Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses

Abstract

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 ångström resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well defined protein environment1. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation2, and slight differences were found in the Mn–Mn distances determined by XRD1, EXAFS3,4,5,6,7 and theoretical studies8,9,10,11,12,13,14. Here we report a ‘radiation-damage-free’ structure of PSII from Thermosynechococcus vulcanus in the S1 state at a resolution of 1.95 ångströms using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn–Mn distances that are shorter by 0.1–0.2 ångströms. The valences of each manganese atom were tentatively assigned as Mn1D(iii), Mn2C(iv), Mn3B(iv) and Mn4A(iii), based on the average Mn–ligand distances and analysis of the Jahn–Teller axis on Mn(iii). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Atomic structure of the OEC by XFEL.
Figure 2: Comparison of the OEC structures obtained by XFEL and SR.
Figure 3: Possible mechanisms for the oxygen-evolving reaction.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the structure determination have been deposited in the Protein Data Bank with accession codes 4UB6 and 4UB8 for data sets 1 and 2, respectively.

References

  1. Umena, Y., Kawakami, K., Shen, J.-R. & Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55–60 (2011)

    ADS  CAS  Article  Google Scholar 

  2. Yano, J. et al. X-ray damage to the Mn4Ca complex in single crystals of photosystem II: a case study for metalloprotein crystallography. Proc. Natl Acad. Sci. USA 102, 12047–12052 (2005)

    ADS  CAS  Article  Google Scholar 

  3. Yano, J. et al. High-resolution Mn EXAFS of the oxygen-evolving complex in photosystem II: structural implications for the Mn4Ca cluster. J. Am. Chem. Soc. 127, 14974–14975 (2005)

    CAS  Article  Google Scholar 

  4. Yano, J. et al. Where water is oxidized to dioxygen: structure of the photosynthetic Mn4Ca cluster. Science 314, 821–825 (2006)

    ADS  CAS  Article  Google Scholar 

  5. Dau, H. & Haumann, M. The manganese complex of photosystem II in its reaction cycle — basic framework and possible realization at the atomic level. Coord. Chem. Rev. 252, 273–295 (2008)

    CAS  Article  Google Scholar 

  6. Dau, H., Grundmeier, A., Loja, P. & Haumann, M. On the structure of the manganese complex of photosystem II: extended-range EXAFS data and specific atomic-resolution models for four S-states. Phil. Trans. R. Soc. Lond. B 363, 1237–1243 (2008)

    CAS  Article  Google Scholar 

  7. Glöckner, C. et al. Structural changes of the oxygen-evolving complex in photosystem II during the catalytic cycle. J. Biol. Chem. 288, 22607–22620 (2013)

    Article  Google Scholar 

  8. Ames, W. et al. Theoretical evaluation of structural models of the S2 state in the oxygen evolving complex of Photosystem II: protonation states and magnetic interactions. J. Am. Chem. Soc. 133, 19743–19757 (2011)

    CAS  Article  Google Scholar 

  9. Galstyan, A., Robertazzi, A. & Knapp, E. W. Oxygen-evolving Mn cluster in photosystem II: the protonation pattern and oxidation state in the high-resolution crystal structure. J. Am. Chem. Soc. 134, 7442–7449 (2012)

    CAS  Article  Google Scholar 

  10. Grundmeier, A. & Dau, H. Structural models of the manganese complex of photosystem II and mechanistic implications. Biochim. Biophys. Acta 1817, 88–105 (2012)

    CAS  Article  Google Scholar 

  11. Isobe, H. 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)

    CAS  Article  Google Scholar 

  12. Luber, S. et al. S1-state model of the O2-evolving complex of photosystem II. Biochemistry 50, 6308–6311 (2011)

    CAS  Article  Google Scholar 

  13. Pantazis, D. A., Ames, W., Cox, N., Lubitz, W. & Neese, F. Two interconvertible structures that explain the spectroscopic properties of the oxygen-evolving complex of photosystem II in the S2 state. Angew. Chem. Int. Edn Engl. 51, 9935–9940 (2012)

    CAS  Article  Google Scholar 

  14. Blomberg, M. R. A., Borowski, T., Himo, F., Liao, R. Z. & Siegbahn, P. E. M. Quantum chemical studies of mechanisms for metalloenzymes. Chem. Rev. 114, 3601–3658 (2014)

    CAS  Article  Google Scholar 

  15. Zouni, A. et al. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409, 739–743 (2001)

    ADS  CAS  Article  Google Scholar 

  16. Kamiya, N. & Shen, J.-R. Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-Å resolution. Proc. Natl Acad. Sci. USA 100, 98–103 (2003)

    ADS  CAS  Article  Google Scholar 

  17. Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J. & Iwata, S. Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831–1838 (2004)

    ADS  CAS  Article  Google Scholar 

  18. Guskov, A. et al. Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride. Nature Struct. Mol. Biol. 16, 334–342 (2009)

    CAS  Article  Google Scholar 

  19. Southworth-Davies, R. J., Medina, M. A., Carmichael, I. & Garman, E. F. Observation of decreased radiation damage at higher dose rates in room temperature protein crystallography. Structure 15, 1531–1541 (2007)

    CAS  Article  Google Scholar 

  20. Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752–757 (2000)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  24. Holton, J. M. & Frankel, K. A. The minimum crystal size needed for a complete diffraction data set. Acta Crystallogr. D 66, 393–408 (2010)

    CAS  Article  Google Scholar 

  25. Hirata, K. et al. Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL. Nature Methods 11, 734–736 (2014)

    CAS  Article  Google Scholar 

  26. Shoji, M. et al. Theoretical modeling of biomolecular systems I. Large scale QM/MM calculations of hydrogen bonding networks of oxygen evolving complex of photosystem II. Mol. Phys. http://dx.doi.org/10.1080/00268976.2014.960021 (published online, 29 September 2014)

  27. Cox, N. et al. Effect of Ca2+/Sr2+ substitution on the electronic structure of the oxygen-evolving complex of photosystem II: a combined multifrequency EPR, 55Mn-ENDOR, and DFT study of the S2 state. J. Am. Chem. Soc. 133, 3635–3648 (2011)

    CAS  Article  Google Scholar 

  28. Rapatskiy, L. 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)

    CAS  Article  Google Scholar 

  29. Siegbahn, P. E. 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)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

  33. Murray, J., Garman, E. & Ravelli, R. X-ray absorption by macromolecular crystals: the effect of wavelength and crystal composition on absorbed dose. J. Appl. Crystallogr. 37, 513–522 (2004)

    CAS  Article  Google Scholar 

  34. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank T. Ishikawa, M. Yabashi, K. Tono and Y. Inubushi for help in using the SACLA beamline, T. Tsukihara and S. Yoshikawa for suggestions and comments on the experiments, F. Seno for assistance with sample preparation, and K. Kawakami and Y. Umena for suggestions in the initial stage of the project. F. H. M. Koua participated in the initial stage of this work. This work was supported by an X-ray Free Electron Laser Priority Strategy Program (The Ministry of Education, Culture, Sports, Science and Technology of Japan, MEXT) (H.A. and J.-R.S.), a JST/CREST grant (K.H.), a grant-in-aid for Specially Promoted Research no. 24000018 (J.-R.S.) and KAKENHI grant no. 26840023 (M.S.) from JSPS, MEXT, Japan. The XFEL experiments were performed at beamline 3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos 2012A8011, 2012B8040, 2013A8047, 2013B8052 and 2014A8036), and we thank staff at SACLA for their help.

Author information

Authors and Affiliations

Authors

Contributions

H.A. and J.-R.S. planned and organized the experiments, F.A. and Y.N. prepared the samples, F.A. made the crystals, G.U., H.M., K.H., H.A. and M.Y. designed and established the experimental set-up, M.S., F.A., T.S., Y.N., G.U., H.M., K.H., H.A., M.Y. and J.-R.S. conducted the diffraction experiments, K.Y. performed scaling of raw diffraction images, M.S. performed the structural analysis, M.S. and J.-R.S. wrote the manuscript, and all authors discussed and commented on the results and the manuscript.

Corresponding authors

Correspondence to Masaki Yamamoto, Hideo Ago or Jian-Ren Shen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Diffraction experiment using the XFEL beam at SACLA.

a, Schematic drawing of the diffraction experiment. Still diffraction images were recorded from different points on large PSII crystals. The crystals were rotated by 0.2° between each image over a range of 180°. Adjacent irradiation points were separated by 50 µm in the horizontal direction for any rotational angle. Translation in the vertical direction was varied depending on the rotational angle, so that the irradiation points were also separated by 50 µm in the vertical direction. b, A picture of the PSII crystal in a cryo-loop after the XFEL diffraction experiment. Note that the path where the XFEL beam passed through became hollowed out, and resulted in footprints of the irradiation points, which were well separated.

Extended Data Figure 2 A diffraction image from a PSII crystal obtained with the XFEL beam.

A typical diffraction image is shown; the boxed area at the right is shown enlarged in the inset, where diffraction spots at the maximum resolution are visible.

Extended Data Figure 3 Anomalous signals of the metal ions obtained with the XFEL beam.

Shown are anomalous difference Fourier maps contoured at 5σ distributions. a, Manganese atoms of the OEC. b, The non-haem iron in the acceptor side between QA and QB.

Extended Data Figure 4 Coordination environment of Mn1D, Mn2C, Mn3B and Mn4A.

The ligand environment of a, Mn1D, b, Mn2C, c, Mn3B and d, Mn4A are drawn in stereo view. The ligand bonds involving O5 coordination are slightly longer than the others in both Mn1D and Mn4A. Note that Mn1D is in a pseudo-five-coordinated, trigonal bipyramidal geometry with additional weak interaction to O5. Colour code: grey, manganese; green, carbon; blue, nitrogen; red, oxo-oxygen; yellow, O5; orange, water.

Extended Data Figure 5 Jahn-Teller axes on Mn1D and Mn4A.

The ligand environments of Mn1D and Mn4A are drawn in a stereo view. Based on the longer ligand bonds involving the O5 coordination as shown in Extended Data Fig. 4a, d, two possible Jahn–Teller axes were assigned which are approximately parallel. Colour codes are the same as those in Extended Data Fig. 4. Light blue lines indicate two Jahn–Teller axes found in the OEC in the S1 state. a, View direction almost orthogonally oriented relative to the two Jahn–Teller axes. b, View direction rotated by 90° from a.

Extended Data Figure 6 Scaling of the raw images.

Standard deviations of the average pixel values calculated in the group belonging to the same crystal before and after scaling are plotted. X and Y axes indicate standard deviations obtained before and after scaling, respectively. Red line in the plot represents the relationship Y = X. a, Plot of the images collected during the first half of the beam time (including the first half of the images for data set 1). b, Plot of the images collected during the last half of the beam time (including the rest of the images for data set 1 and all of the images for data set 2).

Extended Data Table 1 Data collection and refinement statistics with the XFEL beam (data set 1 and data set 2) in comparison with the SR data
Extended Data Table 2 B-factor values of the individual atoms in the OEC of the XFEL structures
Extended Data Table 3 Mn–Mn, Mn–Ca, Mn–O, Mn–water, Ca–O, Ca–water, O5–water and ligand distances of the OEC in each PSII monomer of the two XFEL structures
Extended Data Table 4 Assignment of valences of individual manganese atoms in the OEC in the S1 state based on average ligand distance and distribution of Jahn–Teller axes

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Suga, M., Akita, F., Hirata, K. et al. Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 517, 99–103 (2015). https://doi.org/10.1038/nature13991

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13991

Further reading

Comments

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing