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

Inspired by the period-four oscillation in flash-induced oxygen evolution of photo system 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 (S-0, S-1, S-2 and S-3) and one transient S-4 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 femto second 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 angstrom). In addition, we report structures of two transient states at 150 and 400 mu s, revealing notable structural changes including the binding of one additional 'water', Ox, during the S-2 -> S-3 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 S-3 state between Ca and Mnl 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 O-2 release. Thus, our results exclude peroxo-bond formation in the S-3 state, and the nucleophilic attack of W3 onto W2 is unlikely.