Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

Abstract

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Extended Data Figure 1. Photosystem II crystal growth and characterization

a, Scheme of free interface diffusion enhanced sedimentation method for growth of photosystem II nano/microcrystals. b, Large photosystem II crystals (average size 300 µm) suitable for X-ray data collection at synchrotron sources. c, Optical image of nano/microcrystals of photosystem II grown by free interface diffusion used for the TR-SFX data collection at LCLS. The crystallinity must be confirmed by other methods such as SONICC (see (e) for the SONICC image of the crystals) because nanocrystals look similar to amorphous precipitate. d, DLS results of the crystals shown in c indicate an average Stokes radius of 1 µm. e, SONICC image of the photosystem II microcrystals shown in c. f, g, Panels showing the EPR analysis of S-states yield of PSII after double-excitation. f, X-band EPR spectra (10 K) of photosystem II protein solution used for crystallization exposed to 0 (dark adapted sample, no flash NF), one (1F) or two (2F) saturating laser excitation flashes at room temperature. The samples were flash frozen after illumination. For comparison we also show the EPR spectra of dark adapted photosystem II subjected to continuous illumination at 190 K (NF, illu). At low temperature, the S-state cycle stops in the S₂ state which means that this conditions corresponds to the maximal yield of multi-line signal. Three individual samples of each type were analysed and the same MLS intensities were consistently found for similar samples. g, Fit of the quantified S₂ state multiline signal (MLS) oscillations to the Kok model of the S-state transition cycle. Please note that the MLS yield after the second and third flash is nearly constant in the measurements, whereas the fit predicts a decline after the third flash. This is expected as we have not added quinones or artificial electron acceptors to the sample, so that there is no terminal electron acceptor present after PQH₂ has left the Q_B binding site after the second flash.

Extended Data Figure 2. Background corrected diffraction pattern of a photosystem II microcrystal

a, b, From the dark (S₁) data set (a) and the double-flash data set (b) collected at the CXI instrument at LCLS. The resolution is indicated by red and yellow rings corresponding to resolution shells in Å 10, 9, 8 (red), 7 (orange), 6, 5, 4 (yellow). The right panel shows an enlarged view of the diffraction patterns (see blue box).

Extended Data Figure 3. Distribution of photosystem II unit cell constants of 4 different femtosecond crystallography data sets

Row 1 (top row) shows unit cell constants of the dark data set (S₁ state) collected at the CXI instrument in January 2012 (experiment (A)). Row 2 shows unit cell constants of the double-flash data set (putative S₃ state) collected at the CXI instrument in January 2012 (experiment (A)). Row 3 shows unit cell constants of the dark data set (S₁ state) collected at the CXI instrument in June 2012 (experiment (B)) (quinone PQ_decyl was added to these crystals to allow replacement of the quinone for triple excitation). Row4 shows unit cell constants of the triple-flash data set (putative S₄ state) collected at the CXI instrument in June 2012 (experiment (B)). The comparison of unit cell constants shows that significant changes in the unit cell constants are observed after double-flash excitation of photosystem II. These changes are fully reversed when photosystem II is excited by three laser flashes. Although the number of indexed patterns currently available does not yet allow for the determination of an accurate structure of the PSII after triple excitation, the data allows extraction of information on the hit rates, indexing rates and unit cell constants, showing that the unit cell constants are identical for the dark S₁ and triple-flash state.

Extended Data Figure 4. R_split as a function of resolution bins and number of indexed patterns

a, R_split as a function of the resolution shell (in total 20 bins) for dark state data (blue) and double-pumped state data (red). b, R_split as a function of resolution bins for dark S₁ state, R_split decreases indicating better data quality with increase in number of indexed patterns from 3,300 to 34,000 images. c, R_split as a function of resolution bins for the dark and double-flash states, the R_split decreases indicating better quality with increase in number of indexed patterns from 1,800 to 18,800 images.

Extended Data Figure 5. The arrangement of the transmembrane helices in the photosystem II dimer

a, b, An overview of the arrangement of transmembrane helices in photosystem II. The protein subunits are named according to their genes so PsbA is subunit A, PsbB is subunit B, etc. The identification of the location of subunits with more than one transmembrane helix is facilitated by ovals, which are labelled using the same colour code as the corresponding subunit. a, Top view from the stromal side of the arrangement of transmembrane helices in the middle of the membrane. The assignment of helices to different protein subunits is based on the structural assignments of ref. . The 5 transmembrane helices of the core subunits of the reaction centre are PsbA (blue) and PsbB (red). b, The picture shows the omit maps (2F_o−F_c) of the dark and double-flash states at the contour level of 1.5 σ in the same view direction as shown in Extended Data Fig. 5a. c–f, These panels show that most of the alpha helices in the middle of the membrane are well matched between the dark and double-flash states, in the reaction centre core (PsbA and PsbB) as well as in the peripheral parts of photosystem II (for example PsbZ). The view and colour coding of helices are the same as in Extended Data Fig. 5a. c, d, Omit maps of the dark (green) and double-flash (white) states of PSII show a cut through the central region of photosystem II at 1.5 σ. e, The superposition of omit maps indicates a good general overlay of the transmembrane helices and the lumenal loop regions in the two omit maps featuring the reaction centre core subunits PsbA (blue) and PsbB (red) as well as the peripheral subunit X (cyan), and the subunits M(pink) and L (grey) in the dimerization domain of the photosystem II dimer. The electron density is shown at the contour level of 1.5 σ. f, The structural model is also shown with same colour codes as in Extended Data Fig. 5a.

Extended Data Figure 6. Omit map of the dark and double-flash states of the most peripheral photosystem II membrane integral subunits and the chlorophylls of the primary electron donor P680

a–d, This picture features the peripheral subunits PsbZ (grey-green), PsbK (brown), PsaY (grey) and the core-antenna protein CP43 (PsbC) (cyan). The omit electron density map at the contour level of 1.5 σ for the dark (S₁) state is depicted in green (a) and the double-flash (putative S₃) state is depicted in white (b). c, The overlay of the two omit maps is shown at the contour level of 1.5 σ. The globular densities between PsbC and PsbK correspond to antenna chlorophylls. The figure shows that even the electron density for the two most peripheral helices that belong to subunit PsbZ are well defined. We also note the good match of the strongly kinked helix of PsbK between the S₁ and S₃-state electron density maps. d, The subunits are labelled according to their genes in the view of the structural model. e–h, The figure features the surroundings of the two chlorophylls of P680 and the accessory chlorophyll of the active electron transfer branch of photosystem II (see Fig. 2c). The omit electron density map at the contour level of 1.5 σ for the dark (S₁) state is depicted in green (e) and the double-flash (putative S₃) state is depicted in white (f). g, The figure also shows the overlay of the two omit maps at the contour level of 1.5 σ. h, Model of the chlorophylls of the primary electron donor P680 without electron density map.

Extended Data Figure 7. The electron acceptor side of photosystem II

Omit map electron density and structural model of the dark and double-flash state of photosystem II, the view from the stromal side onto the membrane plane. a–d, The loops that coordinate the non-haem iron and cover the quinone binding sites looking from the stromal side onto the membrane plane. The omit electron density map at the contour level of 1.5 σ for the dark (S₁) state is depicted in green (a) and the double-flash (putative S₃) state is depicted in white (b). c, The overlay of the two omit maps at 1.5 σ. d, The structural model indicates the positions of PQ_A and PQ_B as well as the non-haem iron located below the loops. We note that the electron densities of the loop regions at the electron acceptor side show significant differences between the dark and the double-flash states. The electron density of both states may suggest a conformation of the loops that could differ in their backbone trace from the model derived from the 1.9 Å structure from ref. . e–h, The side view of the acceptor side of photosystem II along the plane in the membrane. The omit electron density map at the contour level of 1.5 σ for the dark (S₁) state is depicted in green (e) and the double-flash (putative S₃) state is depicted in white (f). g, The overlay of the two omit maps featuring the changes in the position of the non-haem iron and loop regions at the contour level of 1.5 σ. h, Model of the electron acceptor side of photosystem II. The protein subunits are colour coded as in Extended Data Fig. 5a of the main text; the non-haem iron is depicted as a red sphere. The tightly bound plastoquinone PQ_A is shown in white, the mobile plastoquinone PQ_B is depicted in cyan.

Extended Data Figure 8. Simulated annealed omit map of the Mn₄CaO₅ cluster of photosystem II

The electron density of the dark state of photosystem II. This figure shows the superimposed SA-omit maps for the dark (S₁) (blue) state of the Mn₄CaO₅ cluster. We use a different colour scheme for the SA-omit maps and the ‘regular 2F_o−F_c’ omits maps to allow the reader a better orientation of the type of map shown in each figure. The electron density is shown at the contour level of 3.0 σ to ensure that it solely features the metal Mn₄CaO₅ cluster. The X-ray structure at 1.9 Å from ref. is placed inside the SA-omit map for comparison. The nomenclature for the Mn atoms proposed in ref. is used for the colour-coding of the individual Mn atoms in the cluster. The Mn ions that form the distorted Mn₃O_xCa cubane (Mn1, Mn2 and Mn3) are depicted in light pink, while Mn4 (violet) (referred to as the dangler Mn) is located outside the cubane. This figure shows that the dangler Mn sticks out of the SA-omit map electron density, which indicates that this Mn atom may be located in closer proximity to the Mn₃O_xCa cubane in the dark S₁ state that is not influenced by X-ray damage.

Figure 1. Experimental schemes for the time-resolved serial femtosecond crystallography experiments on photosystem II

a, S-state scheme of the oxygen-evolving complex depicting changes the oxidation state of the 4 manganese ions of the Mn₄CaO₅ cluster in the S-state cycle (*note that the oxidation states of the Mn atoms in the S₄ state are still under debate). The scheme also indicates the reduction of the plastoquinone (PQ) to plastoquinol (PQH₂) in the Q_B site. The blank boxes represent the unoccupied PQ_B binding site. b, Experimental setup. The crystal-stream of photosystem II, was exposed to two subsequent optical laser pulses at 527 nm before interacting with the femtosecond X-ray FEL pulses. With a FEL frequency of 120 Hz and triggering of the laser at 60 Hz, X-ray diffraction patterns from crystals in the dark state and ‘light’ double-flash state alternate. c, Laser excitation scheme. The first 527 nm laser pulse excited the crystals 110 µs after the trigger pulse. The delay time between the first and second 527 nm laser pulse was 210 µs, with X-ray diffraction data collected 570 µs after the second laser pulse.

Figure 2. Overall structure and omit map electron density of photosystem II

a, Transmembrane helices and cofactors in photosystem II (stromal view density map). The proteins are named according to their genes and labelled with coloured letters. b, Side view of PSII at its longest axis along the membrane plane. c, Electron transport chain of PSII (P680 (blue), accessory chlorophylls (olive-green), pheophytins (yellow) and plastoquinones PQ_A (white) and PQ_B (cyan)); atoms of the OEC are depicted as spheres (Mn purple, Ca green, Olight red). d–f, Omit map electron densities (view as in b) at 1.5 σ for the dark state (S₁) (green) (d), double-flash state (putative S₃ state) (white) (e) and overlay of the two omit maps (f). g–i, Omit maps (1.5 σ) of the electron acceptor side of photosystem II for the dark (S₁) (green) (g), double-flash (putative S₃ state) (white) (h) and overlay of the two omit maps (i). Note that changes include a shift of the electron density of the non-haem iron.

Figure 3. The OEC simulated annealed omit maps

a, b, At 1.5 σ for dark and double-flash states of the Mn₄CaO₅ cluster of PSII for the dark S₁-state (blue) (a) and double-flash, putative S₃ state (b) with the 1.9 Å structural model (3ARC) from ref. . Mn in the distorted Mn₃O_xCa cubane (Mn-1 to Mn-3) (light-pink), dangler manganese (Mn-4) (violet), calcium (green) and oxygen (red). c, 1.9 Å crystal structure of the Mn₄CaO₅ cluster with ligands from ref. (PDB accession code: 3ARC). d, Proposed model of the S₃ state based on DFT calculations by Isobe et al. (reproduced with permission of The Royal Society of Chemistry). Larger diversions in the SA-omit map of the double-flash (putative S₃ state) include potential movement of the loop connecting transmembrane helices C/D (CD loop) with D170 and AB loop (with D61), and an increase of the distance between the dangler Mn and the Mn₃O_xCa cubane (violet arrow).