Abstract

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC)1,2,3. The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, ‘distorted-chair’ form4. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’5 structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously6,7.

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Acknowledgements

We thank K. Kawakami and N. Kamiya for sharing unpublished information, H. Mino for information on the flash illumination conditions and H. Ago and K. Yamaguchi for discussions. This work was supported by a program for promoting the enhancement of research universities at Okayama University, JSPS KAKENHI Grant Nos JP15H01642, JP16H06162, JP16H06296 (M. Suga), JP16K21181 (F.A.), JP15H05588 (Y.U.), JP15H03841, JP15H01055 (M.K.) and JP24000018 (J.-R.S.), an X-ray Free Electron Laser Priority Strategy Program (J.-R.S., S.Iw.) from MEXT, Japan, an Asahi Glass Foundation (F.A.), a Kato Memorial Bioscience Foundation (F.A.), an Inamori Foundation (M. Suga), the Research Acceleration Program from Japan Science and Technology agency (JST) (S.Iw.), PRESTO from JST (M.K. and F.A.), a grant from Pioneering Project ‘Dynamic Structural Biology’ of RIKEN (M.K.), and the Strategic Priority Research Program of CAS (XDB17030100) (J.-R.S.). The XFEL experiments were performed at beamline 3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos 2013B1259, 2014A1243, 2014A6927, 2014B1281, 2014B6927, 2014B8048, 2015A1108, 2015A6522, 2015B2108, 2015B6522, 2015B8044, 2016A2542, 2016A6621 and 2016A8033), and we thank the staff at SACLA for their help. We also acknowledge computational support from the SACLA HPC system and the Mini-K supercomputer system.

Author information

Author notes

    • Michihiro Suga
    • , Fusamichi Akita
    • , Michihiro Sugahara
    • , Minoru Kubo
    •  & Yoshiki Nakajima

    These authors contributed equally to this work.

Affiliations

  1. Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan

    • Michihiro Suga
    • , Fusamichi Akita
    • , Yoshiki Nakajima
    • , Yasufumi Umena
    • , Makoto Nakabayashi
    • , Takahiro Yamane
    • , Takamitsu Nakano
    • , Shinichiro Yonekura
    • , Long-Jiang Yu
    • , Tomohiro Sakamoto
    • , Taiki Motomura
    • , Jing-Hua Chen
    •  & Jian-Ren Shen
  2. Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

    • Fusamichi Akita
    •  & Minoru Kubo
  3. RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan

    • Michihiro Sugahara
    • , Minoru Kubo
    • , Keitaro Yamashita
    • , Mamoru Suzuki
    • , Tetsuya Masuda
    • , Shigeyuki Inoue
    • , Tetsunari Kimura
    • , Takashi Nomura
    • , Takaki Hatsui
    • , Eriko Nango
    • , Rie Tanaka
    • , Hisashi Naitow
    • , Yoshinori Matsuura
    • , Ayumi Yamashita
    • , Masaki Yamamoto
    • , Makina Yabashi
    • , Tetsuya Ishikawa
    •  & So Iwata
  4. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

    • Takanori Nakane
    •  & Osamu Nureki
  5. Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan

    • Mamoru Suzuki
  6. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

    • Tetsuya Masuda
  7. Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    • Shigeyuki Inoue
  8. Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan

    • Tetsunari Kimura
  9. Department of Picobiology, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan

    • Taiki Motomura
    •  & Jian-Ren Shen
  10. Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, No.20 Nanxincun, Xiangshan, Beijing 100093, China

    • Jing-Hua Chen
    •  & Jian-Ren Shen
  11. Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

    • Yuki Kato
    •  & Takumi Noguchi
  12. Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan

    • Kensuke Tono
    • , Yasumasa Joti
    • , Takashi Kameshima
    •  & Makina Yabashi
  13. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

    • Eriko Nango
    •  & So Iwata

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Contributions

J.-R.S. conceived the project; M. Suga, F.A., M.Sugah., M.K., Y.U. and J.-R.S. contributed to the design of the experiment; F.A., Y.N., M.N. and T. Nakano grew the cells and purified the PSII samples; F.A. and Y.N. prepared the PSII crystals; M.Sugah., E.N. and R.T. developed the sample delivery system; F.A., Y.N., M.N., Y.U. and M.Sugah. tested and optimized buffer and crystal suspension conditions for injection; M.Suz., T.Ma., S.In., T.Mo., J.-H.C., H.N., Y.M. and A.Y. operated the injector; K.T., Y.J., T.Ka., T.H., M.Yab., T.I. and S.Iw. developed the diffraction instrumentation; M.K., T.Ki. and T.Nom. designed and optimized the laser excitation scheme and aligned the lasers; M. Suga, F.A., M.Sugah., Y.N., T. Nakane, K.Y., M.N., Y.U., M.Suz., T.Ma., S.In., S.Y., L.-J.Y., T.Mo., J.-H.C., R.T., H.N., Y.M., A.Y. and J.-R.S. participated in collection of the X-ray diffraction data at SACLA; T. Nakane, K.Y., M.Yam., O.N. and S.Iw. developed the data evaluation and/or hit-finding programs; Y.K., F.A. and T.Nog. performed FTIR analysis; M. Suga, T.Y. and T.S. analysed the femtosecond crystallography X-ray diffraction data; M. Suga refined the structure, calculated the electron density maps and made the figures; M. Suga and J.-R.S. wrote the manuscript, and all the authors participated in the discussion of the results and writing of the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to So Iwata or Jian-Ren Shen.

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

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https://doi.org/10.1038/nature21400

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