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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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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.
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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 4.0σ, 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.

Mentions: As a positive control of the methodology, we first analyzed 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 Å (9.76 keV), about 100 eV above the Zn edge (9.66 keV). Diffraction was observed out to the corners of the detector (1.50 Å) and the integrated intensities were merged to obtain a dataset to 1.80 Å resolution (Table 1, 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, 18 σ 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 sulfur of one of the methionine residues (Figs. 5A, B and Supplementary Fig. 7).


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)

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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4151126&req=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.
Mentions: As a positive control of the methodology, we first analyzed 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 Å (9.76 keV), about 100 eV above the Zn edge (9.66 keV). Diffraction was observed out to the corners of the detector (1.50 Å) and the integrated intensities were merged to obtain a dataset to 1.80 Å resolution (Table 1, 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, 18 σ 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 sulfur of one of the methionine residues (Figs. 5A, B and Supplementary Fig. 7).

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