Abstract

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3,4,5,6,7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

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Data availability

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the following accession codes: 6DHE for the 0F, 6DHF for the 1F, 6DHO for the 2F, 6DHG for the 2F(150μs), 6DHH for the 2F(400μs) and 6DHP for the 3F data.

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Acknowledgements

This work was supported by the Director, Office of Science, Office of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences, and Biosciences (CSGB), Department of Energy (DOE) (J.Y., V.K.Y.), by National Institutes of Health (NIH) grants GM055302 (V.K.Y.), GM110501 (J.Y.) GM126289 (J.K.), GM117126 (N.K.S.), GM124149 and GM124169 (J.M.H.), the Ruth L. Kirschstein National Research Service Award (GM116423-02, F.D.F.), and Human Frontiers Science Project RGP0063/2013 (J.Y., U.B., A.Z.). We acknowledge the DFG-Cluster of Excellence “UniCat” coordinated by T.U. Berlin and Sfb1078, TP A5 (A.Z., H.D.); the Artificial Leaf Project (K&A Wallenberg Foundation 2011.0055) and Vetenskapsrådet (2016-05183) (J.M.); Diamond Light Source, Biotechnology and Biological Sciences Research Council (grant 102593) and a Strategic Award from the Wellcome Trust (A.M.O.). This research used NERSC, supported by DOE, under Contract No. DE-AC02-05CH11231. Synchrotron facilities at the ALS, Berkeley and SSRL, Stanford, were funded by DOE OBES. The SSRL Structural Molecular Biology Program is supported by the DOE OBER, and NIH (P41GM103393). LCLS and SSRL, SLAC National Accelerator Laboratory, are supported by DOE, OBES under Contract No. DE-AC02-76SF00515. We thank the staff at LCLS/SLAC and SSRL (BL 6-2, 7-3) and ALS (BL 5.01, 5.0.2, 8.2.1, 8.3.1).

Author information

Author notes

    • Thomas Fransson

    Present address: Interdisciplinary Center for Scientific Computing, University of Heidelberg, Heidelberg, Germany

  1. These authors contributed equally: Ruchira Chatterjee, Iris D. Young, Franklin D. Fuller.

Affiliations

  1. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    • Jan Kern
    • , Ruchira Chatterjee
    • , Iris D. Young
    • , Franklin D. Fuller
    • , Louise Lassalle
    • , Sheraz Gul
    • , Aaron S. Brewster
    • , Lacey Douthit
    • , Ernest Pastor
    • , Nigel W. Moriarty
    • , James M. Holton
    • , Paul D. Adams
    • , Nicholas K. Sauter
    • , Junko Yano
    •  & Vittal K. Yachandra
  2. Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany

    • Mohamed Ibrahim
    • , Rana Hussein
    • , Miao Zhang
    • , Julia Wersig
    • , Ina Seuffert
    • , Holger Dobbek
    •  & Athina Zouni
  3. Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    • Thomas Fransson
    •  & Uwe Bergmann
  4. LCLS, SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    • Roberto Alonso-Mori
    • , Clemens Weninger
    • , Raymond G. Sierra
    • , Mengning Liang
    • , Alexander Batyuk
    • , Jason E. Koglin
    • , Sergio Carbajo
    •  & Sébastien Boutet
  5. Institutionen för Kemi, Kemiskt Biologiskt Centrum, Umeå Universitet, Umeå, Sweden

    • Casper de Lichtenberg
    • , Dmitry Shevela
    •  & Johannes Messinger
  6. Department of Chemistry—Ångström, Molecular Biomimetics, Uppsala University, Uppsala, Sweden

    • Casper de Lichtenberg
    • , Mun Hon Cheah
    •  & Johannes Messinger
  7. SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    • Dimosthenis Sokaras
    • , Thomas Kroll
    •  & James M. Holton
  8. Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, UK

    • Pierre Aller
    • , Agata Butryn
    •  & Allen M. Orville
  9. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA

    • James M. Holton
  10. Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA

    • Paul D. Adams

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Contributions

U.B., V.K.Y. and J.Y. conceived the experiment; R.A.-M., A.Z., J.M., U.B., N.K.S., J.K., V.K.Y. and J.Y. designed the experiment; R.C., M.I., L.L., R.H., M.Z., L.D., J.W., I.S., A.Z. and J.K. prepared samples; A. Batyuk, M.L., S.B., R.A.-M., J.E.K. and S.C. operated the MFX instrument; F.D.F., S.G., E.P., P.A., A.M.O., J.M. and J.K. developed, tested and ran the sample delivery system; M.H.C., D. Shevela, R.C., C.d.L., J.Y. and J.M. performed and analysed O2 evolution and EPR measurements; R.C., F.D.F., S.G., M.I., C.d.L., M.H.C., I.D.Y., A.S.B., R.A.-M., R.H., M.Z., L.L., L.D., D. Sokaras, E.P., C.W., T.F., T.K., R.G.S., P.A., A. Butryn, A. Batyuk, M.L., S.B., J.E.K., S.C., A.M.O., A.Z., J.M., U.B., N.K.S., J.K., V.K.Y. and J.Y. performed the LCLS experiment; I.D.Y., A.S.B., N.W.M., J.M.H., P.D.A. and N.K.S. developed new software for data processing; I.D.Y., A.S.B., L.L., F.D.F., C.W., T.F., P.A., H.D., J.M.H., N.K.S. and J.K. processed and analysed XFEL data; J.M., J.K., V.K.Y. and J.Y. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Athina Zouni or Johannes Messinger or Junko Yano or Vittal K. Yachandra.

Extended data figures and tables

  1. Extended Data Fig. 1 Overview of the PSII structure and electron density maps of the 3F state.

    a, Structure of the native PSII homodimer. In the left monomer the location of cofactors for the initial charge separation (P680, PheoD1), and for the electron transfer leading to the reduction of the plastoquinone (QA, QB) at the acceptor side and to the oxidation of the OEC at the donor side by P680+ are indicated. In the right monomer, the locations of the protein subunits are displayed. bd, 2mFobs − DFcalc map (blue, 1.5σ contour) obtained from the room temperature 3F data set. b, Density around the main chain and a chlorophyll. c, Well-resolved ordered water molecules. d, Chlorophyll and pheophytin molecules with well-resolved tails. e, Clear density in hydrophobic regions and along cofactor hydrocarbon tails.

  2. Extended Data Fig. 2 Flash-induced S-state turnover of PSII micro crystals.

    a, Change of the first moment of the in situ-measured Mn Kβ XES as a function of flashes and fit to the data. b, Flash-induced O2 yield as measured by MIMS as a function of flash number and fit to the data. c, The estimated S-state population (%) for each of the flash states from fitting of the XES data and of the flash-induced O2 evolution pattern of a suspension of PSII crystals at pH 6.5. Two different fits were performed: a global fit of both O2 and XES data using an equal miss parameter of 22% and 100% S1 population in the 0F sample (black traces in a, b; S-state distribution listed in the columns headed O2), and a direct fit of the XES data using a 8% miss parameter in the S1→S2 and a 27% miss parameter for the S2→S3 and S3→S0 transitions (XES in c). For the XES fit, shifts of −0.06 eV per oxidation state increase for all S states were assumed. The XES raw spectra are published elsewhere36.

  3. Extended Data Fig. 3 Isomorphous difference maps in the second monomer at the QB site.

    ac, Fobs − Fobs maps contoured at 3σ at plastoquinone QB in monomer a. a, 1F − 0F difference map matching reduction of the plastoquinone to a semiquinone and concomitant slight geometry change. b, 2F − 0F difference map matching replacement of the fully reduced quinol with another quinone at the original position. c, 3F − 0F difference map, showing again structural changes similar to the 1F − 0F map, indicating formation of the semiquinone. Similar views are shown for monomer A in Fig. 1d–f and comparison of both monomers indicates similar flash-induced changes in both monomers.

  4. Extended Data Fig. 4 Movement of ligands around the OEC in the different S states.

    a, Overview of the ligand environment of the OEC, showing the dark state (0F) structure. Coordination of the OEC by nearby side chains and water molecules is indicated by dashed lines. bg, Trends for selected individual side chains in both monomers (bd, monomer A; eg, monomer a). Overlays of the refined models at the OEC following least-squares fitting of subunit D1 residues 55–65, 160–190 and 328, subunit CP43 residues 328 and 354–358, and chain D residue 352 of each other model to the 0F model. The largest and most consistent motions of side chains near the OEC through the sequence of illuminated state models are annotated with arrows indicating the trend. A motion observed in only one monomer is indicated by a dashed line.

  5. Extended Data Fig. 5 Impact of the data quality on the resolving power of the maps.

    af, The data quality evidenced by 2F state models and 2mFobs − DFcalc maps contoured at 1.5σ. a, 5TIS (2F, 2.25 Å) model and map. Overlays indicate atom numbering in the OEC and the identities of selected coordinating sidechains. b, Current 2F model and map cut to 2.25 Å. c, Current 2F model and map at the full 2.07 Å resolution. Emergence of locations of O4 with improved data quality is indicated by bold arrow. d, As in a from a different angle and with mFobs − DFcalc density at 3σ indicating the lack of sufficient evidence for inserting an additional O atom at a chemically reasonable position. e, f, As in b, c from the same direction as d and with mFobs − DFcalc density at 3σ shown to 2.25 and 2.05 Å, respectively, after omitting the inserted Ox atom. Centring of the refined Ox position within the omit density gives a clear indication of the position of the inserted water in the S3 state with the current, higher-quality data, even when artificially cut to the same resolution as the previous data set. g, mFobs − DFcalc maps of the 2F data that compare the O6 model from Suga et al.9 and the Ox model from the current study. Map shows the mFobs − DFcalc density calculated with our current 2F data and our model adding the O6 position of Suga et al.9 (with the occupancy of 0.7 and B-factor of 30) (g-1), and with our Ox model (g-2). We see clearly a positive density for the missing Ox and a negative density at the O6 position in g-1. Schematics of the O6 and Ox S3 models are shown on the left.

  6. Extended Data Fig. 6 Isomorphous difference maps in the second monomer at the OEC.

    Isomorphous difference density OEC sites in monomer a. Fobs − Fobs difference densities between the various illuminated states and the 0F data are contoured at +3σ (blue) and −3σ (orange). The model for the 0F data is shown in light grey whereas carbons are coloured as follows: 1F (cyan), 2F (150 μs) (green), 2F (400 μs) (yellow) and 2F (0.2 s) (blue).

  7. Extended Data Fig. 7 Water environment of the OEC.

    a, Extended schematic of the hydrogen bonding network connecting the OEC to the solvent-exposed surface of PSII and identification of several channels for either possible water movement or proton transfer. Top right, locations of four selected channels in the PSII monomer. be, Movements within the water networks across monomers. Coloured spheres are shown for each ordered water or chloride ion across the four metastable states, 0F through 3F, and for both monomers, with the stronger colour matching the first (A) monomer and the lighter colour matching the second (a) monomer. For ordered solvent, residue number is shown; for OEC atoms, the atom identifier is shown; and for the Cl2 site, the Cl 680 label is shown. b, The O1 water chain. Positional disagreement between monomers is visible especially near waters 77 (2F) and 27 (3F) and is on the same scale as changes between illuminated states, both of which may indicate a more dynamic water channel. c, The O4 water chain. With the notable exception of water 20, most water positions are stable across monomers and illuminated states. Water 20 is highly unstable in position in the two states (0F, 3F) in which it is modelled, and there is not sufficient density in the remaining states to model a water 20 position. d, The Cl1 site water channel with no notable movements. e, The Cl2 site water channel with no notable movements. f, Indication of a split position of W3 in the S0 state. mFobs − DFcalc difference density (green mesh) in the 3F state suggests an alternate position near W3 (W3b in Fig. 4d). g, h, Possible access to W3/Ox side from the Cl1 or the O1 channel. The surface of the protein is shown in grey to visualize the extent of the cavities around the OEC, and Van der Waals radii are indicated for selected residues or atoms by dotted spheres. Shown are two different views for each channel. The direction of the Cl1 channel is indicated by a green arrow and the O1 channel by a pink arrow. Water W2 is shown in purple, W3 in cyan and Ox in orange. Yellow spheres indicate other waters. Mn are shown in magenta, other bridging oxygens as red spheres.

  8. Extended Data Table 1 Merging and refinement statistics for (a) the refined data sets including all lattices or (b) for the additional data sets containing only lattices with unit cells within 1% of the target unit cell
  9. Extended Data Table 2 Interatomic distances at the OEC in each merged data set, in each monomer (A/a), in Å
  10. Extended Data Table 3 Channel nomenclature in the literature

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https://doi.org/10.1038/s41586-018-0681-2

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