with reduced Mn1(III), which lacks the additional sixth water molecule O6. As the O^•- formation at O6 and the short O5···O6 distance (1.9 Å) have been reported in another 2F-XFEL structure with reduced Mn4(III), two distinct oxygen-radical conformations exist in the 2F-XFEL crystals.