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

Nature volume 517, pages 99–103 (2015)Cite this article

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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.

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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.

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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.

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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.

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Author notes

  1. Michihiro Suga and Fusamichi Akita: These authors contributed equally to this work.

Authors and Affiliations

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

    Michihiro Suga, Fusamichi Akita, Yoshiki Nakajima, Tetsuya Shimizu & Jian-Ren Shen

  2. RIKEN SPring-8 Center, 1-1-1 Kouto Sayo, Hyogo 679-5148, Japan ,

    Kunio Hirata, Go Ueno, Hironori Murakami, Keitaro Yamashita, Masaki Yamamoto & Hideo Ago

  3. Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Saitama 332-0012, Japan ,

    Kunio Hirata

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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.

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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
Full size table
Extended Data Table 2 B-factor values of the individual atoms in the OEC of the XFEL structures
Full size table
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
Full size table
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
Full size table

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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

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