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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.



  1. 1.

    The structure of photosystem II and the mechanism of water oxidation in photosynthesis. Annu. Rev. Plant Biol. 66, 23–48 (2015)

  2. 2.

    & Mn4Ca cluster in photosynthesis: where and how water is oxidized to dioxygen. Chem. Rev. 114, 4175–4205 (2014)

  3. 3.

    et al. Manganese compounds as water-oxidizing catalysts: From the natural water-oxidizing complex to nanosized manganese oxide structures. Chem. Rev. 116, 2886–2936 (2016)

  4. 4.

    , , & Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55–60 (2011)

  5. 5.

    et al. Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 517, 99–103 (2015)

  6. 6.

    A structure-consistent mechanism for dioxygen formation in photosystem II. Chemistry 14, 8290–8302 (2008)

  7. 7.

    Structures and energetics for O2 formation in photosystem II. Acc. Chem. Res. 42, 1871–1880 (2009)

  8. 8.

    , & Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism. Photochem. Photobiol. 11, 457–475 (1970)

  9. 9.

    et al. Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature. Science 340, 491–495 (2013)

  10. 10.

    et al. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser. Nature 513, 261–265 (2014)

  11. 11.

    et al. Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy. Nat. Commun. 5, 4371 (2014)

  12. 12.

    et al. Structure of photosystem II and substrate binding at room temperature. Nature 540, 453–457 (2016)

  13. 13.

    et al. Grease matrix as a versatile carrier of proteins for serial crystallography. Nat. Methods 12, 61–63 (2015)

  14. 14.

    , , & . Photosystem II and the unique role of bicarbonate: a historical perspective. Biochim. Biophys. Acta 1817, 1134–1151 (2012)

  15. 15.

    et al. Detection of the water-binding sites of the oxygen-evolving complex of Photosystem II using W-band 17O electron-electron double resonance-detected NMR spectroscopy. J. Am. Chem. Soc. 134, 16619–16634 (2012)

  16. 16.

    et al. Possible mechanisms for the O-O bond formation in oxygen evolution reaction at the CaMn4O5(H2O)(4) cluster of PSII refined to 1.9 Å X-ray resolution. Chem. Phys. Lett. 511, 138–145 (2011)

  17. 17.

    , & Energetics of proton release on the first oxidation step in the water-oxidizing enzyme. Nat. Commun. 6, 8488 (2015)

  18. 18.

    , , & pKa of a proton-conducting water chain in photosystem II. J. Phys. Chem. Lett. 7, 1925–1932 (2016)

  19. 19.

    , , & Chemical equilibrium models for the S3 state of the oxygen-evolving complex of photosystem II. Inorg. Chem. 55, 502–511 (2016)

  20. 20.

    Water oxidation mechanism in photosystem II, including oxidations, proton release pathways, O-O bond formation and O2 release. Biochim. Biophys. Acta 1827, 1003–1019 (2013)

  21. 21.

    et al. Photosynthesis. Electronic structure of the oxygen-evolving complex in photosystem II prior to O-O bond formation. Science 345, 804–808 (2014)

  22. 22.

    , , , & Recent developments in biological water oxidation. Curr. Opin. Chem. Biol. 31, 113–119 (2016)

  23. 23.

    , & QM/MM study of the S2 to S3 transition reaction in the oxygen-evolving complex of photosystem II. Chem. Phys. Lett. 636, 172–179 (2015)

  24. 24.

    et al. Theoretical illumination of water-inserted structures of the CaMn4O5 cluster in the S2 and S3 states of oxygen-evolving complex of photosystem II: full geometry optimizations by B3LYP hybrid density functional. Dalton Trans. 41, 13727–13740 (2012)

  25. 25.

    , & Mechanism of tyrosine D oxidation in Photosystem II. Proc. Natl Acad. Sci. USA 110, 7690–7695 (2013)

  26. 26.

    & Infrared detection of a proton released from tyrosine YD to the bulk upon its photo-oxidation in photosystem II. Biochemistry 54, 5045–5053 (2015)

  27. 27.

    & Binding and functional properties of two new extrinsic components, cytochrome c-550 and a 12-kDa protein, in cyanobacterial photosystem II. Biochemistry 32, 1825–1832 (1993)

  28. 28.

    & Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus. Biochemistry 39, 14739–14744 (2000)

  29. 29.

    & Flash-induced FTIR difference spectra of the water oxidizing complex in moderately hydrated photosystem II core films: effect of hydration extent on S-state transitions. Biochemistry 41, 2322–2330 (2002)

  30. 30.

    , & pH dependence of the flash-induced S-state transitions in the oxygen-evolving center of photosystem II from Thermosynechoccocus elongatus as revealed by Fourier transform infrared spectroscopy. Biochemistry 44, 1708–1718 (2005)

  31. 31.

    & Flash-induced Fourier transform infrared detection of the structural changes during the S-state cycle of the oxygen-evolving complex in photosystem II. Biochemistry 40, 1497–1502 (2001)

  32. 32.

    , & Monitoring proton release during photosynthetic water oxidation in photosystem II by means of isotope-edited infrared spectroscopy. J. Am. Chem. Soc. 131, 7849–7857 (2009)

  33. 33.

    et al. Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser. J. Synchrotron Radiat. 22, 532–537 (2015)

  34. 34.

    et al. Development of an X-ray pixel detector with multi-port charge-coupled device for X-ray free-electron laser experiments. Rev. Sci. Instrum. 85, 033110 (2014)

  35. 35.

    et al. Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data. J. Appl. Crystallogr. 47, 1118–1131 (2014)

  36. 36.

    et al. Data processing pipeline for serial femtosecond crystallography at SACLA. J. Appl. Crystallogr. 49, 1035–1041 (2016)

  37. 37.

    et al. CrystFEL: a software suite for snapshot serial crystallography. J. Appl. Crystallogr. 45, 335–341 (2012)

  38. 38.

    Indexing in single-crystal diffractometry with an obstinate list of reflections. J. Appl. Crystallogr. 25, 92–96 (1992)

  39. 39.

    , , , & iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011)

  40. 40.

    Processing of X-ray snapshots from crystals in random orientations. Acta Crystallogr. D Biol. Crystallogr. 70, 2204–2216 (2014)

  41. 41.

    & Assessing and maximizing data quality in macromolecular crystallography. Curr. Opin. Struct. Biol. 34, 60–68 (2015)

  42. 42.

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)

  43. 43.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010)

  44. 44.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004)

  45. 45.

    et al. A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354, 1552–1557 (2016)

Download references


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.


  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


  1. Search for Michihiro Suga in:

  2. Search for Fusamichi Akita in:

  3. Search for Michihiro Sugahara in:

  4. Search for Minoru Kubo in:

  5. Search for Yoshiki Nakajima in:

  6. Search for Takanori Nakane in:

  7. Search for Keitaro Yamashita in:

  8. Search for Yasufumi Umena in:

  9. Search for Makoto Nakabayashi in:

  10. Search for Takahiro Yamane in:

  11. Search for Takamitsu Nakano in:

  12. Search for Mamoru Suzuki in:

  13. Search for Tetsuya Masuda in:

  14. Search for Shigeyuki Inoue in:

  15. Search for Tetsunari Kimura in:

  16. Search for Takashi Nomura in:

  17. Search for Shinichiro Yonekura in:

  18. Search for Long-Jiang Yu in:

  19. Search for Tomohiro Sakamoto in:

  20. Search for Taiki Motomura in:

  21. Search for Jing-Hua Chen in:

  22. Search for Yuki Kato in:

  23. Search for Takumi Noguchi in:

  24. Search for Kensuke Tono in:

  25. Search for Yasumasa Joti in:

  26. Search for Takashi Kameshima in:

  27. Search for Takaki Hatsui in:

  28. Search for Eriko Nango in:

  29. Search for Rie Tanaka in:

  30. Search for Hisashi Naitow in:

  31. Search for Yoshinori Matsuura in:

  32. Search for Ayumi Yamashita in:

  33. Search for Masaki Yamamoto in:

  34. Search for Osamu Nureki in:

  35. Search for Makina Yabashi in:

  36. Search for Tetsuya Ishikawa in:

  37. Search for So Iwata in:

  38. Search for Jian-Ren Shen in:


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.

Extended data

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.