Young, ID; Ibrahim, M; Chatterjee, R; Gul, S; Fuller, FD; Koroidov, S; Brewster, AS; Tran, R; Alonso-Mori, R; Kroll, T; Michels-Clark, T; Laksmono, H; Sierra, RG; Stan, CA; Hussein, R; Zhang, M; Douthit, L; Kubin, M; de Lichtenberg, C; Vo Pham, L; Nilsson, H; Cheah, MH; Shevela, D; Saracini, C; Bean, MA; Seuffert, I; Sokaras, D; Weng, TC; Pastor, E; Weninger, C; Fransson, T; Lassalle, L; Bräuer, P; Aller, P; Docker, PT; Andi, B; Orville, AM; Glownia, JM; Nelson, S; Sikorski, M; Zhu, D; Hunter, MS; Lane, TJ; Aquila, A; Koglin, JE; Robinson, J; Liang, M; Boutet, S; Lyubimov, AY; Uervirojnangkoorn, M; Moriarty, NW; Liebschner, D; Afonine, PV; Waterman, DG; Evans, G; Wernet, P; Dobbek, H; Weis, WI; Brunger, AT; Zwart, PH; Adams, PD; Zouni, A; Messinger, J; Bergmann, U; Sauter, NK; Kern, J; Yachandra, VK; Yano, J
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 centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.