Structure of photosystem II and substrate binding at room temperature

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment-protein complex, couples the one-electron photochemistry at the reaction center with the four-electron redox chemistry of water oxidation at the Mn(4)CaO(5) cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S(0) to S(4))(1), where S(1) is the dark stable state and S(3) is the last semi-stable state before O-O bond formation and O(2) evolution(2,3). A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site(4-6), is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) structures of dark-adapted (S(1)), two-flash illuminated (2F; S(3)-enriched), and ammonia-bound two-flash illuminated (2F-NH(3); S(3)-enriched) PS II. Although the recent 1.95 Å structure of PS II(7) at cryogenic temperature using an XFEL provided a damage-free view of the S(1) state, RT measurements are required to study the structural landscape of proteins under functional conditions(8,9), and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn(4)CaO(5) cluster in the S(2) and S(3) states(10). Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site(10-13). Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms