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Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

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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Figure 1: TR-SFX of PSII at SACLA–XFEL.
Figure 2: Isomorphous difference Fourier map between the 2F-state and the dark-state of the pre-flashed samples.
Figure 3: Isomorphous difference Fourier map and structural changes around the QB/non-haem iron-binding site and the OEC.
Figure 4: Possible mechanism of O=O bond formation and proton transfer pathways from the OEC.

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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.

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Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to So Iwata or Jian-Ren Shen.

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The authors declare no competing financial interests.

Additional information

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 figures and tables

Extended Data Figure 1 Micro-sized crystals of PSII, its diffraction image and quality of the diffraction data sets.

a, Micro-sized crystals of PSII used for TR-SFX. b, A diffraction image from a micro-sized PSII crystal obtained with a single pulse from SACLA-XFEL. cf, Quality of the data sets. CC1/2 (c), Rsplit (d), multiplicity (e) and <I/σ(I)> (f) plotted against resolution for the dark data and the 2F data of the pre-flashed samples.

Extended Data Figure 2 Measurement of the S3 state population upon 2F illumination by FTIR.

Top, ATR–FTIR difference spectra upon the first (a) and second (b) flashes of the PSII crystal (red lines) and PSII solution (black lines). Bottom, result of the least-squares fitting analysis of the second flash spectrum of the PSII crystal in the symmetric COO region (1,470–1,300 cm−1). The spectrum of the PSII crystal (red line) was fit with a linear combination of the first and second flash spectra of the solution sample, F1(ν) and F2(ν), respectively (black lines in the top panel). The resulting fitting spectrum (grey line) and the two components, 0.27F1(ν) (blue line) and 0.64F2(ν) (green line), are also shown.

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

ac, Anomalous difference Fourier maps for OEC atoms (a), a region around the OEC (b), and a newly identified Ca-binding site in CP43 (c), of the non-pre-flashed dark data. The maps are shown in cyan and magenta contoured at 4σ and 12σ, respectively (a), grey contoured at 3σ (b) and cyan contoured at 4σ (c). d, Histogram analysis of the anomalous map of the non-pre-flashed dark data.

Extended Data Figure 4 Electron density maps and structural changes around YD between the non-pre-flashed 2F and dark data sets.

a, Isomorphous difference Fourier map between the non-pre-flashed 2F state and dark state data sets in green (positive) and red (negative) contoured at ±5σ, superimposed with the non-pre-flashed dark structure and 2F structure. The non-pre-flashed dark and 2F structures are coloured yellow and green, respectively. The two different positions of W508 observed in the 1.95 Å resolution XFEL structure (W508I and W508II) are shown as black dots. b, c, 2mFo − DFc map and mFo − DFc map of the non-pre-flashed dark state (b) and 2F state (c). The 2mFo − DFc map is coloured grey, contoured at 1.5σ, and the mFo − DFc map is colored cyan (positive) and brown (negative), contoured at ±4σ.

Extended Data Figure 5 Electron density maps for the O4–water chain.

The mFo − DFc maps calculated for the O4–water chain by omitting the four water molecules W567, W665, W542 and W546 are shown for the non-pre-flashed dark state (a, b) and 2F state (c, d). The maps shown are for monomer A (a, c) and monomer B (b, d) in cyan (positive) and orange (negative) contoured at ±3σ.

Extended Data Figure 6 Distribution of the unit cell parameters and crystal packing of PSII for the non-pre-flashed samples.

ac, Distributions of unit cell parameters for the dark state images (a) and 2F state images (b) from the non-pre-flashed samples and the dark images when the post-crystallization procedure was not adequate (c) are shown with their average lengths and standard deviations. No large differences were observed between the dark images and the 2F images, whereas a longer c axis was observed in c. df, Comparison of the crystal packing between the dark state and the data set with a longer c axis is shown in a view direction perpendicular to the bc plane. Colour codes: PSII and the crystallographic symmetric molecules in the dark state, cyan and blue, respectively; PSII, PsbY subunits in PSII and the crystallographic symmetric molecules in the data set with a long c axis, grey, red and khaki, respectively. Note that PsbY has considerable steric hindrance with the adjacent PSII molecule in the crystal packing of the dark data (e).

Extended Data Table 1 Statistics of the diffraction data and structural refinements for the dark state and 2F state data sets of both pre-flashed and non-pre-flashed samples, collected at SACLA (a), and the Riso values between the different data sets (b)
Extended Data Table 2 Statistics for the processing of the diffraction data for the pre-flashed dark state (a), pre-flashed 2F state (b), non-pre-flashed dark state (c), and non-pre-flashed 2F state (d) collected at SACLA
Extended Data Table 3 List of the top 30 anomalous peaks identified in the non-pre-flashed dark state and 2F state data sets

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Suga, M., Akita, F., Sugahara, M. et al. Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543, 131–135 (2017). https://doi.org/10.1038/nature21400

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