Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy

Abstract

The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O₂ formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2-flash (2F) and 3-flash (3F) photosystem II samples, and of a transient 3F' state (250 μs after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn reduction, does not yet occur within 250 μs after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.

ARTICLE

Received 20 Feb 2014 | Accepted 10 Jun 2014 | Published 9 Jul 2014

DOI: 10.1038/ncomms5371

Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy

Jan Kern1,2, Rosalie Tran1, Roberto Alonso-Mori2, Sergey Koroidov3, Nathaniel Echols1, Johan Hattne1, Mohamed Ibrahim4,5, Sheraz Gul1, Hartawan Laksmono6, Raymond G. Sierra6, Richard J. Gildea1,w, Guangye Han1, Julia Hellmich4,5, Benedikt Lassalle-Kaiser1,w, Ruchira Chatterjee1, Aaron S. Brewster1, Claudiu A. Stan6, Carina Glckner5, Alyssa Lampe1, Drte DiFiore5, Despina Milathianaki2, Alan R. Fry2,M. Marvin Seibert2,w, Jason E. Koglin2, Erik Gallo7, Jens Uhlig7, Dimosthenis Sokaras8, Tsu-Chien Weng8, Petrus H. Zwart1, David E. Skinner9, Michael J. Bogan2,6, Marc Messerschmidt2, Pieter Glatzel7,Garth J. Williams2, Sbastien Boutet2, Paul D. Adams1, Athina Zouni4,5, Johannes Messinger3,Nicholas K. Sauter1, Uwe Bergmann2, Junko Yano1 & Vittal K. Yachandra1

The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photo-system II reaction centre is excited by three single-turnover ashes. Here we present combined X-ray emission spectra and diffraction data of 2-ash (2F) and 3-ash (3F) photosystem II samples, and of a transient 3F state (250 ms after the third ash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial OO bond formation, coupled to Mn reduction, does not yet occur within 250 ms after the third ash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no signicant structural changes at the present resolution of 4.5 . This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.

1 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. 2 LCLS, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA. 3 Institutionen fr Kemi, Kemiskt Biologiskt Centrum, Ume Universitet, 90187 Ume, Sweden. 4 Institut fr Biologie, Humboldt-Universitat zu Berlin, D-10099 Berlin, Germany. 5 Max-Volmer-Laboratorium fr Biophysikalische Chemie, Technische Universitat, D-10623 Berlin, Germany. 6 PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA. 7 European Synchrotron Radiation Facility, F-38043 Grenoble, France. 8 SSRL, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA. 9 National Energy Research Scientic Computing Center, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. w Present addresses: Diamond Light Source, Harwell Science and Innovation

Campus, Didcot, Oxfordshire OX11 0DE, UK (R.J.G.); Synchrotron SOLEIL, F-91192 Gif-Sur-Yvette, France (B.L.-K.); Department of Cell and Molecular Biology, Uppsala Universitet, 751 24 Uppsala, Sweden (M.M.S.). Correspondence and requests for materials should be addressed to U.B.(email: mailto:[email protected]

Web End [email protected] ) or to J.Y. (email: mailto:[email protected]

Web End [email protected] ) or to V.K.Y. (email: mailto:[email protected]

Web End [email protected] ).

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Aerobic life on earth is supported by the constant regeneration of dioxygen through photosynthetic water oxidation in green plants, algae and cyanobacteria. This

reaction takes place in photosystem II (PS II), a multi-subunit membrane protein complex. PS II couples the one-electron photochemistry of the primary charge separation at the reaction center with the four-electron redox chemistry of water oxidation at the Mn4O5Ca cluster of the oxygen evolving complex (OEC) at the lumenal side of the protein complex, using the spatial and temporal organization of the electron donor and acceptor cofactors in PS II1,2. This well-controlled electron and proton ow results in the high quantum efciency of PS II.

During the water oxidation reaction, the OEC functions as a redox capacitor by storing four oxidizing equivalents before the release of molecular oxygen. Starting from the dark stable S1 state, the oxidation state of the OEC is increased by one on each light excitation of PS II until the highest oxidized stable intermediate state, S3, is reached. Following the next light excitation, the OEC is oxidized one more time to form the transient S3YZox and S4 states that lead to dioxygen formation, which converts the OEC to its most reduced state, S0 (ref. 3). The fourth light excitation sets the OEC back to the S1 state, and thereby completes the cycle (Fig. 1a).

Much structural and mechanistic information about PS II, the OEC and the OO bond formation was gained through mass spectrometric4, various spectroscopic49, crystallographic1013 and theoretical1416 studies over the past decade. In particular, the most recent structure, inferred from X-ray diffraction (XRD) data, has provided detailed geometric information of the OEC, including ligands and bound water molecules13. Most of the experimental studies, however, are carried out at cryogenic temperatures and represent a static picture of the system in a frozen state. Although the stable intermediate states, S0 through

S3, can be trapped and studied at cryogenic temperatures, the critical S3-S3YZox-S4-S0 stepwhere dioxygen is formed, two protons and O2 are released, and where at least one substrate water bindsonly occurs under ambient conditions and has no intermediates that can be cryotrapped. To date, there has been only one transient X-ray spectroscopy study of the S3-S3YZox-

S4-S0 transition performed at room temperature (RT)17. More detailed investigations of the transient states by X-ray spectroscopy and by kinetic crystallography have been hampered due to the severe radiation damage, especially to the Mn4CaO5 cluster that is signicantly faster at RT as compared with cryogenic conditions. However, X-ray-induced changes, particularly at the redox-active metal site, have even been an issue for experiments carried out at cryogenic temperatures1820.

We have recently introduced a combined spectroscopy and diffraction data collection methodology at RT21 using the probe before destroy method2224 made possible by the ultra-short (fs) and bright X-ray pulses of an X-ray free electron laser (XFEL). In this approach, XRD data and Mn Kb-X-ray emission spectra (XES), sensitive to the metal charge density25,26, are measured simultaneously from micrometre-sized crystals of PS II, thereby obtaining information about the geometric and the electronic structure of the active site, under identical conditions. Owing to the ultra-short fs X-ray pulse duration, the sample is probed before the manifestation of X-ray induced changeswhich predominantly take place on the picosecond time scale (for damage to the atomic structure)even under ambient conditions. One should note that with conventional synchrotron X-ray sources the main source of radiation damage is via the generation of radicals from the solvent (water). Subsequent diffusion of these radicals leads to specic damage (for example, reduction of metal sites) and modication of amino acid side chains (for example, decarboxylations). Such events are diffusion controlled and occur on a longer time scale (4picoseconds) and seem not to be dependent on the dose rate. Earlier work21,27 showed that the approach of using ultrafast (o50 fs) and ultra-bright (1012 photons per pulse) X-ray pulses permits the collection of XES and XRD data from intact PS II, and we reported results from the dark-adapted (S1) and the one ash (S2) samples with an XRD resolution limited to 5.5 .

Here we present XES and XRD data from the last step of the Kok cycle, where O2 is evolved, with an improved resolution of4.5 . This step, triggered by the third ash given to dark-adapted PS II samples, advances the PS II complex from the S3 to the S0 state, via the transient S3YZox and S4 states (S3-S3YZox-S4-S0 transition). Furthermore, we observe an anomalous signal for the Mn atoms in the OEC from all the states, including the transient S3YZox state. This observation supports the quality of our XRD data and also the data analysis protocols, and we envision that the Mn anomalous signal could be used as a sensitive probe for monitoring changes of the atomic positions of Mn in the OEC during the catalytic cycle in future studies at higher resolution.

ResultsXES at different time points in the catalytic cycle. PS II was advanced through its reaction cycle in situ, using a ow/illumination scheme (Fig. 1b) employing an electrospun liquid jet28. The protocol consisted of visible-laser illumination using three optical bres directly attached to the sample delivery capillary, and an additional laser for illumination of the sample in the jet

a

b

1

Sample injector

2F

e

1F

S2

S1

Visible laser 2

3

0.5 s

0.5 s

e, H+

H2O

3F 4F

H+,O2

H2O

~ 1 ms

H+ Mn(III)3(IV)

e, H+

O

O

0.5 s

S3 [S3YzOX] [S4] S0

~250 s

Mn(IV)4

e

XRD

XES

LCLS X-ray pulses

t

Visible laser 4

Figure 1 | Flash-induced changes in PS II and experimental set-up used at LCLS. (a) Kok cycle describing the different stable intermediate states of the catalytic water oxidation reaction in PS II. (b) Scheme for the illumination set-up used to advance PS II in the catalytic cycle and measure simultaneously the XRD and XES signal at LCLS. Lasers 2 and 3 were used to generate 2F samples, lasers 1, 2 and 3 for 3F samples and lasers 2, 3 and 4 to generate the 3F samples.

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a

(see Methods). The temporal frequency for illumination was chosen to match the sample ow rate, so that each volume segment was illuminated by each bre once while passing through the capillary. The set-up also allows enough time (B0.5 s) for complete PS II turnover between consecutive illuminations, which takes into account the slower acceptor side reactions7,2931, while being rapid enough to avoid signicant decay of the S-states, that are stable for on the order of several tenth of seconds. The fourth laser (labelled laser 4 in Fig. 1b) illuminated the sample in the jet, to study transients during the S3 to S0 transition by changing the timing between the third visible-laser pulse and the X-ray probe pulse.

O2 detection via membrane-inlet mass spectrometry (MIMS) was used for optimizing the conditions for S-state turnover in the capillary ow sample delivery system, using a facsimile of the ow/ illumination set-up employed at Linac Coherent Light Source (LCLS) (see Methods). One of the most important factors in the illumination scheme is the required light intensity for efcient turnover through the Si state cycle. Too low intensities can lead to only partial turnover of the samples, while too high intensities increase the miss parameter via light scattering along the capillary, and may also inactivate the sample. The optimal light intensity can be found by the quality of the O2 oscillation pattern, and also by the total O2 produced per PSII complex and ash number. The former method should normally be sufcient, but a small uncertainty remains whether there can be a certain part of the sample that never sees any light, and thus does not contribute to the oscillation pattern. To address this question, the latter method needs to be employed (see Methods), which requires the absolute calibration of the MIMS signals. The amount of 0.73 O2/RC after three ashes (Fig. 2a) shows that the light conditions used for illumination are optimal for saturating all PSII reaction centres in the sample. The O2 evolution patterns obtained from PS II solutions and PS II microcrystals (Fig. 2b) show light-induced turnover of the catalytic cycle as expected. Analysis of the ash pattern indicates that the S3 state is the majority component (Z55%) in the samples given two visible-laser ashes (2F) with virtually no S0 state present. In contrast, the largest component in the 3F samples is the S0 state (Z40%). Therefore, the difference between the 3F and 2F samples is dominated by the formation of the S0 state at the expense of the S3 state.

We measured XES on PS II solutions at the Coherent X-ray Imaging (CXI) instrument32 at LCLS (see Methods). As shown in Fig. 3, a clear shift between the 2F (S3-enriched) and 3F (S0-enriched) spectra is observable. Calculation of the rst moment (see Methods) revealed that the 3F spectrum is shifted B0.1 eV to higher energies indicating a reduction of Mn26, as expected for the transition of the OEC from the highly oxidized S3 to the most reduced S0 state, in which the formal oxidation states are assigned as Mn4IV and Mn3IIIMnIV, respectively1,2,6,33,34. Comparing these data to synchrotron radiation (SR) data collected at cryogenic temperature from Thermosynechococcus elongatus PS II and previously recorded data from spinach PS II35 shows a very similar trend (Fig. 3c and Supplementary Fig. 1).

In addition to the 2F and 3F spectra, we measured the XES at a time point 250 ms after the third ash (3F0) using lasers 2, 3 and 4 (Fig. 1). The XES for this transient state is similar in position to the 2F spectrum (Fig. 3a,b), but its shape is different with broadening towards the lower energy side. Although such broadening could be caused by oxidation of a fraction of the lower S-states (S1 and S2) in our sample, it could also be due to light-induced changes in the electronic structure of the S3 fraction. Nevertheless, the result shows that there is no signicant reduction or oxidation of the Mn taking place within the 250-ms time span between the third visible-laser excitation pulse and the X-ray probe pulse.

XRD in the higher S-states. XRD data from 2F (S3-enriched), 3F (S0-enriched) and 3F0 (S3YZox-enriched; 250 ms after the third ash) PS II crystals, as well as in the dark state (S1), were collected. Microcrystals of PS II were prepared using a new seeding protocol (see Methods). Clear Bragg spots were observed to a resolution of B4.1 , with thermal diffuse scattering extending well beyond this to B3.0 , indicative of correlated atomic motion in the crystal. For the 2F data, a total of 16,973 indexed patterns were merged resulting in a data set of 4.5 resolution (see Table 1 and Supplementary Tables 1 and 2 for details). The resolution cutoff for the merged data sets was chosen based on the resolution dependence both of the multiplicity and of CC1/2,

the correlation coefcient of semi-data sets merged from oddand even-numbered images36; that is, completeness 490%, multiplicity 46 and CC1/2430%. Likewise, data sets of 3F, 3F and 0F states were obtained with resolutions of 4.6 (13,094 lattices), 5.2 (7,850 lattices) and 4.9 (6,695 lattices), respectively (Table 1 and Supplementary Tables 1 and 35). Electron density maps for all four states are shown in Fig. 4 and Supplementary Figs 2 and 3. A comparison with the SR data cut to the same resolution shows that the level of detail visible is as expected for this resolution range (Supplementary Fig. 4). The occupancy for selected non-protein molecules was set to zero and the simulated annealing omit maps were computed for all data sets, to remove potential model bias arising from phasing with a complete, high-resolution starting model (pdb: 3bz1)12. The

1.0

O 2yield per PSII

0.5

0.0

1 2 3 4 Flash number

b

100

PSII solution PSII crystals

80

Relative oxygen yield (%)

60

40

20

0

1 2 3 4

Flash number

Figure 2 | Oxygen production by PSII. (a) O2 yield per PSII reaction centre as detected by MIMS as a function of ash number (measurement shown is for PS II solutions, ow rate 0.5 ml min 1, frequency 4 Hz, light intensity was 7 mJ for each bre). (b) O2 yield measured by MIMS as a function of ash number from PS II solutions (black) and PS II microcrystals (red).

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2F (S3 enriched)

3F (S0 enriched)

3F (S3YZox enriched)

Intensity (a.u.)Intensity (a.u.)

3F - 2F

3F - 2F

Intensity (a.u.)

3F - 2F (SR)

6,485 6,489 6,493 6,497

Energy (eV)

Figure 3 | Mn Kb XES of PS II. (a) XES recorded with o50 fs X-ray pulses at LCLS. Spectra were measured 0.5 s after two laser ashes (2F, black;

lasers 2 and 3 on), or 0.5 s after three laser ashes (3F, blue; lasers 1, 2 and 3 on), and B250 ms after three laser ashes (3F, red; lasers 2, 3 and 4 on), respectively. (b) Difference between the Mn Kb XES of PS II, blue: 3F2F;

red: 3F2F. Before calculating the difference curves, spectra were smoothed by moving average (dotted line) or cubic polynomial tting (solid line, similar to the procedure used for analysing the synchrotron data). (c) The 3F2F difference spectrum (green) from SR data collected at 15 K.

result clearly shows the electron density of the Mn4O5Ca cluster, the non-haem Fe, the chlorophyll and even partially for the quinone cofactors (Supplementary Fig. 3) in the mFo DFc

difference maps. The regions around the OEC, the acceptor-side quinones and non-haem iron, where the largest changes are expected, were inspected for changes between the different states. No statistically signicant changes were observed in the 2mFo DFc maps of the individual data sets (Fig. 4a,b and

Supplementary Figs 2 and 3) and in the isomorphous difference maps (mFo mFo) between the different data sets (Fig. 4c,d and

Supplementary Fig. 5). This shows that any structural changes related to the S-state transitions are smaller than what we can detect at the current resolution. However, it should be noted that the mFo DFc Fourier maps contain several features that are

observed consistently in both monomers and all ash states; namely, an electron density peak at the position of the OEC when viewed at a contour level of 3s, a small peak 10 distant that

appears to be coordinated by residues Glu 333 and Asp 61 of the

D1 polypeptide, and other nearby peaks. Smaller negative peaks are seen at the 3s contour, for example, close to Val 185 and

Phe 182 of the D1 protein (Supplementary Fig. 6). In general, we observe these low-intensity peaks at the same positions in both monomers and across all four illuminated states. This suggests that they are not artefacts of the Fourier transform, and are rather due to structural differences between SR data collected at cryogenic temperature and the RT data presented here. However, the current resolution does not allow them to be fully modelled in our nal atomic coordinate sets.

Measurement of anomalous XRD signal from Mn in PS II. Accurate determination of the Bragg spot intensities and the derived structure factors is challenging for single-shot crystal-lography at XFELs21,23,24. As a control to validate the data quality and our analysis protocol, we investigated whether small anomalous differences could be detected in the recorded Bragg spot intensities. Such differences between inversion-related Bragg spots (Bijvoet pairs) arise from the collection of diffraction data at energies above an absorption edge and are often only in the order of B1% of the total signal intensity. We used an incident energy of 7.1 keV in our current XES/XRD data collection, which is close to the Mn edge (6.54 keV), and favours observing the anomalous signal from Mn in the OEC.

As a positive control of the methodology, we rst analysed microcrystal diffraction data from a model system, thermolysin, which natively binds one Zn and several Ca ions37. Data from thermolysin microcrystals were collected at 1.27 (9.76 keV), B100 eV above the Zn edge (9.66 keV). Diffraction was observed out to the corners of the detector (1.50 ) and the integrated intensities were merged to obtain a data set to 1.80 resolution (Table 1 and Supplementary Table 6). Analysis of the Bijvoet pairs in the merged data showed a clear anomalous signal contribution, and anomalous difference maps showed a clear maximum, 18s above the mean, located at the position of the Zn ion as well as lower maxima for three of the four Ca ions and for the sulphur of one of the methionine residues (Fig. 5a,b and Supplementary Fig. 7).

In PS II, a clear anomalous signal (Fig. 5c,d, Fig. 6 and Supplementary Figs 8 and 9) from Mn in the OEC is also detected in all four data sets (0F, 2F, 3F and 3F0) (Supplementary

Table S1). Figure 5c,d shows the anomalous difference map from the 3F data after omitting the OEC and performing simulated annealing renement. It is evident from the overview shown in Fig. 5c that the largest peak (s46) in the anomalous density is located at the position of the OEC. The density covers the Mn ions in the cluster and does not include the Ca (Fig. 5d) as expected from the weaker anomalous contribution of Ca at7.1 keV (f 00 of 1.6 for Ca compared with 3.4 for Mn at 7.1 keV). Similar results were obtained for the other PS II data sets for both monomers in the PS II dimer (Fig. 6 and Supplementary Figs 8 and 9). It should be noted however that the anomalous difference Patterson maps did not reveal peaks above the noise level attributable to Mn. This result is expected as also the anomalous data measured at SR sources at 3.5 resolution10 did not yield any peaks in the Patterson map above the noise level, due to the large protein mass and the low number of anomalous scatterers per unit cell volume.

DiscussionThe quality of the PS II XRD data reported here for the S1 state is improved compared with the previously obtained XFEL data:4.5 versus 5.7 21. Owing to the inherent uctuations in pulse intensity, crystal size and crystal quality in single-shot microcrystal experiments at an XFEL, the signal strength varies

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Table 1 | Statistics for processed data and rened structures.

Dark (S1) 2-ash (2F) 3-ash 250 ms (3F) 3-ash 500 ms (3F) Thermolysin

Wavelength () 1.77 1.27 Resolution range () 72.934.9 (5.084.9) 72.974.5 (4.664.5) 68.415.2 (5.395.2) 72.964.6 (4.764.6) 34.271.80 (1.861.80) Space group P 212121 P 212121 P 212121 P 212121 P 6122

Unit cell dimensions () 132.9 229.0

307.7

132.3

228.7

308.0

132.6

229.3

306.8

132.4

228.8

307.9

93.093.0 130.4

Unique reections 41,292 (4,013) 52,965 (5,008) 34,679 (3,378) 49,771 (4,812) 31,458 (3,075) Completeness (%) 99.7 (98.6) 99.5 (95.8) 99.7 (98.1) 99.7 (98.2) 100.0 (100.0) Wilson B-factor 172 153 176 159 16.4 R-work 0.281 (0.363) 0.276 (0.367) 0.271 (0.347) 0.278 (0.371) 0.208 (0.349) R-free 0.292 (0.337) 0.284 (0.393) 0.289 (0.378) 0.284 (0.346) 0.232 (0.368) Number of non-hydrogen atoms 50,244 2,740 Macromolecules 41,052 2,415 Ligands 9,192 5 Waters 0 324 Protein residues 5,214 315RMS (bonds) () 0.005 0.005 0.005 0.005 0.005 RMS (angles) () 0.75 0.75 0.77 0.75 0.92

Ramachandran favoured (%) 91 91 91 91 95 Ramachandran outliers (%) 1.2 1.2 1.1 1.2 0 Clashscore 9.43 9.45 9.50 9.34 1.72 Average B-factor (2) 207 174 208 180 19.6

Statistics for the highest-resolution shell are shown in parentheses. All unit cell angles are 90 for photosystem II structures, and a b 90, g 120 for thermolysin.

Tyr161

Asp170

Tyr161

Asp170

Asp61

Asp61

His332

His332

Glu189

Glu189

Asp170

Asp170

Asp61

Glu333

Asp61

Glu333

His332

His332

Figure 4 | Electron density maps obtained for PS II. (a) 2mFo DFc maps for the dark and (b) the 2F data of PS II are shown in grey contoured at

1.0s, mFo DFc maps after omitting the OEC are shown in green and red, contoured at 5.0s. (c) mFo mFo isomorphous difference maps for the 2Fdark

data and (d) the 3F2F data are shown for both monomers and are contoured at 3s (bright green, monomer I; pale green, monomer II) and 3s

(red, monomer I; salmon, monomer II) together with the model for the 2F data (Mn shown as magenta spheres, Ca as white sphere).

from shot to shot. Therefore, we expect a distribution of diffraction images with different maximum resolution. To avoid adding noise into the diffraction data, an individual resolution cutoff was computed for each diffraction image based on the signal strength (see Methods). The observed distribution of the resolution for PS II as well as for thermolysin explains why

the multiplicity in both cases (Supplementary Tables 26) decreases steadily in the higher resolution shells.

Recently, the rst observation of an anomalous signal from femtosecond diffraction experiments with microcrystals at an XFEL38 and the rst successful de novo phasing of lysozyme at2.1 resolution using the anomalous signal of gadolinium

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Figure 5 | Anomalous signal in the XFEL data sets. (a) Anomalous difference map of the thermolysin data after simulated annealing with the occupancy for Zn and Ca set to zero to minimize model bias. The map is contoured at 4.0s, extending over the entire thermolysin molecule. The position of the highest peak in the map (Zn atom) is highlighted. (b) The same anomalous difference map of thermolysin shown in the region of the natively bound Zn ion (magenta sphere), contour level at 3.0s. (c) Anomalous difference map obtained from the 3F data of PS II, shown for one monomer, location of the strongest peak is highlighted, contour level at 4.0s. (d) Enlarged view of the 3F anomalous density for the region of the OEC (contoured at 4.0s;

Mn shown as magenta spheres, Ca as white sphere). All maps shown are anomalous difference simulated annealing omit maps.

Tyr161

His 332 His 332

His 332

Asp170 Asp170

Glu189

Figure 6 | Anomalous signal from Mn for different illumination states of PS II. (a) Anomalous map of the OEC in PS II is shown for the 2F data (magenta) in monomer I. (b) Anomalous map of the 3F data in monomer I. (c) Anomalous map of the 2F (cyan) and 3F (magenta) data in monomer I, orientation is rotated by 90 around horizontal and vertical axis compared with the view in a. (d) Anomalous map for monomer II, 2F (cyan) and 3F (magenta) data are shown, view direction is similar to c. All maps shown are anomalous difference simulated annealing omit maps contoured at 3s.

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Tyr161

Asp170 Asp170

Glu189

Glu189

His 332

Glu189

Ala344

Ala344

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obtained in an XFEL experiment were reported39. In the gadolinium phasing experiment of lysozyme, the anomalous signal strength was around 515%39. In comparison, we expect an anomalous signal of o1% for Mn in PS II and of B1.5% for Zn in thermolysin. The observation of the very strong anomalous peak for Zn in the thermolysin data indicates that the Bragg peak intensities were determined with sufcient accuracy to extract the weak anomalous difference (see ref. 40 for a report on determining the anomalous Zn signal of thermolysin from SR measurements). Furthermore, the presence of the anomalous density for Mn in all of the PS II data sets, despite the expected low signal strength, conrms the quality of the data and implies that structure factors can be extracted reliably from the current PS II data sets. In this regard, it should be noted that even the high-resolution shell of the data still contains a considerable amount of anomalous signal as can be seen in Supplementary Fig. 9B.

As described above, the electron density of PS II shows the level of detail expected at the specied resolutions (4.55.2 , depending on number of collected images per S-state; Supplementary Fig. 4). The quinone co-factors (QA in the dark, 2F and 3F data; QB in the 3F data), that were not visible in the previous XFEL data due to limited resolution, are now partially visible in the mFo DFc difference maps (Supplementary Fig. 3).

In the earlier SR XRD structures of PS II10,4144 with a resolution lower than 3.0 , it was difcult to locate them with condence (especially the mobile QB) due to partial occupancy and quinone mobility.

The native XRD data indicate that there are no large-scale rearrangements of the Mn4O5Ca cluster and its protein environment between the different states (dark, 2F, 3F and 3F0) in PS II. This is in line with Mn EXAFS data, which suggests that the largest possible changes in MnMn distances on S-state transitions are o0.5 (this estimate is based on the changes proposed in the S2 to S3 transition, if di-m-oxo bridged Mn becomes mono-m-oxo bridged)20, which are well below the sensitivity of our current XRD measurements. To test the level of change detectable using our data, we simulated a shift of the Mn4CaO5 cluster by 1 and 0.4 compared with the starting model based on SR data. In both cases, a strong positive and negative peak corresponding to the shift was visible in the isomorphous difference density (Supplementary Fig. 10A,B), indicating that shifts of that magnitude should be detectable within the signal to noise level of the current data. In contrast, when only perturbing the position of a single Mn within the Mn4CaO5 cluster by 0.5 , no clear peaks were observed in the difference density (Supplementary Fig. 10C), indicating that a structural change of such order can not be resolved at the present resolution. We furthermore evaluated the noise level in the current electron densities by computing the difference in position of the Mn4CaO5 cluster between the two monomers in each data set. For this approach, we superimposed the mFo DFc Fourier

omit maps after simulated annealing (see Methods) for the two monomers and evaluated the differences between the OEC peak in these maps. The values are in the range of 0.30.6 , indicating that changes in Mn positions larger than B0.5 should be visible in our data.

The height and volume of the anomalous XRD difference map peak of the OEC reects the amount of data available for each S-state data set. Supplementary Fig. 9A shows that after scaling the anomalous difference densities of the 2F, 3F and 3F0 data sets individually at a s-value of 80% of the peak maxima, the extent of the anomalous peak at the OEC is roughly similar for all data sets. It is also evident from this plot that the peak position of the anomalous signal for all data sets is in the vicinity of the centre of mass of the four manganese, as expected for a signal originating from Mn. Before attempting to interpret the visible differences in

the anomalous signal for the different S-states in terms of structural differences of the Mn4CaO5 cluster, it should be noted that at the present resolution the difference of the anomalous signal between two monomers in one PS II dimer is larger than the differences for the Mn4CaO5 cluster in the same monomer in different S-states. We quantied this by evaluating the difference in the peak position for the OEC between the two monomers for each of the data sets and found differences in the order of above 1 . This indicates that the noise level in our present anomalous data precludes the observation of the small changes expected between the different illumination states. Therefore, analysis of detailed structural changes based on the anomalous signal of the OEC has to await higher data quality, and in the light of the present data interpretations of structural changes can only be made using the isomorphous omit maps, as these are derived from the full data for each ash state instead of only the anomalous data and consequently show lower noise levels.

We have used fs XES to follow the changes in the oxidation state of the Mn4CaO5 cluster on advancing from the S3 to the S0 state. The peak shift that was observed between the 2F and the 3F sample reproduced the shift in oxidation state between the highest oxidized (S3 state) and most reduced state (S0 state) found earlier35. Interestingly, the peak position of the spectrum observed at the transient time point, 250 ms after the third ash, is very similar to that of the 2F spectrum. The observation that there is no signicant change in the oxidation state of Mn within this time span is consistent with the kinetics of Mn oxidation/ reduction in the S3-S3YZox-S4-S0 transition based on earlier time-resolved ultravioletvisible45,46, electron paramagnetic resonance47, infrared spectroscopy7 and Mn K-edge XAS17 studies. It was inferred from these studies that a de-protonation step, forming a transient state S3YZOx,(also refered to as S30)7,46 occurs before Mn redox chemistry7,46. This lag phase before the onset of Mn redox chemistry was reported to be in the range of 100250 ms7,17. Our results provide direct evidence that Mn redox chemistry does not occur within the rst 250 ms after illumination of the S3 state. This implies that the formation of a MnV species that has been invoked for a nucleophilic attack mechanism2, or the formation of a peroxide intermediate that will result in Mn reduction1, does not occur within the rst 250 ms after the third ash. The formation of an oxygen radical species4,6 within this time period cannot be excluded by our data (as no Mn oxidation would be involved), but is unlikely on the basis of the earlier ultravioletvisible data45. The long delay before the onset of Mn redox chemistry suggests that the formation of the S3YZox state is not a simple deprotonation step. It is rather likely to be accompanied by slower structural changes of the Mn4CaO5 cluster and/or the protein framework7 that are required to stabilize the deprotonated S3YZox state of the OEC in the conformation necessary for subsequent OO bond formation. In line with this conclusion, a recent report46 underlined the importance of the exact structure of the H-bonding network for efcient turn over in the S3-S3YZox-S4-S0 transition by demonstrating that Ca/Sr exchange in the OEC perturbs the H-bonding network and results in a signicant slowing of the S3-S3YZox transition4,46.

In summary, we have investigated the S-state intermediates and a transient state with fs XES/XRD by following the S-state transitions under ambient conditions. Advancement of S-states by in situ photoexcitation was conrmed by the O2 evolution pattern and the XES spectral shifts. The XES data indicate that the Mn oxidation state does not change within 250 ms after the illumination of the S3 state. The most probable explanation for this observation is that the deprotonation process of the OEC proceeds before the electron transfer, and the OO bond formation occurs 4250 ms after the third photo-excitation, in

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agreement with previous studies7,17,46,47. Structural changes that are large enough to access with the current XRD resolution of4.55.2 were not observed in the OEC, surrounding amino acid residues, or the quinone sides on the S3, S3YZox and S0 state

formation, which implies that the structural changes in the OEC are within the order of r0.5 . Interestingly, our RT structural data clearly show the presence of several featuresalthough not interpretable at the present resolutionin the mFoDFc difference electron density maps, indicating structural differences in the RT XFEL data compared with the previous SR cryogenic structural models. Future improvements in the crystal quality and data, especially the anomalous signal, will also allow us to use the XFEL approach to resolve the sequence of the important structural and electronic changes during the S3-S3YZox-S4-S0 transition, providing unprecedented experimental insights into the mechanism of photosynthetic water oxidation.

Methods

Sample preparation. PS II was puried from T. elongatus as described elsewhere48. Crystals that were obtained as described in ref. 48 and a seed kit (Hampton Research, CA, USA) were used to produce a PS II seed stock solution in buffer A (100 mM PIPES, pH 7, 5 mM CaCl2, 6% (w/w) PEG 2000, 0.03% bdodecyl maltoside) for microcrystallization of PS II. Microcrystals of PS II were obtained by mixing aliquots of the PS II seed stock solution with PS II solution (chlorophyll (Chl) concentration 4 mM, corresponding to a protein concentration ofB40 mg ml 1) in a 1:4 ratio. Box-shaped crystals (510 mm in the longest dimension, 5 mm in the shorter dimension) were suspended in buffer C (100 mM MES, pH 6.5, 5 mM CaCl2, 10% (w/v) PEG 2000, 30% (w/v) glycerol). The nal concentration of the crystal suspension was determined by measuring Chl concentration of small aliquots of the suspension, dissolved in 80% acetone49. The Chl concentration was adjusted between 0.3 and 0.5 mM, corresponding to a protein concentration of 8.514 mM (35 mg ml 1). For solution samples, the puried PS II was resuspended in buffer D (100 mM MES, pH 6.5, 5 mM CaCl2,

0.015% b-dodecyl maltoside, 1.3 M sucrose) to a nal protein concentration of 8090 mg ml 1.

Thermolysin was obtained from Hampton Research (CA, USA). Microcrystals of thermolysin were obtained as described previously28 using PEG2000 as a precipitant.

MIMS measurements. Sample suspensions of PS II from T. elongatus of 8 mg ml 1 Chl were diluted to 7 mg ml 1 Chl with H218O (98%) to give a nal enrichment of H218O of B12% and nal salt concentrations of 4.4 mM CaCl2, 85 mM MES and 1.1 M sucrose. No electron acceptors were added. The 18O-enriched samples were loaded into a gas-tight Hamilton syringe and pumped through a silica capillary (inner diameter (ID) 50 mm, outside diameter (OD)

160 mm) into another gas tight Hamilton syringe that collected the sample. Both syringes were placed on separate syringe pumps. Samples were kept in darkness or very dim green light during all steps, except when illuminated inside the capillary with laser light travelling through one to four optical bres (400 mm core diameter)

directly attached to a region of the capillary with the polyimide coating removed. This set-up directly mirrors the in-capillary illumination set up for the CXI experiment (see Fig. 1a and below).

The oscillation pattern of PS II crystals was obtained in the same way, but the experimental details were as follows: the PS II crystal suspension was concentrated to 3.2 mM Chl and was then diluted with H218O (98%) to 2.5 mM Chl to give a nal enrichment of 21.5%. The nal concentrations of other additions were 5 mM CaCl2, 80 mM MES, 1.2 M sucrose and 11% PEG 2000. The capillary that was used to conduct these experiments had an ID of 100 mm and an OD of 360 mm.

A Nd:YAG laser (Continuum Inlite II-20, 532 nm, 7 ns pulse width) was used for sample illumination. To obtain a stable output intensity of 7 mJ per bre (intensities of individual ashes may vary by 5%), the laser was operated continuously at 20 Hz. The illumination periods were set with the help of a fast shutter (SH05 operated with SC10 Controller; both from Thorlabs), while the ash frequency was controlled via the Q-switch divider (20 or 10 Hz).

The oxygen produced was quantied by injecting the illuminated sample into a membrane-inlet cell containing 600 ml water, connected via a Si membrane (Mem 213) and a cooling trap (dry ice/ethanol) to an isotope ratio mass spectrometer (DELTA V, ThermoFinnigan)50. The O2 formed during illumination was detected with excellent S/N ratio as the non-labelled 16O16O, the mixed labelled 16O18O and double-labelled 18O18O species. To obtain a ash pattern, the light-induced yields for O2 production (detected at m/z 34) obtained with (x 1) illuminations were

subtracted from that with x illuminations (Fig. 2b). For the rst ash, the background 34O2 signal of a non-illuminated sample was subtracted. Each measurement was repeated twice (deviation of the points was within 10%).

Determination of the total O2 produced per PS II complex. Determination of the total O2 produced per PS II complex and ash number requires the absolute calibration of the MIMS signals. This calibration was achieved by the injection of known volumes of air-saturated water into the MIMS cell. This value was used to determine the micromoles of O2 produced by PS II by the illumination with 3 ashes using 7 mJ per bre measured in a silica capillary (ID 75 mm, OD 160 mm),

with a ow rate of 0.5 ml min 1 and a ash frequency of 4 Hz. To account for diffusion losses of 34O2 out of the capillary during the ow of the sample, a loss factor was determined separately by measuring the O2 content of a PS II sample that was illuminated inside a gas tight syringe by 50 consecutive Xenon lamp ashes (2 Hz frequency, in the presence of acceptors) and either directly injected into the MIMS cell or rst owed through the capillary set-up used for the ash measurements into the collection syringe and then injected into the MIMS cell.

After correcting the O2 amount obtained in the 3F experiment by the loss factor, this number was then divided by the mmole of PS II reaction centre, which resulted in B0.73 O2/RC (Fig. 2a). As three consecutive ashes are required to produce one molecule of oxygen in a dark-adapted PS II reaction centre, a maximum of one O2/reaction centre can be expected under these conditions. The above number of 73% oxygen yield directly translates into light saturation. For 100% light saturation, we would expect a value of 73% (0.93), as even under stationary conditions an average miss of 10% occurs due to charge equillibria within PS II.

Sample injection and illumination at CXI. Samples were injected into the CXI instrument chamber32 using an electrospun liquid microjet28. Aliquots of 50 150 ml of sample were placed in a microcentrifuge tube placed inside the pressurized cell with a Pt-electrode and the end of the injector capillary submerged in the sample. Pressures of 1720 psi against the CXI chamber pressure(10 4 Torr) and voltages of around 3,000 V were applied depending on the buffer composition and crystal concentration. The injector capillary was a clear silica capillary with an ID of 75 or 100 mm and an OD of 150 or 360 mm, respectively.

The ow rate was in the range of 0.251.0 ml min 1 (for the 75 mm ID capillary) and 1.23.5 ml min 1 (for the 100 mm ID capillary) depending on the sample viscosity and the backing pressure. We measured the ow rate from the mass of sample consumed divided by the run time (data quoted here was obtained this way), and in several cases we also estimated the ow rate from the velocity of crystals owing in the capillary by using in-capillary visualization of the ow via a microscope-mounted camera. To ensure that the samples were in the dark stable S1 state before injection, all sample handling and storage was performed in darkness or under dim green light. For visualization of the jet, an infrared laser diode (Coherent Lasiris, 785 nm, 15 mW) was used (see ref. 28 for details). The wavelength was chosen to be outside the absorption spectrum of PS II.

Sample illumination for advancement into higher S-states was conducted using the output of a frequency-doubled Nd:YLF laser at 527 nm (Coherent Evolution) with 150 ns pulse duration. The light from the laser was coupled into multi-mode bre light guides with a core diameter of 400 mm. Three of these light guides (laser 1, 2 and 3 in Fig. 1a) were directly coupled onto the silica capillary of the sample injector and an additional laser (laser 4 in Fig. 1a) illuminated the sample in the jet. The output of all three bres was equalized to 10 mJ per pulse (1.4 times the power necessary for saturation of the oxygen production in our ofine O2 f1ash yield experiments, see above) using wave plates, and the transmission prole and output power of all bres was measured before the experiment and after each change to the experimental set-up. The centrecentre distance between the bres was1.98 mm and typical illumination parameters for a 75-mm ID capillary (1 ml min 1 ow rate, 8.9 Hz illumination frequency) result in a delay between the pump laser ashes on the same volume segment of 0.52 s. To generate 2F samples (enriched in the S3 state), lasers 2 and 3 were used, and the sample reached the X-ray interaction region B0.5 s after the second pump laser ash. To generate 3F samples (enriched in the S0 state), lasers 1, 2 and 3 were used and the sample reached the X-ray interaction region B0.5 s after the third pump laser ash. Finally, to study transients during the S3 to S0 transition, lasers 2 and 3 (for advancing the sample from the S1 to the S3 state) in combination with laser 4 (to start the transition from the S3 to the S0 state) were used. In this experiment, the time delay of the X-ray probe with respect to the laser 4 was set to 250 ms, giving rise to the 3F0 data (enriched in the S3YZox state).

CXI instrument and X-ray parameters. The CXI instrument of LCLS32 was used in the 1-mm focus setting with the beam being focused to 1.5 1.5 mm2 full-width

half maximum using KirkpatrickBaez mirrors51. The pulse length used was B45 fs and the repetition rate was 120 Hz. The energy was varied between 7.1 and9.7 keV with 36 1011 photons per pulse. The dose therefore varied between

50 and 300 MGy. XES was measured using 7.1 keV excitation, due to the higher cross section for the Mn transition at this energy. XRD of PS II was measured at both energies but no difference in diffraction quality between the two energies was observed. The majority of the PS II XRD data was obtained with incident energy of7.1 keV. XRD of thermolysin was collected at 9.7 keV (above the Zn edge).

X-ray emission spectroscopy setup. X-ray emission spectra were recorded in a shot-by-shot mode using a custom-built spectrometer in the von Hamos

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geometry25,52. The crystal analyser array was located 500 mm from the interaction point and with an angle of 81 between the centre of the array and the X-ray beam. A set of 16 Si(440) crystal analysers was used and the Bragg angle range was 85.9 to 83.4, equivalent to an energy range from 6,473.8 to 6,500 eV (limited by the detector size). The signal was recorded on a 140 k CSPAD53,54, located below the interaction region. The set-up was calibrated by recording spectra from solutions of MnIICl2 as previously described25. The rst moment values of the emission spectra were calculated between 6,486 and 6,494 eV as described35. Before computing difference spectra, the ash state spectra were smoothed by using either a moving average over a 0.5-eV window or by tting a cubic polynomial of 3 eV width to each data point.

SR XES spectra were recorded at European Synchrotron Radiation Facility (ESRF) beamline ID 26 using the 440 reection of ve spherically bent(R 1,000 mm) Si crystal analysers in combination with a silicon drift detector

aligned in a Rowland geometry. The overall energy bandwidth of the X-ray emission spectrometer was 0.8 eV. The sample was kept at 15 K in a liquid He cryostat surrounded by He as a heat-exchange gas. The ESRF storage ring was run in 16 bunch mode with ring currents between 60 and 90 mA. The incident beam was monochromatized and tuned to 6.75 keV using the 111 reection of a pair of cryogenically cooled Si crystals. The beam size was 1.0 (h) 0.2 (v) mm2 and the

beam position on the sample was changed after 1 s of X-ray illumination.

Computational facilities. Over a 5-day period, 114-TB data were collected at LCLS, grouped into ve 12-h shifts. Data were processed immediately to assess their completeness and quality. However, as the data size exceeded the processing capacity of the 480-core Linux cluster available at LCLS, arrangements were made to access an additional 1,000 Linux cores at the National Energy Research Scientic Computing Center (NERSC). Transfer of the data from SLAC to NERSC was made over the Energy Sciences Network at a maximum sustained rate of 7.5 Gb s 1.

XRD data processing. XRD data were recorded using the large CSPAD at LCLSs CXI instrument32, and processed using cctbx.xfel55,56. A dark-current image (pedestal) was subtracted from each image before data reduction. An initial triage step was evaluated, retaining only those images containing 16 or more strong, low-resolution Bragg spots as determined by the Spotnder procedure56,57. However, it was found that this step rejected some useful data; thus, it was ultimately omitted from the data processing protocol. Indexing (determination of the unit cell and crystal orientation) was performed with the LABELIT implementation58 of the Rossmann DPS algorithm59,60, and was guided by supplying the known unit cell61,62. Where more than one crystal was exposed in the same shot, indexing was attempted on the two most dominant lattices56. The number of images or lattices retained after each processing step is detailed in Supplementary Table 1.

Crystal orientations determined by LABELIT were optimized by minimizing the positional difference between the observed Bragg spots and those predicted by the model. Orientational models were further rened so that minimal perturbations were needed to exactly t the observed Bragg spots to Braggs law, under the simplifying assumptions of a perfect crystal lattice and a monochromatic beam. Differences between this idealized model and the actual set of observations then allowed us to estimate crystal properties such as mosaicity and the average size of coherently scattering mosaic blocks, leading to a realistic model of Bragg spot positions suitable for signal integration.

Intensities were integrated by summation within a spot mask derived from nearby strong spots atop a planar background63,64 and corrected for polarization65. Intensity variances, s2(I), were derived by counting statistics66 and a coarse estimate of the detector gain. Error estimates from each diffraction pattern were then inated by assuming that negative values of I/s(I) are actually decoy measurements (noise only) with a Gaussian distribution centred at zero and with an s.d. of 1, thus providing a lower bound on modelling errors. A separate resolution cutoff was determined for each image based on a Wilson plot (average intensity versus binned resolution).

Integrated, non-negative intensities from separate images were then scaled to intensities derived from isomorphous reference structures (PDB codes 3bz1 and 3bz2 for PS II12 and PDB code 2tli for thermolysin37), without separately accounting for the partiality fraction of each observation. Images whose intensities correlated poorly (r10%) with those of the reference model were rejected, as were images that deviated from the reference unit cell lengths (10%) or angles (2), or that did not obey the expected symmetry. Multiple measurements with the same Miller index were merged by averaging and the error was modelled by propagating the s(I) values in quadrature. The resolution cutoff for the merged data sets was determined from the resolution dependence both of the multiplicity and of CC1/2,

the correlation coefcient of semi-data sets merged from odd- and even-numbered images36. The expected contribution of the anomalous signal to |F| was estimated using the Web server of the Biomolecular Structure Center at University of Washington (http://skuld.bmsc.washington.edu/scatter/AS_index.html

Web End =http://skuld.bmsc.washington.edu/scatter/AS_index.html).

Phasing and renement. As a starting PSII model, we used PDB IDs 3bz1 and 3bz2 (ref. 12, modied to include all atoms in the OEC based on the high-resolution structure 3arc13, and re-rened against the 3bz1/3bz2 deposited amplitudes in phenix.rene67. The structure was then reduced to a single copy of

the PS II complex and the processed data sets were phased by molecular replacement in Phaser68. Renement of coordinates and B-factors was performed in phenix.rene using tight restraints, including two-fold non-crystallographic symmetry, with the distances between heavy atoms in the OEC restrained to the values determined by EXAFS20. Simulated annealing omit maps were generated with the OEC atoms set to zero occupancy, with harmonic restraints69 applied to the OEC and surrounding atoms; the default parameters of a starting temperature of 5,000 K and 100 K steps were used. Isomorphous difference maps were generated using phenix.fobs_minus_fobs_map. Structures and maps were aligned usingthe PHENIX structure comparison tool. All structure gures were created in PyMOL 1.2.

The thermolysin structure was solved by molecular replacement using PDB ID 2tli37 with metals and waters removed, rebuilt using the PHENIX AutoBuild wizard70 and rened in phenix.rene. Simulated annealing omit maps were generated as for PS II, with Zn and Ca occupancies set to zero.

References

1. Renger, G. Light induced oxidative water splitting in photosynthesis: energetics, kinetics and mechanism. J. Photochem. Photobiol. B 104, 3543 (2011).

2. Brudvig, G. W. Water oxidation chemistry of photosystem II. Phil. Trans. R. Soc. B 363, 12111218 (2008).

3. Kok, B., Forbush, B. & McGloin, M. Cooperation of charges in photosynthetic oxygen evolution. I. A linear four step mechanism. Photochem. Photobiol. 11, 457475 (1970).

4. Cox, N. & Messinger, J. Reections on substrate water and dioxygen formation. Biochim. Biophys. Acta 1827, 10201030 (2013).

5. Yano, J. et al. Where water is oxidized to dioxygen: structure of the photosynthetic Mn4Ca cluster. Science 314, 821825 (2006).

6. Yano, J. & Yachandra, V. K. Where water is oxidized to dioxygen: structure of the photosynthetic Mn4Ca cluster from X-ray spectroscopy. Inorg. Chem. 47, 17111726 (2008).

7. Noguchi, T., Suzuki, H., Tsuno, M., Sugiura, M. & Kato, C. Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution. Biochemistry 51, 32053214 (2012).

8. Haddy, A. EPR spectroscopy of the manganese cluster of photosystem II. Photosynth. Res. 92, 357368 (2007).

9. Cox, N., Pantazis, D. A., Neese, F. & Lubitz, W. Biological water oxidation. Acc. Chem. Res. 46, 15881596 (2013).

10. Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J. & Iwata, S. Architecture of the photosynthetic oxygen-evolving center. Science 303, 18311838 (2004).

11. Loll, B., Kern, J., Saenger, W., Zouni, A. & Biesiadka, J. Towards complete cofactor arrangement in the 3.0 resolution structure of photosystem II. Nature 438, 10401044 (2005).

12. Guskov, A. et al. Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride. Nat. Struct. Mol. Biol. 16, 334342 (2009).

13. Umena, Y., Kawakami, K., Shen, J. R. & Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 angstrom. Nature 473, 5560 (2011).

14. Siegbahn, P. E. M. Structures and energetics for O2 formation in photosystemII. Acc. Chem. Res. 42, 18711880 (2009).15. Sproviero, E. M., McEvoy, J. P., Gascon, J. A., Brudvig, G. W. & Batista, V. S. Computational insights into the O2-evolving complex of photosystem II.

Photosynth. Res. 97, 91114 (2008).16. Yamaguchi, K. et al. Full geometry optimizations of the mixed-valence CaMn4O4X(H2O)4 (X OH or O) cluster in OEC of PS II: Degree of symmetry

breaking of the labile Mn-X-Mn bond revealed by several hybrid DFT

calculations. Int. J. Quantum Chem. 113, 525541 (2013).17. Haumann, M. et al. Photosynthetic O2 formation tracked by time-resolved X-ray experiments. Science 310, 10191021 (2005).

18. 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, 1204712052 (2005).

19. Grabolle, M., Haumann, M., Mller, C., Liebisch, P. & Dau, H. Rapid loss of structural motifs in the manganese complex of oxygenic photosynthesis by x-ray irradiation at 10-300 K. J. Biol. Chem. 281, 45804588 (2006).20. Glckner, C. et al. Structural changes of the oxygen evolving complex in photosystem II during the catalytic cycle. J. Biol. Chem. 288, 2260722620 (2013).

21. Kern, J. et al. Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature. Science 340, 491495 (2013).

22. Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752757 (2000).

23. Chapman, H. N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 7377 (2011).

NATURE COMMUNICATIONS | 5:4371 | DOI: 10.1038/ncomms5371 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 9

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5371

24. Boutet, S. et al. High-resolution protein structure determination by serial femtosecond crystallography. Science 337, 362364 (2012).

25. Alonso-Mori, R. et al. Shot-by-shot energy-dispersive X-ray emission spectroscopy using an X-ray free electron laser. Proc. Natl Acad. Sci. USA 109, 1910319107 (2012).

26. Glatzel, P. & Bergmann, U. High resolution 1s core hole X-ray spectroscopy in 3d transition metal complexes - electronic and structural information. Coord. Chem. Rev. 249, 6595 (2005).

27. Kern, J. et al. Room temperature femtosecond X-ray diffraction of photosystem II microcrystals. Proc. Natl Acad. Sci. USA 109, 97219726 (2012).

28. Sierra, R. G. et al. Nanoow electrospinning serial femtosecond crystallography. Acta Cryst. D 68, 15841587 (2012).

29. de Wijn, R. & van Gorkom, H. J. Kinetics of electron transfer from Q(a) to Q(b) in photosystem II. Biochemistry 40, 1191211922 (2001).

30. Krivanek, R., Kern, J., Zouni, A., Dau, H. & Haumann, M. Spare quinones in the Q(B) cavity of crystallized photosystem II of Thermosynechococcus elongatus. Biochim. Biophys. Acta 1767, 520527 (2007).

31. Shevela, D. & Messinger, J. Probing the turnover efciency of photosystem II membrane fragments with different electron acceptors. Biochim. Biophys. Acta 1817, 12081212 (2012).

32. Boutet, S. & J Williams, G. The Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). New J. Phys. 12, 035024 (2010).

33. Iuzzolino, L., Dittmer, J., Drner, W., Meyer-Klaucke, W. & Dau, H. X-ray absorption spectroscopy on layered photosystem II membrane particles suggests manganese-centered oxidation of the oxygen-evolving complex for the S0-S1, S1-S2, and S2-S3 transitions of the water oxidation cycle. Biochemistry 37, 1711217119 (1998).

34. Kulik, L. V., Epel, B., Lubitz, W. & Messinger, J. Electronic structure of the Mn4OxCa cluster in the S0 and S2 states of the oxygen-evolving complex of photosystem II based on pulse Mn55-ENDOR and EPR spectroscopy. J. Am. Chem. Soc. 129, 1342113435 (2007).

35. Messinger, J. et al. Absence of Mn-centered oxidation in the S2 -4 S3

transition: implications for the mechanism of photosynthetic water oxidation.J. Am. Chem. Soc. 123, 78047820 (2001).36. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 10301033 (2012).

37. English, A. C., Done, S. H., Caves, L. S. D., Groom, C. R. & Hubbard, R. E. Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol. Proteins 37, 628640 (1999).

38. Barends, T. R. et al. Anomalous signal from S atoms in protein crystallographic data from an X-ray free-electron laser. Acta Cryst. D 69, 838842 (2013).39. Barends, T. R. et al. De novo protein crystal structure determination from X-ray free-electron laser data. Nature 505, 244247 (2013).

40. Ferrer, J. L., Larive, N. A., Bowler, M. W. & Nurizzo, D. Recent progress in robot-based systems for crystallography and their contribution to drug discovery. Expert Opin. Drug Discov. 8, 835847 (2013).

41. Zouni, A. et al. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 angstrom resolution. Nature 409, 739743 (2001).

42. Biesiadka, J., Loll, B., Kern, J., Irrgang, K. D. & Zouni, A. Crystal structure of cyanobacterial photosystem II at 3.2 angstrom resolution: a closer look at the Mn-cluster. Phys. Chem. Chem. Phys. 6, 47334736 (2004).

43. Kamiya, N. & Shen, J. R. Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7- resolution. Proc. Natl Acad. Sci. USA 100, 98103 (2003).

44. Kern, J. et al. Cyanobacterial photosystem II at 3.2 resolution - the quinone binding pockets. Photosynth. Res. 84, 153159 (2005).

45. Rappaport, F., Blancharddesce, M. & Lavergne, J. Kinetics of electron-transfer and electrochromic change during the redox transitions of the photosynthetic oxygen-evolving complex. Biochim. Biophys. Acta 1184, 178192 (1994).

46. Rappaport, F., Ishida, N., Sugiura, M. & Boussac, A. Ca2 determines the entropy changes associated with the formation of transition states during water oxidation by photosystem II. Energ. Environ. Sci. 4, 25202524 (2011).

47. Razeghifard, M. R. & Pace, R. J. EPR kinetic studies of oxygen relaese in thylakoids in PSII membranes: A kinetic intermediate in the S3 to S0 transition.

Biochemistry 38, 12521257 (1999).48. Kern, J. et al. Purication, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor. Biochim. Biophys. Acta 1706, 147157 (2005).

49. Porra, R. J., Thompson, W. A. & Kriedemann, P. E. Determination of accurate extinction coefcients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verication of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384394 (1989).

50. Beckmann, K., Messinger, J., Badger, M. R., Wydrzynski, T. & Hillier, W. On-line mass spectrometry: membrane inlet sampling. Photosynth. Res. 102, 511522 (2009).

51. Siewert, F. et al. Ultra-precise characterization of LCLS hard X-ray focusing mirrors by high resolution slope measuring deectometry. Opt. Express 20, 45254536 (2012).

52. Alonso-Mori, R. et al. A multicrystal wavelength dispersive X-ray spectrometer. Rev. Sci. Instrum. 83, 073114 (2012).

53. Hart, P. et al. The CSPAD megapixel x-ray camera at LCLS. Proc. SPIE 8504, 85040C85041C (2012).

54. Herrmann, S. et al. Cspad-140k: a versatile detector for LCLS experiments. Nucl. Instrum. Meth. A 718, 550553 (2013).

55. Sauter, N. K., Hattne, J., Grosse-Kunstleve, R. W. & Echols, N. New Python-based methods for data processing. Acta Cryst. D 69, 12741282 (2013).

56. Hattne, J. et al. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers. Nat. Methods 11, 545548 (2014).

57. Zhang, Z., Sauter, N. K., van den Bedem, H., Snell, G. & Deacon, A. M. Automated diffraction image analysis and spot searching for high-throughput crystal screening. J. Appl. Crystallogr. 39, 112119 (2006).

58. Sauter, N. K., Grosse-Kunstleve, R. W. & Adams, P. D. Robust indexing for automatic data collection. J. Appl. Crystallogr. 37, 399409 (2004).

59. Steller, I., Bolotovsky, R. & Rossmann, M. G. An algorithm for automatic indexing of oscillation images using Fourier analysis. J. Appl. Crystallogr. 30, 10361040 (1997).

60. Rossmann, M. G. & van Beek, C. G. Data processing. Acta Cryst. D 55, 16311640 (1999).

61. Paithankar, K. S. et al. Simultaneous X-ray diffraction from multiple single crystals of macromolecules. Acta Cryst. D 67, 608618 (2011).

62. White, T. A. et al. Crystallographic data processing for free-electron laser sources. Acta Cryst. D 69, 12311240 (2013).

63. Otwinowski, Z. & Minor, W. Processing of X-Ray Diffraction Data Collected in Oscillation Mode Vol. 276 Macromolecular Crystallography, Part A (Academic Press, 1997).

64. Leslie, A. G. Integration of macromolecular diffraction data. Acta Cryst. D 55, 16961702 (1999).

65. Kahn, R. et al. Macromolecular crystallography with synchrotron radiation -photographic data-collection and polarization correction. J. Appl. Crystallogr. 15, 330337 (1982).

66. Leslie, A. G. The integration of macromolecular diffraction data. Acta Cryst. D 62, 4857 (2006).

67. Afonine, P. V. et al. Towards automated crystallographic structure renement with phenix.rene. Acta Cryst. D 68, 352367 (2012).

68. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007).

69. Hodel, A., Kim, S. H. & Brunger, A. T. Model bias in macromolecular crystal-structures. Acta Cryst. A 48, 851858 (1992).

70. Terwilliger, T. C. et al. Iterative model building, structure renement and density modication with the PHENIX AutoBuild wizard. Acta Cryst. D 64, 6169 (2008).

Acknowledgements

This work was supported by NIH Grant GM055302 (V.K.Y) for PS II biochemistry, structure and mechanism; the Director, Ofce of Science, Ofce of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences and Biosciences (CSGB) of the Department of Energy (DOE) under Contract DE-AC02-05CH11231 (J.Y. and V.K.Y.) for X-ray methodology and instrumentation; by NIH grant P41GM103393 for part of the XES instrumentation and support of U.B.; an LBNL Laboratory Directed Research and Development award to N.K.S; and NIH grants GM095887 and GM102520 (N.K.S.) for data processing methods. The DFG-Cluster of Excellence UniCat coordinated by the Technische Universitat Berlin and Sfb1078, TP A5 (A.Z.); the Alexander von Humboldt Foundation (J.K.); the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no 283745 (CRISP) (P.G.); the Solar Fuels Strong Research Environment (Ume University), the Articial Leaf Project (K&A Wallenberg Foundation), Energimyndigheten (J.M.) and VR (M.M.S., J.M.), and the Human Frontiers Science Project (J.Y., U.B. and A.Z.) are acknowledged for supporting this project. The injector work was supported by DOE Ofce of Basic Energy Sciences, Chemical Sciences Division, under Contract DE-AC02-76SF00515 (H.L., C.A.S. and M.J.B.), LCLS (R.G.S.), the Human Frontiers Science Project Award RPG005/2011 (H.L.) and the SLAC Laboratory Directed Research and Development Program (C.A.S. and M.J.B.). We thank Professor Ken Sauer for continuing scientic discussions. We thank Tom Terwilliger, Randy Read, Nigel Moriarty, Ralf Grosse-Kunstleve, Pavel Afonine and Jeffrey Headd for helpful discussions and technical assistance regarding XRD data processing; Matthew Latimer for support with the XES set-up; Don Schaefer, Alan Miahnahri and William White for support with development of the laser illumination and injector set-up; Christopher Kenney, Ryan Herbst, Jack Pines, Philip Hart, John Morse, Gunther Haller and Sven Herrmann for support with the CSPAD detectors; Amedeo Perazzo and Igor Gaponenko (SLAC) for computing support; Gregory Bell (Energy Sciences Network (ESnet)) for arranging network access for data transfer; and Shane Canon (NERSC) for arranging computing access. We thank the staff at LCLS/SLAC and the staff at SSRL, ALS, APS and ESRF for support of synchrotron experiments. Portions of this research

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were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. LCLS is an Ofce of Science User Facility operated for the U.S. Department of Energy Ofce of Science by Stanford University. Data processing was performed in part at the National Energy Research Scientic Computing Center, supported by the DOE Ofce of Science, contract number DE-AC02-05CH11231. Testing of crystals and various parts of the set-up were carried out at synchrotron facilities that were provided by the Advanced Light Source (ALS), BL 5.0.2, in Berkeley, and Stanford Synchrotron Radiation Lightsource (SSRL), BL 6-2, in Stanford, funded by DOE OBES. The SSRL Biomedical Technology programme is supported by NIH, the National Center for Research Resources and the DOE Ofce of Biological and Environmental Research. SR PS II XES data were recorded at the European Synchrotron Radiation Facility (ESRF), Grenoble, France.

Author contributions

U.B., V.K.Y. and J.Y. conceived the experiment; U.B., J.Y.,V.K.Y., J.K., R.A.-M., J.M., A.Z., N.K.S., G.J.W., S.B., A.R.F., D.M. and M.J.B. designed the experiment; R.T.,J. Hellmich, G.H., R.C., D.D., M.I., C.G., J.K., A.L., B.L.-K., S.G. and A.Z. prepared samples; S.B., J.E.K., M.M., M.M.S. and G.J.W. operated the CXI instrument; M.J.B., H.L., R.G.S., J.K., J.M., B.L.-K., S.G., R.T. and C.G., J.Hellmich and G.J.W. developed, tested and ran sample delivery system; S.K. and J.M. conceived, set up and performed O2 evolution measurements; R.C., R.A.-M., E.G., J.U. and P.G. collected SR XES of PS II; R.A.-M., J.K., R.T., B.L.-K., S.G., T.-C.W., D.S. and J.Y. developed and tested XES set-up; R.A.-M., A.B., U.B., M.J.B., S.B., R.C., R.J.G., P.G., C.G., S.G., G.H., J.Hattne., J.Hellmich,

J.K., J.E.K., A.L., H.L., B.L.-K., D.M., M.M., J.M., N.K.S., M.M.S., R.G.S., C.A.S., D.S., R.T.,T.-C.W., G.J.W., V.K.Y., J.Y. and A.Z. performed the LCLS experiment; J. Hattne, N.E., R.J.G., A.B., R.A.-M., J.K., C.A.S., P.H.Z., M.M., P.D.A. and N.K.S. developed new software and/or processed and analysed data; D.E.S. arranged computer access; J.K., J.Y., J.M., N.K.S., U.B. and V.K.Y. wrote the manuscript with input from all authors.

Additional information

Accession codes: The X-ray crystallographic coordinates and structure factors for structures reported in this study have been deposited at the Protein Data Bank (PDB), under deposition numbers 4TNL (thermolysin), 4TNH (PS II dark state), 4TNJ (PS II 2F), 4TNI (PS II 3F) and 4TNK (PS II 3F). These data can be obtained free of charge from http://www.pdb.org

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Kern, J. et al. Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy. Nat. Commun. 5:4371 doi: 10.1038/ncomms5371 (2014).

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