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

Kern J, Tran R, Alonso-Mori R, Koroidov S, Echols N, Hattne J, Ibrahim M, Gul S, Laksmono H, Sierra RG, Gildea RJ, Han G, Hellmich J, Lassalle-Kaiser B, Chatterjee R, Brewster AS, Stan CA, Glöckner C, Lampe A, DiFiore D, Milathianaki D, Fry AR, Seibert MM, Koglin JE, Gallo E, Uhlig J, Sokaras D, Weng TC, Zwart PH, Skinner DE, Bogan MJ, Messerschmidt M, Glatzel P, Williams GJ, Boutet S, Adams PD, Zouni A, Messinger J, Sauter NK, Bergmann U, Yano J, Yachandra VK - Nat Commun (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA [2] LCLS, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA.

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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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.
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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.

Mentions: XRD data from 2F (S3-enriched), 3F (S0-enriched), and 3F′ (S3YZox -enriched; 250 μs after the 3rd flash) 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 ~4.1 Å, with thermal diffuse scattering extending well beyond this to ~3.0 Å, 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 Å resolution (see Table 1, 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 coefficient of semi-datasets merged from odd- and even-numbered images36; i.e. completeness > 90%, multiplicity > 6, and CC1/2 > 30%. Likewise, data sets of 3F, 3F′ and 0F states were obtained with resolutions of 4.6 Å (13,094 lattices), 5.2 Å (7,850 lattices) and 4.9 Å (6,695 lattices), respectively (Table 1, Supplementary Tables 1, 3–5). Electron density maps for all four states are shown in Fig. 4, 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 result clearly shows the electron density of the Mn4O5Ca cluster, the non-heme 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-heme iron, where the largest changes are expected, were inspected for changes between the different states. No statistically significant changes were observed in the 2mFo–DFc maps of the individual data sets (Fig. 4A,B, Supplementary Fig. 2 and 3) and in the isomorphous difference maps (mFo-mFo) between the different data sets (Fig. 4C, D, 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 flash states; namely an electron density peak at the position of the OEC when viewed at a contour level of +3σ, a small peak 10 Å 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 −3σ contour, for example close to Val 185 and Phe 182 of the D1 protein (Supplementary Fig. 6). We observe these low intensity peaks at the same positions generally in both monomers and across all four illuminated states. This suggests that they are not artifacts of the Fourier transform, and are rather due to structural differences between SR data collected at cryogenic temperature and the room temperature data presented here. However, the current resolution does not allow them to be fully modeled in our final atomic coordinate sets.


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

Kern J, Tran R, Alonso-Mori R, Koroidov S, Echols N, Hattne J, Ibrahim M, Gul S, Laksmono H, Sierra RG, Gildea RJ, Han G, Hellmich J, Lassalle-Kaiser B, Chatterjee R, Brewster AS, Stan CA, Glöckner C, Lampe A, DiFiore D, Milathianaki D, Fry AR, Seibert MM, Koglin JE, Gallo E, Uhlig J, Sokaras D, Weng TC, Zwart PH, Skinner DE, Bogan MJ, Messerschmidt M, Glatzel P, Williams GJ, Boutet S, Adams PD, Zouni A, Messinger J, Sauter NK, Bergmann U, Yano J, Yachandra VK - Nat Commun (2014)

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.
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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.
Mentions: XRD data from 2F (S3-enriched), 3F (S0-enriched), and 3F′ (S3YZox -enriched; 250 μs after the 3rd flash) 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 ~4.1 Å, with thermal diffuse scattering extending well beyond this to ~3.0 Å, 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 Å resolution (see Table 1, 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 coefficient of semi-datasets merged from odd- and even-numbered images36; i.e. completeness > 90%, multiplicity > 6, and CC1/2 > 30%. Likewise, data sets of 3F, 3F′ and 0F states were obtained with resolutions of 4.6 Å (13,094 lattices), 5.2 Å (7,850 lattices) and 4.9 Å (6,695 lattices), respectively (Table 1, Supplementary Tables 1, 3–5). Electron density maps for all four states are shown in Fig. 4, 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 result clearly shows the electron density of the Mn4O5Ca cluster, the non-heme 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-heme iron, where the largest changes are expected, were inspected for changes between the different states. No statistically significant changes were observed in the 2mFo–DFc maps of the individual data sets (Fig. 4A,B, Supplementary Fig. 2 and 3) and in the isomorphous difference maps (mFo-mFo) between the different data sets (Fig. 4C, D, 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 flash states; namely an electron density peak at the position of the OEC when viewed at a contour level of +3σ, a small peak 10 Å 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 −3σ contour, for example close to Val 185 and Phe 182 of the D1 protein (Supplementary Fig. 6). We observe these low intensity peaks at the same positions generally in both monomers and across all four illuminated states. This suggests that they are not artifacts of the Fourier transform, and are rather due to structural differences between SR data collected at cryogenic temperature and the room temperature data presented here. However, the current resolution does not allow them to be fully modeled in our final atomic coordinate sets.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA [2] LCLS, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA.

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.

Show MeSH
Related in: MedlinePlus