Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2,3. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site4,5,6. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10,11,12,13. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.
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This work was supported by the Director, Office of Science, Office of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences, and Biosciences (CSGB) of the Department of Energy (DOE) (J.Y., V.K.Y.) for X-ray methodology and instrumentation; National Institutes of Health (NIH) grants GM055302 (V.K.Y.) for PS II biochemistry, structure and mechanism, GM110501 (J.Y.) for instrumentation development for XFEL experiments, GM102520 and GM117126 (N.K.S.) for development of computational protocols for XFEL data; the Ruth L. Kirschstein National Research Service Award (GM116423-02, F.D.F.); and the Human Frontiers Science Project Award No. RGP0063/2013 310 (J.Y., U.B., P.W., A.Z.). The DFG-Cluster of Excellence “UniCat” coordinated by T.U. Berlin and Sfb1078 (Humboldt Universität Berlin), TP A5 (A.Z., H.D.), the Solar Fuels Strong Research Environment (Umeå University), the Artificial Leaf Project (K&A Wallenberg Foundation 2011.0055) and Energimyndigheten (36648-1) (J.M.) are acknowledged for support. H.L. and C.A.S. acknowledge support from the US DOE, OBES, CSGB Division. W.I.W. and A.T.B. acknowledge support from an HHMI Collaborative Innovation Award. D.G.W. is funded by industrial income received by CCP4. This research used resources of NERSC, a User Facility supported by the Office of Science, DOE, under Contract No. DE-AC02-05CH11231. Portions of this work were supported by a BNL/US DOE, LDRD grant (11-008; A.M.O.); and NIH/NCRR grant 2-P41-RR012408, NIH/NIGMS grants 8P41GM103473-16 and P41GM111244 and the US DOE, OBER grant FWP BO-70 (A.M.O., B.A.). A.M.O and P.T.D were supported in part by the Diamond Light Source, and A.M.O acknowledges support from a Strategic Award from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (grant 102593). P.B. was supported by a Wellcome Trust DPhil studentship. Testing of crystals and various parts of the setup were carried out at synchrotron facilities that were provided by the Advanced Light Source (ALS) in Berkeley and Stanford Synchrotron Radiation Lightsource (SSRL) in Stanford, funded by DOE OBES under contract DE-AC02-05CH11231 (ALS) and DE-AC02-76SF00515 (SSRL). The SSRL Structural Molecular Biology Program is supported by the DOE OBER and by the NIH (P41GM103393). Use of the LCLS and SSRL, SLAC National Accelerator Laboratory, is supported by the US DOE, Office of Science, OBES under Contract No. DE-AC02-76SF00515. We thank M. Bommer for discussions and help regarding structure refinement, crystallographic model building and validation, J. Hattne for his contributions to the development of XFEL diffraction data processing, A. Boussac for discussions on ammonia binding and his contributions to the substrate water exchange measurements of the S3 state in the presence of ammonia, and the previous CXI beamline scientist, G. Williams, for his support during the initial stages of this project. We thank the support staff at LCLS/SLAC and at SSRL (BL 6-2, 7-3) and ALS (BL 5.01, 5.0.2, 8.2.1).
The authors declare no competing financial interests.
Reviewer Information Nature thanks J. Murray, C. Yocum 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 Schematic of the reaction centre and OEC in PS II and packing of the dimeric complex in the crystal lattice.
a, The reaction centre is shown with cofactors labelled as Pheo (pheophytin), Chl (chlorophyll), PQ (plastoquinone), QA, QB (primary and secondary acceptor plastoquinones bound to PS II, respectively). The numbering of Mn (purple spheres), oxygen (red/grey spheres) atoms and metal-bound waters in the OEC follows the convention of ref. 21. Upon illumination of PS II, an electron is transferred ~35 Å across the membrane from the excited primary electron donor P680 to the final electron acceptor QB via ChlD1, PheoD1, QA, and a non-haem FeII. After accepting two electrons and undergoing protonation, plastoquinol QBH2 is released from PS II into the membrane matrix. The photo-generated radical cation P680•+ is reduced by a tyrosine residue (TyrZ) to generate a neutral tyrosine radical TyrZ•, which acts as an oxidizing agent for water at the OEC. b, c, d, Packing of the dimeric complex observed in the room temperature data for three different view directions. The unit cell is indicated by a wire frame and axes are labelled. Dimers related by translation are coloured identically. e–g, Packing observed in the cryogenic structure in ref. 16 (PDB: 4PJ0) in the same orientations as in b–d. h–j, Packing observed in the cryogenic XFEL structure in ref. 7 (PDB: 4UB6). The space group is the same in all three cases, but the unit cell dimensions and packing are different. Whereas the a and b dimensions are very similar between 4PJ0 and the current room temperature data, the c axis is elongated. This results in a very similar arrangement of dimers in rows along the a axis (compare d and g), whereas there is a larger spacing between two dimer rows at the cytoplasmic side of the complex (compare black ellipse in b and e) owing to the elongation of the c axis in the room temperature packing. The structure in ref. 7 has a very different arrangement of the dimers and no closely packed rows of dimers are visible (see c and f compared with i, and d and g compared with j).
Extended Data Figure 2 Electron density omit maps of the lumenal CD helix and part of the loop region of subunit D1.
a–h, Obtained from the room tempearture dark (a, b), 2F (c, d), and 2F-NH3 (e–h) datasets. For all maps residues 165–190 of subunit D1 (shown as grey sticks) were omitted followed by three rounds of coordinate and real space refinement of the model with (g, h) or without (a–f) simulated annealing in phenix.refine. a, 2mFo − DFc map (blue, 1.5σ contour) of the dark dataset. b, Polder mFo − DFc map (green, 4σ contour) of the dark dataset. c, 2mFo − DFc map (blue, 1.5σ contour) of the 2F dataset. d, Polder mFo−DFc map (green, 4σ contour) of the 2F dataset. e, 2mFo−DFc map (blue, 1.5σ contour) of the 2F-NH3 dataset. f, Polder mFo−DFc map (green, 4σ contour) of the 2F-NH3 dataset. g, Standard mFo−DFc omit map (green, 3σ contour) of the 2F-NH3 dataset after simulated annealing. h, Polder mFo−DFc map (green, 4σ contour) of the 2F-NH3 dataset after simulated annealing.
Extended Data Figure 3 Comparison of dark room temperature structure with cryogenic structures from refs
7 and 16 . Our room temperature dark state structure is shown in grey, the Suga XFEL7 in cyan and the Hellmich16 cryogenic structure in purple. a, Overlay of the room temperature and Suga7 structures. b, Overlay of the room temperature and Hellmich16 structures. A large-scale rigid body motion of the two monomers with respect to each other and an in-plane expansion of each PS II monomer in the room temperature structure are visible. c–e, Comparison of TMH 2 of subunit CP47 between the dark state and the cryogenic XFEL structure7 (c), the dark state and the Hellmich16 cryogenic structure (d) and between the two cryogenic structures (e). The two cryogenic datasets reflect crystals with different packing. View is from the cytoplasmic side (top) or along the membrane plane (bottom). Despite the different packing, only a small shift of 0.1–0.2 Å is observed between the two cryogenic structures. In contrast, the room temperature structure exhibits a larger shift of 0.5–0.6 Å in the positions of TMH 2 with respect to the cryogenic structures in both crystal forms, well above the error margin in our data. The cryogenic structures were superposed onto our dark structure model in PyMol (Schrödinger, LLC) using monomer 1 for alignment.
Extended Data Figure 4 Comparisons of the cofactor–cofactor distances in the crystal structures collected at cryogenic temperature and room temperature.
a, Distances for central cofactors; the histogram shows the deviation of the cofactor–cofactor distances in the Hellmich cryogenic SR dark state structure (4PJ016, highlighted in blue) and in our dark and 2F room temperature structures (highlighted in yellow) from those of the cryogenic XFEL structure reported by Suga et al.7 (4UB6). Changes are indicated by bars for monomers 1 and 2 (m1, m2), and colour coding of bars matches the colouring of the associated pair of cofactors in the diagram. Among the differences, there is a consistent elongation in the distances involving ChlZ as well as in the QB–Cyt b-559 and OEC–Cyt c-550 distances of both monomers in the room temperature data. In other cases, expansion of individual cofactor distances is observed in both room temperature structures and 4PJ0 relative to 4UB6 (for example, PD2–ChlD2, QB–PheoD2), and in the case of PD1–PD2 on average the elongation is more pronounced at room temperature than in the cryogenic structures. Changes in Chl positions between the room temperature dark structure and 4UB6 (b), between the room temperature dark structure and 4PJ0, which have the same dimer–dimer packing (c), and between 4PJ0 and 4UB6 (d). The distances of the Chl ring centres from the membrane normal passing through the centre between PheoD1 and PheoD2 are computed and relative changes with respect to the values obtained from 4UB6 or 4PJ0 are shown as colour coding on a rainbow scale from green (0.2 Å contraction) to red (1.0 Å expansion). For selected Chl–Chl pairs, distances are given with the absolute change in parentheses.
a, Water positions refined in the dark room temperature structure of the PS II dimer are indicated by blue and yellow spheres. View is along the membrane plane with the cytoplasm on top and lumen on the bottom. Waters whose positions coincide with waters located in the cryogenic XFEL structure7 (4UB6) are coloured in yellow and waters in other positions are coloured in dark blue. b, Enlarged view of the lumenal region showing the OEC of one monomer (magenta, red and green spheres for Mn, O and Ca) as well as the two Cl− (pink spheres) located close to the OEC. c, Waters located at the lumenal side of one monomer. View is from the lumenal side onto the membrane plane, with colour coding as in a and b. d, Polder omit maps (2.5σ contour, green mesh) for waters in the direct vicinity of the OEC in the dark state. Waters included in the refined model are indicated as red spheres, additional waters placed based on polder maps as orange spheres, and waters from 4UB6 are shown in light cyan. e, Possible water networks next to the OEC. Waters included in the refined model of the 2F state are indicated as red spheres and waters from 4UB6 are shown in light cyan. Polder omit maps (2.0σ contour, green mesh, carved at 2 Å around water positions from 4UB6) confirm the positions of the refined waters and indicate the presence of additional waters (for example, A571, A588), but no omit map density was observed at the position of water C665. The starting points of three water/proton channels postulated in ref. 21 are indicated by dashed red, yellow, and blue lines.
a, The 2mFo−DFc electron density (top) contoured at 4.0σ (green) and 8.0σ (blue mesh) and the polder mFo−DFc electron density (middle) of the dark dataset after omitting the OEC and real space refinement contoured at 8.0σ (green) and 14.0σ (blue mesh). At the bottom, the mFo−DFc electron density after omitting individual metal atoms and refining with simulated annealing is shown contoured at 4.0σ (Ca), 7.0σ (Mn1, Mn2), 8.0σ (Mn3) and 4.0σ (Mn4). The model of the OEC is shown with Mn as magenta, Ca as green and oxygen as red spheres overlaid with yellow spheres indicating the centres of the obtained omit densities. b, The 2mFo−DFc electron density (top) contoured at 3.0σ (green) and 6.0σ (blue mesh) and the polder mFo−DFc electron density (middle) of the 2F dataset after omitting the OEC and real space refinement contoured at 8σ (green) and 14σ (blue mesh). At the bottom, the mFo−DFc electron density after omitting individual metal atoms and refining with simulated annealing is shown contoured at 12.0σ (Ca, Mn1, Mn3), 13.0σ (Mn2) and 10.0σ (Mn4), with colour coding as in a. c, The 2mFo−DFc electron density (top) contoured at 5.0σ (green) and 8.0σ (blue mesh) and the polder mFo−DFc electron density (middle) of the 2F-NH3 dataset after omitting the OEC and real space refinement contoured at 11σ (green) and 16σ (blue mesh). At the bottom, the mFo − DFc electron density after omitting individual metal atoms and refining with simulated annealing is shown contoured at 5.0σ (Ca), 10.0σ (Mn1), 8.0σ (Mn2, Mn4) and 11.0σ (Mn3), with colour coding as in a.
Extended Data Figure 7 Characterization of PS II samples and substrate water exchange rates in the S3 state.
a, Flash-induced O2 evolution pattern (FIOP) of a suspension of the native PS II core complexes (PSIIcc) at pH 6.5. Fit parameter: 100% S1 in the dark, miss 20%, double hit 4%, damping 2%, fit done on first five flashes. In the 2F sample, if double hits are excluded, ~60% of the S3 population was calculated, with ~30% of S2 and ~4% of S1. b, FIOP of a suspension of the PS IIcc (TRIS, pH 7.5, 100 mM (NH4)2SO4) with and without PPBQ measured with 12 s between flashes using the thin layer MIMS setup (see Methods) and 532 nm laser flash illumination: an increase in O2 yield by 15% was observed if PPBQ was added. c, Light saturation in the thin layer MIMS set up resembling the illumination conditions of the DOT approach. The ratio of the oxygen yield of the third flash over that of the fourth flash is plotted as a qualitative measure for the miss parameter, which is minimal when the ratio is large. d, EPR spectra of native (pH 6.0 and 7.5) and ammonia-treated (pH 7.5) PS II solutions after continuous illumination at 195 K for 1 min followed by annealing to 260 K for 30 s. Spectrometer condition: microwave frequency, 9.23 GHz; field modulation amplitude, 32 G at 100 KHz; microwave power, 20 mW. The spectra were collected at 7 K. e, EPR spectra of ammonia-treated (pH 7.5) PS II solution after applying one (red) or two (black) flashes. Spectrometer conditions are as in a. f, O2-flash pattern of PS II crystals at pH 7.5 (TRIS, (NH4)2SO4) measured by MIMS with the replica set up for jet illumination described above. g, FIOP (Joliot-type electrode) of PSIIcc at pH 7.5 and 20 °C in TRIS buffer with either 100 mM (NH4)2SO4 or 100 mM Na2SO4 addition. The O2 yields for each sample were normalized to the O2 yields induced by flashes 3–6 (Y3–6). No artificial electron acceptors were added. The flash frequency of the Xe-flash lamp was 2 Hz, and the Chl concentration 0.4 mM. Data are the average of three technical replicates. From the data, a miss parameter of 23–25% and an S3 state population of 50–53% can be extracted for both sample types. Double hits, caused by the Xe flash lamp, are 3–6%, and are absent under laser flash illumination used during the XFEL experiments. The total O2 yield of the ammonia-containing sample was 66% of the Na2SO4 control, and 52% of the FIOP at pH 6.5 (a). h, FIOP of PS II core sample incubated in TRIS and 100 mM (NH4)2SO4 for 5 min at pH 7.6 versus one that was incubated for 4 h at room temperature. No degradation of the sample was observed over time (O2 yields of both FIOPs normalized to Y3–6 of 4-min trace). i, FIOP of PS II core sample containing TRIS and 100 mM (NH4)2SO4 versus one containing MOPS and 100 mM (NH4)2SO4. Both FIOPs normalized to Y3–6 of TRIS containing sample. j, k, Substrate water exchange was measured for the S3 state of PS II core complexes at pH 7.6/20 °C as described2,4,12 in HEPES buffer containing 100 mM NH4Cl (j) or 100 mM NaCl (k). The left panels show the biphasic rise of the mass 34 peak (16O18O), while the right side shows the simultaneously recorded monophasic rise of the 36 peak of the double exchanged 18O18O. Red symbols represent the individually measured data points, while the blue lines are the kinetic simulations. Nearly identical rates for the exchange of the fast (kfast) and slow (kslow) substrates were found with and without ammonia.
The models are grouped into four classes (a–d, see below). Mn are numbered (1–4) as in the main text. a, Models with an inserted water (highlighted in yellow) on the left side (closed cubane proposed as a transient S3-state in ref. 64 in teal; ref. 65 in light orange) shift Mn4 the furthest out. b, Models with an inserted water (highlighted in yellow) on the right side (model in ref. 64 in yellow; ref. 66 in pink; ref. 67 in dark blue) closely resemble models with an open cubane, and were proposed initially by Li and Siegbahn23. c, Other models with no inserted water: from ref. 67 in marine blue; 4UB67 in green; from ref. 67 in lavender; from ref. 65 in dark orange). d, Only one proposed model featured a closed cubane with no inserted water (ref. 68 in yellow-green). Note that, except for the type a structure (a complete cubane plus mono-μ-oxo bridged Mn4) and the type d structure (a closed cubane with no water inserted), the Mn atomic positions are very similar in all models within 0.26 Å. Even between the type a and other models, the Mn4 positions differ only by 0.73 Å. On the other hand, the O5 position is expected to differ among the models by at most 1.52 Å.
Extended Data Figure 9 The electron density of the Cl− binding sites and environment of the W1 and W2 sites at the OEC in the 2F-NH3 samples in the 2F dataset.
a, The Cl− binding site 1 with Cl− (green sphere) at its refined position. The 2mFo − DFc map (blue mesh) is shown at 1.5σ, and the mFo − DFc map (green/red) is shown at ± 3σ. b, Cl− binding site 1 with ammonia (blue sphere) instead of Cl− included in the model. The lack of difference density at the Cl− position in the refined model and the positive difference density observed when ammonia is substituted for Cl− indicate that ammonia does not account for the electron density and that Cl− is a good model for the observed density. c, d, Comparison of the positions of W1 and W2 among the 2F-NH3 (red), 2F (light pink) and cryogenic S1-XFEL7 (light cyan) structures in two different orientations. The outcome of two different alignment procedures (to optimize overlap of either OEC Mn atoms or the surrounding protein ligands) are shown, illustrating the error in these alignments. A small shift of both W1 and W2 upon transition from the cryogenic S1-XFEL to the 2F structure is visible. In the 2F-NH3 model, W1 is shifted slightly further along the same direction as the dark–2F difference. In contrast, the displacement of W2 in the 2F-NH3 model is significantly larger than that between the S1-XFEL and 2F structures. e, 2mFo − DFc electron density (blue, 1.0σ) of the 2F-NH3 dataset around Mn4, and polder omit maps at 3.5σ (green). Red spheres, water refined in the current model; orange spheres, water placed in the polder maps but not included in the refined model; cyan spheres, water positions from the cryogenic XFEL structure. f, When calculating the mFo − DFc electron density using the refined positions of W1 and W2 (light orange spheres) from the 2F-NH3 data, only a negative peak is observed (orange mesh, −3σ contour) while using W1 and W2 positions (purple spheres) from the S1-XFEL structure yields clear positive and negative peaks (green and purple mesh, ± 3σ contour, colouring of other waters as in e). This indicates that the W2 position from the S1-XFEL structure does not provide a good fit to the observed electron density. However, at the present resolution the observed difference densities may be influenced by other effects, for example, Fourier series truncations and the strong density of H2OA close to W2.
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Young, I., Ibrahim, M., Chatterjee, R. et al. Structure of photosystem II and substrate binding at room temperature. Nature 540, 453–457 (2016). https://doi.org/10.1038/nature20161
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