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. 2014 Jul 9:5:4371.
doi: 10.1038/ncomms5371.

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

Jan Kern  1 , Rosalie Tran  2 , Roberto Alonso-Mori  3 , Sergey Koroidov  4 , Nathaniel Echols  2 , Johan Hattne  2 , Mohamed Ibrahim  5 , Sheraz Gul  2 , Hartawan Laksmono  6 , Raymond G Sierra  6 , Richard J Gildea  7 , Guangye Han  2 , Julia Hellmich  5 , Benedikt Lassalle-Kaiser  7 , Ruchira Chatterjee  2 , Aaron S Brewster  2 , Claudiu A Stan  6 , Carina Glöckner  8 , Alyssa Lampe  2 , Dörte DiFiore  8 , Despina Milathianaki  3 , Alan R Fry  3 , M Marvin Seibert  9 , Jason E Koglin  3 , Erik Gallo  10 , Jens Uhlig  10 , Dimosthenis Sokaras  11 , Tsu-Chien Weng  11 , Petrus H Zwart  2 , David E Skinner  12 , Michael J Bogan  13 , Marc Messerschmidt  3 , Pieter Glatzel  10 , Garth J Williams  3 , Sébastien Boutet  3 , Paul D Adams  2 , Athina Zouni  5 , Johannes Messinger  4 , Nicholas K Sauter  2 , Uwe Bergmann  3 , Junko Yano  2 , Vittal K Yachandra  2
Affiliations

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

Jan Kern et al. Nat Commun. .

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.

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Figures

Figure 1
Figure 1
Flash-induced changes in PS II and experimental setup 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 setup 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, 3 for 3F samples and lasers 2, 3 and 4 to generate the 3F′ samples.
Figure 2
Figure 2
Oxygen production by PSII A) Relative O2 yield per PSII as detected by MIMS as a function of flash number (measurement shown is for PS II solutions, flow rate 0.5 μl/min, frequency 4 Hz, light intensity was 7 μJ for each fiber). B) O2 yield measured by MIMS as a function of flash number from PS IIsolutions (black) and PS II microcrystals (red).
Figure 3
Figure 3
Mn Kβ XES of PS II A) XES recorded with <50 fs X-ray pulses at LCLS. Spectra were measured 0.5 s after two laser flashes (2F, black; lasers 2 and 3 on), or 0.5 s after three laser flashes (3F, blue; lasers 1, 2, and 3 on), and ~250 μs after three laser flashes (3F′, red; lasers 2, 3, and 4 on), respectively. B) Difference between the Mn Kβ XES of PS II, blue: 3F – 2F; red: 3F′ – 2F. Before calculating the difference curves, spectra were smoothed by moving average (dotted line) or cubic polynomial fitting (solid line, similar to the procedure used for analyzing the synchrotron data). (C) the 3F – 2F difference spectrum (green) from SR data collected at 15 K.
Figure 4
Figure 4
Electron density maps obtained for PS II A) 2mFo–DFsc maps for the dark and B) the 2F data of PS II are shown in grey contoured at 1.0σ, mFo–DFc maps after omitting the OEC are shown in green and red, contoured at +/−5.0σ. C) mFo-mFo isomorphous difference maps for the 2F – dark data and D) the 3F – 2F data are shown for both monomers and are contoured at +3σ (bright green, monomer I; pale green, monomer II) and −3σ (red, monomer I; salmon, monomer II) together with the model for the 2F data.
Figure 5
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 4.0σ, 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, contour level at 3.0σ. 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 4.0σ. D) Enlarged view of the 3F anomalous density for the region of the OEC (contoured at 4.0σ). All maps shown are anomalous difference simulated annealing omit maps.
Figure 6
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 to the view in A. (D) Anomalous map for monomer II, 2F (cyan) and 3F (magenta) data are shown, view direction is similar to panel C. All maps shown are anomalous difference simulated annealing omit maps contoured at 3σ.
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Comment in

  • Lights, X-rays, oxygen!
    Jez JM, Blankenship RE. Jez JM, et al. Cell. 2014 Aug 14;158(4):701-703. doi: 10.1016/j.cell.2014.07.037. Cell. 2014. PMID: 25126779

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