Abstract

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)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2,3. 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 site4,5,6. 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 XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8,9, 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 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10,11,12,13. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.

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Acknowledgements

This work was supported by the Director, Office of Science, Office of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences, and Biosciences (CSGB) of the Department of Energy (DOE) (J.Y., V.K.Y.) for X-ray methodology and instrumentation; National Institutes of Health (NIH) grants GM055302 (V.K.Y.) for PS II biochemistry, structure and mechanism, GM110501 (J.Y.) for instrumentation development for XFEL experiments, GM102520 and GM117126 (N.K.S.) for development of computational protocols for XFEL data; the Ruth L. Kirschstein National Research Service Award (GM116423-02, F.D.F.); and the Human Frontiers Science Project Award No. RGP0063/2013 310 (J.Y., U.B., P.W., A.Z.). The DFG-Cluster of Excellence “UniCat” coordinated by T.U. Berlin and Sfb1078 (Humboldt Universität Berlin), TP A5 (A.Z., H.D.), the Solar Fuels Strong Research Environment (Umeå University), the Artificial Leaf Project (K&A Wallenberg Foundation 2011.0055) and Energimyndigheten (36648-1) (J.M.) are acknowledged for support. H.L. and C.A.S. acknowledge support from the US DOE, OBES, CSGB Division. W.I.W. and A.T.B. acknowledge support from an HHMI Collaborative Innovation Award. D.G.W. is funded by industrial income received by CCP4. This research used resources of NERSC, a User Facility supported by the Office of Science, DOE, under Contract No. DE-AC02-05CH11231. Portions of this work were supported by a BNL/US DOE, LDRD grant (11-008; A.M.O.); and NIH/NCRR grant 2-P41-RR012408, NIH/NIGMS grants 8P41GM103473-16 and P41GM111244 and the US DOE, OBER grant FWP BO-70 (A.M.O., B.A.). A.M.O and P.T.D were supported in part by the Diamond Light Source, and A.M.O acknowledges support from a Strategic Award from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (grant 102593). P.B. was supported by a Wellcome Trust DPhil studentship. Testing of crystals and various parts of the setup were carried out at synchrotron facilities that were provided by the Advanced Light Source (ALS) in Berkeley and Stanford Synchrotron Radiation Lightsource (SSRL) in Stanford, funded by DOE OBES under contract DE-AC02-05CH11231 (ALS) and DE-AC02-76SF00515 (SSRL). The SSRL Structural Molecular Biology Program is supported by the DOE OBER and by the NIH (P41GM103393). Use of the LCLS and SSRL, SLAC National Accelerator Laboratory, is supported by the US DOE, Office of Science, OBES under Contract No. DE-AC02-76SF00515. We thank M. Bommer for discussions and help regarding structure refinement, crystallographic model building and validation, J. Hattne for his contributions to the development of XFEL diffraction data processing, A. Boussac for discussions on ammonia binding and his contributions to the substrate water exchange measurements of the S3 state in the presence of ammonia, and the previous CXI beamline scientist, G. Williams, for his support during the initial stages of this project. We thank the support staff at LCLS/SLAC and at SSRL (BL 6-2, 7-3) and ALS (BL 5.01, 5.0.2, 8.2.1).

Author information

Author notes

    • Tsu-Chien Weng

    Present address: Center for High Pressure Science & Technology Advanced Research, Pudong, Shanghai 201203, China.

    • Iris D. Young
    • , Mohamed Ibrahim
    •  & Ruchira Chatterjee

    These authors contributed equally to this work.

Affiliations

  1. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Iris D. Young
    • , Ruchira Chatterjee
    • , Sheraz Gul
    • , Franklin D. Fuller
    • , Aaron S. Brewster
    • , Rosalie Tran
    • , Tara Michels-Clark
    • , Lacey Douthit
    • , Claudio Saracini
    • , Mackenzie A. Bean
    • , Ernest Pastor
    • , Louise Lassalle
    • , Nigel W. Moriarty
    • , Dorothee Liebschner
    • , Pavel V. Afonine
    • , Petrus H. Zwart
    • , Paul D. Adams
    • , Nicholas K. Sauter
    • , Jan Kern
    • , Vittal K. Yachandra
    •  & Junko Yano
  2. Institut für Biologie, Humboldt-Universität zu Berlin, D-10099 Berlin, Germany

    • Mohamed Ibrahim
    • , Rana Hussein
    • , Miao Zhang
    • , Ina Seuffert
    • , Holger Dobbek
    •  & Athina Zouni
  3. Institutionen för Kemi, Kemiskt Biologiskt Centrum, Umeå Universitet, SE 90187 Umeå, Sweden

    • Sergey Koroidov
    • , Casper de Lichtenberg
    • , Long Vo Pham
    • , Håkan Nilsson
    • , Mun Hon Cheah
    • , Dmitriy Shevela
    •  & Johannes Messinger
  4. LCLS, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Roberto Alonso-Mori
    • , Raymond G. Sierra
    • , James M. Glownia
    • , Silke Nelson
    • , Marcin Sikorski
    • , Diling Zhu
    • , Mark S. Hunter
    • , Thomas J. Lane
    • , Andy Aquila
    • , Jason E. Koglin
    • , Joseph Robinson
    • , Mengning Liang
    • , Sébastien Boutet
    •  & Jan Kern
  5. Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Thomas Kroll
    • , Hartawan Laksmono
    • , Raymond G. Sierra
    • , Claudiu A. Stan
    • , Clemens Weninger
    • , Thomas Fransson
    •  & Uwe Bergmann
  6. SSRL, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Thomas Kroll
    • , Dimosthenis Sokaras
    •  & Tsu-Chien Weng
  7. Institute for Methods and Instrumentation on Synchrotron Radiation Research, Helmholtz Zentrum, 14109 Berlin, Germany

    • Markus Kubin
    •  & Philippe Wernet
  8. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

    • Philipp Bräuer
  9. Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK

    • Philipp Bräuer
    • , Pierre Aller
    • , Peter T. Docker
    • , Allen M. Orville
    •  & Gwyndaf Evans
  10. National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, New York 11973, USA

    • Babak Andi
  11. Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA

    • Artem Y. Lyubimov
    • , Monarin Uervirojnangkoorn
    • , William I. Weis
    •  & Axel T. Brunger
  12. Howard Hughes Medical Institute, Stanford University, California 94305, USA

    • Artem Y. Lyubimov
    • , Monarin Uervirojnangkoorn
    •  & Axel T. Brunger
  13. STFC Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0QX, UK

    • David G. Waterman
  14. CCP4, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0FA, UK

    • David G. Waterman
  15. Department of Photon Science, Stanford University, Stanford, California 94305, USA

    • William I. Weis
    •  & Axel T. Brunger
  16. Department of Structural Biology, Stanford University, Stanford, California 94305, USA

    • William I. Weis
    •  & Axel T. Brunger
  17. Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, USA

    • Paul D. Adams
  18. Department of Chemistry, Molecular Biomimetics, Ångström Laboratory, Uppsala University, SE 75237 Uppsala, Sweden

    • Johannes Messinger

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Contributions

U.B., V.K.Y. and J.Y. conceived the experiment; R.A.-M., S.B., A.Z., J.M., U.B., N.K.S., J.K., V.K.Y. and J.Y. designed the experiment; I.D.Y., M.I., R.C., R.T., M.A.B., R.H., M.Z., L.D., I.S., A.Z. and J.K. prepared samples; M.S.H., A.A., J.E.K., J.R., M.L. and S.B. operated the CXI instrument; R.A.-M., T.J.L., J.E.K., J.R., M.L. and S.B. operated the MFX instrument; R.A.-M., J.M.G., S.N., M.S. and D.Z. operated the XPP instrument; S.G., S.K., F.D.F., H.L., E.P., B.A., A.M.O., R.G.S., C.A.S., C.S., J.M. and J.K. developed, tested and ran the sample delivery system; R.C., S.K., C.d.L., L.V.P., H.N., M.H.C., D.Sh., J.M. and J.Y. performed and analysed O2 evolution and EPR measurements; I.D.Y., M.I., R.C., S.G., S.K., A.S.B., R.A.-M., F.D.F., T.K., T.M.-C., H.L., R.G.S., C.A.S., R.H., M.Z., L.D., M.K., C.d.L., C.S., D.So., T.-C.W., E.P., C.W., T.F., P.A., P.B., B.A., P.T.D., A.M.O., J.M.G., S.N., M.S., D.Z., M.S.H., T.J.L., A.A., J.E.K., J.R., M.L., S.B., P.W., A.Z., J.M., U.B., N.K.S., J.K., V.K.Y. and J.Y. performed the LCLS experiment; I.D.Y., A.S.B, T.M.-C., A.Y.L., M.U., N.W.M., D.L., P.V.A., D.G.W., G.E., W.I.W., A.T.B., P.H.Z., P.D.A. and N.K.S. developed new software for data processing; I.D.Y., A.S.B., F.D.F., C.W., T.F., L.L, P.A., P.B., T.K., T.M.-C., H.D., N.K.S. and J.K. processed and analysed XFEL data; I.D.Y., R.C., J.M., J.K., J.Y. and V.K.Y. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Athina Zouni or Johannes Messinger or Vittal K. Yachandra or Junko Yano.

Reviewer Information Nature thanks J. Murray, C. Yocum and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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