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Ultrafast structural changes within a photosynthetic reaction centre

Nature volume 589, pages 310–314 (2021)Cite this article

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Abstract

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

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Fig. 1: Electron-transfer steps of the photosynthetic reaction centre of B. viridis.
Fig. 2: Light-induced electron density changes in BvRC at the site of photo-oxidation.
Fig. 3: Light-induced electron density changes in BvRC within the QA binding pocket.
Fig. 4: Structural response of BvRC to electron-transfer events.

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Data availability

Atomic coordinates and structure factors have been deposited in the Protein Data Bank. PDB ID codes are as follows: 5O4C, dark conformation (dataset a); 5NJ4, dark conformation (dataset b); 6ZHW, time point Δt = 1 ps; 6ZI4, time point Δt = 5 ps (dataset a); 6ZID, time point Δt = 5 ps (dataset b); 6ZI6, time point Δt = 20 ps; 6ZI5, the time point Δt = 300 ps (dataset a); 6ZI9, time point Δt = 300 ps (dataset b); 6ZIA, time point Δt = 8 μs. Difference Fourier electron density maps and stream files containing X-ray diffraction intensities are deposited at the CXI database (http://www.cxidb.org/) with identification number 161. Source data are provided with this paper.

Code availability

Software used for SVD analysis is available at https://github.com/Neutze-lab/SVD. Code written in MATLAB to analyse difference electron-density amplitudes is available at https://github.com/Neutze-lab/maptool. Software associated with the resampling of X-ray diffraction data is available at https://github.com/Katona-lab/CFEL_tools. The Gromacs 4.5 version linked to TeraChem for QM/MM optimization is available for download from https://github.com/dmmorozo/GromacsTc.

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Acknowledgements

Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. Parts of the sample delivery system used at LCLS for this research were funded by NIH grant P41GM103393, formerly P41RR001209. Complementary studies were performed at BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI, proposal number 2018B8068). R.N. acknowledges funding from the European Commission Marie Curie Training Networks (X-Probe, NanoMem), the European Union’s Horizon 2020 research and innovation programme (grant agreement 789030) and the Swedish Research Council (grants 2015-00560 and 349-2011-6485), the Swedish Foundation for Strategic Research (grant SRL10-0036), and the Knut and Alice Wallenberg Foundation (grant KAW 2012.0284, KAW 2012.0275 and KAW 2014.0275). G.B. acknowledges funding from the Swedish Research Council (grant 2017-06734) and the Swedish Foundation for Strategic Research (grant ID17-0060). G.G., D. Morozov and H.L.L. acknowledge funding from the Academy of Finland (grants 290677 and 304455), the BioExcel CoE project funded by the European Union contracts H2020-INFRAEDI-02-2018-823830 and H2020-EINFRA-2015-1-675728, and the CSC-IT centre in Espoo for access to computing resources. R.N. thanks N. Woodbury for discussions.

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Author notes

  1. These authors contributed equally: Robert Dods, Petra Båth

Authors and Affiliations

  1. Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden

    Robert Dods, Petra Båth, Viktor Ahlberg Gagnér, David Arnlund, Joachim Kübel, Michał Maj, Adams Vallejos, Cecilia Wickstrand, Robert Bosman, Rajiv Harimoorthy, Peter Berntsen, Linda Johansson, Rebecka Andersson, Elin Claesson, Peter Dahl, Greger Hammarin, Cecilia Safari, Amit Sharma, Sebastian Westenhoff, Gergely Katona, Gisela Brändén & Richard Neutze

  2. Nanoscience Center and Department of Chemistry, University of Jyväskylä, Jyväskylä, Finland

    Dmitry Morozov, Hoi Ling Luk & Gerrit Groenhof

  3. Centre for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany

    Kenneth R. Beyerlein, Oleksandr Yefanov & Anton Barty

  4. Department of Physics, Arizona State University, Tempe, AZ, USA

    Garrett Nelson, Chufeng Li & Stella Lisova

  5. Linac Coherent Light Source, Stanford Linear Accelerator Centre (SLAC), National Accelerator Laboratory, Menlo Park, CA, USA

    Mengning Liang, Despina Milathianaki, Joseph Robinson, Sergio Carbajo, Mark S. Hunter, Garth J. Williams, Daniel P. DePonte & Sébastien Boutet

  6. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia

    Peter Berntsen

  7. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Erik Malmerberg

  8. Bridge Institute, USC Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, CA, USA

    Linda Johansson

  9. Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA

    Chelsie E. Conrad

  10. Institut de Biologie Structurale (IBS), Université Grenoble Alpes, CEA, CNRS, Grenoble, France

    Antoine Royant

  11. European Synchrotron Radiation Facility, Grenoble, France

    Antoine Royant

  12. Ångström Laboratory, Department of Chemistry, Uppsala University, Uppsala, Sweden

    Jan Davidsson

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  1. Robert Dods
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R.N. conceived the experiment, which was designed with input from G.B., R.D., J.D., S.B., M.L., S.C., D. Milathianaki, M.S.H., G.J.W., D.P.D. and A.B. Samples were prepared by R.D., P. Båth, D.A. and R.B. Crystallization was supervised by G.B. Time-resolved serial femtosecond crystallography experiments were performed at the LCLS by R.D., P. Båth, D.A., R.B., K.R.B., G.N., M.L., D. Milathianaki, J.R., R.H., P. Berntsen, E.M., L.J., R.A., S.C., E.C., C.E.C., P.D., G.H., M.S.H., C.L., S.L., C.S., A.S., G.J.W., C.W., J.D., D.P.D., A.B., G.B. and R.N. The CXI instrument was set up and run by M.L., M.S.H., G.J.W. and S.B. The photoexcitation laser of 960 nm was operated and aligned by D. Milathianaki, J.R., S.C. and J.D. Sample delivery was performed by K.R.B., G.N., R.H., P. Berntsen, P.D. and D.P.D. Data were analysed by R.D., P. Båth, A.R., O.Y., A.B., G.B. and R.N. Structural refinement was performed by R.D. and P. Båth. Resampling, full-occupancy structural refinement and analysis were performed by V.A.G., G.K. and A.V. Integration within a sphere and statistical tests were performed by C.W., R.N. and P. Båth. SVD analysis was performed by A.V. QM/MM analysis was performed by D. Morozov, H.L.L. and G.G. Time-resolved infrared spectroscopy measurements were performed by J.K., M.M. and S.W. The manuscript was prepared by R.N., P. Båth, R.D. and G.B. with additional input from all authors.

Corresponding author

Correspondence to Richard Neutze.

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The authors declare no competing interests.

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Peer review information Nature thanks Jörg Matysik, Keith Moffat and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Dependence of transient infrared spectra on the pump laser fluence.

a, Time-resolved infrared difference spectra recorded from BvRC in D2O buffer for four time points and variable pump-laser fluence following 960 nm excitation. Spectral changes are consistent with previous reports74,75. Dominant time-dependent features are highlighted and include a negative band at 1,687 cm−1 (9-carbonyl stretch in SPL and SPM) and a broad negative band centred at 1,680 cm−1 (9-carbonyl stretch in BChL). Transient changes from ∆t = 2 ps to 5 ps correlate with the time-dependent photo-oxidation of SP. Grey columns indicate decreasing bands whereas cyan columns indicate increasing bands. b, Time-resolved infrared difference spectra normalized and superimposed. These spectra remain superimposable throughout the analysed pump-laser fluence domain. c, Dependence of the magnitude of the difference infrared signal (calculated as the sum of the absolute value over all pixels) on the pump laser fluence. Below 20 GW cm−2, the absolute signal increases approximately linearly whereas above 60 GW cm−2 the signal flattens out as a plateau is reached. This plateau is consistent with the complete bleaching of the special pair absorption at 960 nm in the photo-excited state and correlates with the phenomenon of hole burning in BvRC42.

Extended Data Fig. 2 Overview of the experimental Fo(light) − Fo(dark) difference Fourier electron density maps calculated between the photo-excited and resting state data.

a, Overview of the structure of BvRC when viewed from the plane of the membrane. Transmembrane helices EL and DM are highlighted in red and gold, respectively. b–h, Difference Fourier electron density maps for the following time points. b, ∆t = 1 ps. c, ∆t = 5 ps (dataset a). d, ∆t = 5 ps (dataset b). e, ∆t = 20 ps. f, ∆t = 300 ps (dataset b). g, ∆t = 300 ps (dataset a). h, ∆t = 8 μs. All maps are contoured at ±4.0σ. Blue, positive difference electron density; gold, negative difference electron density.

Extended Data Fig. 3 Light-induced electron density changes in BvRC at the site of photo-oxidation.

Experimental Fo(light) − Fo(dark) isomorphous difference Fourier electron density maps for the following time points. a, ∆t = 1 ps. b, ∆t = 5 ps (dataset a). c, ∆t = 5 ps (dataset b). d, ∆t = 20 ps. e, ∆t = 300 ps (dataset b). f, ∆t = 300 ps (dataset a). g, ∆t = 8 μs. h, i, Principal components from the SVD analysis of difference Fourier electron density maps for the first four time points, ∆t = 1ps, 5 ps (dataset a), 5 ps (dataset b) and 20 ps (h) and the final three time points, ∆t = 300 ps (dataset a), 300 ps (dataset b) and 8 μs (i). All maps are contoured at ±3.2σ. Blue, positive difference electron density; gold, negative difference electron density. Peak heights listed in Extended Data Table 2 are marked with coloured circles in h and i. j, Representation of a sphere of radius 4.5 Å used to integrate difference electron density above a pedestal of 3.0σ as described previously20. Positive difference density amplitudes (A+) and negative difference density amplitudes (A−) were merged according to \(A({\bf{r}})=\sqrt{{({A}^{+})}^{2}+{({A}^{-})}^{2}}\) and are displayed in Fig. 2f. These integrated difference electron density values are also used in the statistical analyses presented in Extended Data Table 3. k, Simulated difference Fourier electron density maps near the SP calculated from the refined structure for ∆t = 20 ps versus the refined resting state structure. Data are cut at a resolution of 2.8 Å for comparison with experimental data. The simulated map is contoured at ±12.0σ. l, Relative magnitude of the singular values that result from the SVD analysis of the difference Fourier electron density maps for all seven time points. m, First (blue) and second (red) columns of the unitary matrix U resulting from SVD analysis of all seven time points weighted according to their corresponding singular values. The first right singular vector (the principal SVD component) makes a significant and positive contribution to all time points. By contrast, the difference electron density maps for ∆t = 5ps (dataset a) and 300 ps (dataset a) contain strong negative contributions from the second right singular vector, which suggests that differences between the two experimental runs cause systematic differences in the difference Fourier electron density maps that are separated by SVD analysis.

Source data

Extended Data Fig. 4 Electron density changes near the active site of myoglobin.

The Fo(light) − Fo(dark) difference Fourier electron density map for Δt = 10 ps was calculated from data (PDB entries 5CNE minus 5CMV) recorded during TR-SFX studies of the photo-dissociation of carbon monoxide from the active site of myoglobin21. a, Data from 15 Å to 1.8 Å were used to calculate the difference Fourier electron density map. b, Data from 15 Å to 3.0 Å were used to calculate the difference Fourier electron density map. Positive and negative difference electron density features associated with the haem group indicate slight motions of the protoporphyrin-IX. Blue represents positive difference density (contoured at 3.0σ) and gold represents negative density (contoured at −3.0σ). At 1.8 Å, the maximum amplitude of the highlighted difference density features are: a+, 14.5σ; a−, 14.0σ; b+, 4.6σ; b−, 4.0σ; c+, 4.1σ, c−, 4.0σ; d+, 3.8σ. When the map is recalculated after data are cut to a resolution of 3.0 Å the corresponding values are: a+, 11.9σ; a−, 12.4σ; b+, 4.2σ; b−, 4.1σ; c+, 3.7σ, c−, 2.9σ; d+, 3.2σ.

Extended Data Fig. 5 Light-induced electron density changes in BvRC within the QA binding pocket.

Experimental Fo(light) − Fo(dark) difference Fourier electron density maps for the following time points. a, ∆t = 1 ps. b, ∆t = 5 ps (dataset a). c, ∆t = 5 ps (dataset b). d, ∆t = 20 ps. e, ∆t = 300 ps (dataset b). f, ∆t = 300 ps (dataset a). g, ∆t = 8 μs. All seven maps are contoured at ±3.0σ. Blue, positive difference electron density; gold, negative difference electron density). h, i, Principal components from the SVD analysis of difference Fourier electron density maps for the first four time points, ∆t = 1 ps, 5 ps (dataset a), 5 ps (dataset b) and 20 ps (h) and the final three time points, ∆t = 300 ps (dataset a), 300 ps (dataset b) and 8 μs (i). SVD difference Fourier maps are contoured at ±3.2σ. Peak heights listed in Extended Data Table 2 are indicated in i. j, Simulated difference Fourier electron density maps near QA calculated from the refined structure for ∆t = 300 ps (dataset a) versus the refined resting state structure. Data are cut at a resolution of 2.8 Å for comparison with experimental data. The simulated map is contoured at ±12.0σ.

Extended Data Fig. 6 Results of QM/MM energy-minimization calculations.

a–c, QM subsystems used in the QM/MM optimizations of the cofactors. Atoms included in the QM region are shown in ball-and-stick representation; the other atoms of the protein are shown as cartoons. Atoms belonging to the cofactor that are not included in the QM region are shown as sticks. Water molecules that are not part of the QM region are not shown. a, Atoms included in the QM region associated with the SP. b, Atoms included in the QM region associated with the BPhL. c, Atoms included in the QM region associated with the QA. d, Structure of the energy-minimized resting conformation (black, SP and BPhL neutral) and structure after the first electron-transfer step (blue, SP+ and BPhL−) shown near the special pair. e, Structure of the energy-minimized resting conformation (black, SP and BPhL neutral) and structure after the first electron-transfer step (blue, SP+ and BPhL−) shown near BPhL. f, Structure of the energy-minimized resting conformation (black, SP and QA neutral) and structure after the second electron-transfer step (blue, SP+ and QA−) shown near QA. The hydrogen-bond between O1 of the reduced semiquinone and Nδ1 of His217M is predicted to be shortened by 0.17 Å when menaquinone is reduced. g, h, Schematic depiction of the potential energy surfaces for menaquinone in vacuum (g) and within the protein (h) in the resting (neutral) electronic state (black) and the reduced electronic state (red). Vertical electron affinities (VEA), adiabatic electron affinities (AEA) and relaxation energies Er were computed at the PBE0-D3/LANL2DZ level of DFT for isolated QA and at the PBE0-D3/LANL2DZ/Amber03 QM/MM level for QA within the protein.

Extended Data Fig. 7 Recurring movements of Cα atoms quantified by structural refinement.

Recurring movements of Cα atoms quantified by full-occupancy structural refinement against 100 randomly resampled TR-SFX datasets. Recurring movements are represented as error-weighted mean ratios relative to 100 control structural refinements (Methods). Error-weighted means ratios are coloured from grey (<80% of the maximum ratio) to red (≥95% of the maximum ratio). a, Recurring movements of Cα atoms associated with refinements against data for ∆t = 1 ps. b, Recurring movements of Cα atoms for ∆t = 5 ps (dataset a). c, Recurring movements of Cα atoms for ∆t = 5 ps (dataset b). d, Recurring movements of Cα atoms for ∆t = 20 ps. e, Recurring movements of Cα atoms for ∆t = 300 ps (dataset b). f, Recurring movements of Cα atoms for ∆t = 300 ps (dataset a). g, Recurring movements of Cα atoms for ∆t = 8 μs. Transmembrane helices are drawn as rods.

Extended Data Table 1 Crystallographic data and refinement statistics
Full size table
Extended Data Table 2 Difference Fourier electron density peak amplitudes
Full size table
Extended Data Table 3 Two-sample t-tests of integrated electron density amplitudes
Full size table

Supplementary information

Reporting Summary

Video 1

Light-induced electron density changes in RCvir at the site of photo-oxidation. The video initially shows the principal component resulting from SVD analysis of the Fo(light) − Fo(dark) isomorphous difference Fourier electron density maps of the first four measurements (∆t = 1ps, 5 ps (a), 5 ps (b), 20 ps) and is followed by the principal component resulting from SVD analysis of the last three measurements (∆t = 300 ps (a), 300 ps (b), 8 µs). The viewer’s orientation rotates about the special pair. Difference electron density maps are contoured at ± 3.0 σ (blue positive density, gold negative density).

Video 2

Light-induced electron density changes in RCvir within the menaquinone binding pocket. The video initially shows the principal component resulting from SVD analysis of the Fo(light) − Fo(dark) isomorphous difference Fourier electron density maps of the first four measurements (∆t = 1ps, 5 ps (a), 5 ps (b), 20 ps) and is followed by the principal component resulting from SVD analysis of the last three measurements (∆t = 300 ps (a), 300 ps (b), 8 µs). The viewer’s orientation rotates about the menaquinone. Difference electron density maps are contoured at ± 3.0 σ (blue positive density, gold negative density).

Video 3

Light-induced movements of the Cα atoms of RCvir. Colour is used to illustrate the recurring movements of RCvir Cα atoms quantified by full occupancy structural refinement against 100 randomly resampled TR-SFX data sets. White indicates no significant movement whereas red indicates recurring light-induced movements of Cα atoms. The video initially shows structural changes for ∆t = 1 ps, then structural changes for ∆t = 5 ps, and finally structural changes for ∆t = 300 ps. Transmembrane α-helices are drawn as rods whereas all other regions are represented as ribbons.

Supplementary Video 4

Predicted movements of Cα atoms as electrons move between cofactors. Colour is used to illustrate the magnitude of RCvir Cα movements estimated from QM/MM energy minimization. White indicates no significant movement whereas red indicates electron transfer induced movements. The video initially shows structural changes when SP is photo-excited, then structural changes as an electron moves from SP to BPhL, and finally structural changes as an electron moves further to QA. Transmembrane α-helices are drawn as rods whereas all other regions are represented as ribbons.

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Dods, R., Båth, P., Morozov, D. et al. Ultrafast structural changes within a photosynthetic reaction centre. Nature 589, 310–314 (2021). https://doi.org/10.1038/s41586-020-3000-7

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