Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?

In nature, an oxo-bridged Mn4CaO5 cluster embedded in photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn4CaO5 cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S-0-S-4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S-2 state of PSII with structural data collected of the S-1, S-2 and S-3 states by serial crystallography at neutral pH (approximate to 6.5) using an X-ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S-2 state, the room temperature crystallography data can be well-described by just one S-2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.