Structures of the intermediates of Kok's photosynthetic water oxidation clock

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle^1,2. The model comprises four (meta)stable intermediates (S₀, S₁, S₂ and S₃) and one transient S₄ state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn₄CaO₅) cluster in the oxygen-evolving complex^3-7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q_B at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S₂→S₃ state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S₃ state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O₂ release. Thus, our results exclude peroxo-bond formation in the S₃ state, and the nucleophilic attack of W3 onto W2 is unlikely.