Oxygenic photosynthesis converts light energy into chemical energy, thereby sustaining all aerobic life on Earth. The energy-conversion reaction of photosynthesis is carried out by two photosystems (PSs), PSI and PSII, both are large membrane-embedded protein-pigment complexes existing on the thylakoid membranes of plants and various algae. Among these two PSs, PSII uses light energy to extract electrons and protons from water molecules, leading to the oxidation of water and release of dioxygen as a byproduct. This water oxidation reaction is catalyzed by the oxygen-evolving complex (OEC) of PSII and proceeds through a light-driven, five-step Si-state cycle (i=0-4)1-4 (Fig. 1a). In this Si-state cycle, S0 is the ground state, and the OEC progresses to higher Si-states upon its oxidation by YZ+, a tyrosine cation residue generated by P680+, the photoexcited PSII reaction center P680 (Fig. 1b). Dioxygen is released during the S3 ® (S4) ® S0 transition. The OEC in the S1 state is dark-stable, and its structure has been determined to have a chemical composition of Mn4CaO55,6. This structure changes to a Mn4CaO6-cluster in the highest metastable S3 state by incorporation of a new oxygen atom (O67,8 or Ox9) near the unique µ-oxo bridge O5. The interatomic distance between O6 and O5 is 1.9-2.0 Å in the S3 state8,9, which is suitable for an oxyl/oxo type coupling to form dioxygen between them. The detailed mechanism of the water oxidation, however, has not been well understood, in particular with respect to the origin of O6 and the proton exit pathways.
Since the Mn4CaO5 cluster is embedded inside the protein matrix of PSII and covered by a large area of hydrophilic protein regions in the lumenal side of the thylakoid membrane, channels for the inlet of substrate waters and egress of the product protons are important for the water-splitting reaction to proceed properly. The high resolution structure of PSII showed multiple hydrogen-bond networks connecting the site of the Mn4CaO5 cluster to the lumenal surface of PSII complex1,5,6 these channels may therefore function to allow water coming into the catalytic site or protons to be transported to the lumenal solution. Four main such channels have been identified; they are designated O1-channel, O4-channel, Cl1-channel, and Cl2-channel (Fig. 1). The O1-and O4-channels are so called because they start from the oxo-bridge O1 and O4 of the Mn4CaO5 cluster, respectively, whereas the Cl1- and Cl2-channels are mediated by the Cl1 and Cl2 ions in the vicinity of the Mn4CaO5 cluster, respectively. It is not clear which of these channels functions in the water inlet or proton egress, and in the latter case, in which S-state transition.
Pump-probe, time-resolved serial femtosecond X-ray crystallography (TR-SFX) using X-ray free-electron lasers (XFEL) is a powerful method to visualize structural dynamics of light-triggered enzymes10-15, including PSII in different Si-states at an ambient temperature7, 9, 16. XFELs are femtosecond pulses of X-ray with billions of times brilliance than that of conventional synchrotron X-rays, thus enabling collection of the diffraction data before the onset of radiation damage17. For trapping the intermediate Si-states of PSII with this method, a flow of PSII microcrystals is illuminated by a desired number of pump flashes to generate the higher Si-states (one, two, or three flashes generate S2, S3, or S0 states, respectively), followed by detection with an XFEL pulse with a temporal delay time after the flash illumination. One of the most critical factors for the success of this experiment is the selection of an optimal light excitation condition (intervals, the boundary of excitation region, and power of the excitation laser, etc.) under particular sample delivery conditions (crystal size, flow diameter, flow rate, overall sample consumption, etc.). A generally applicable way to determine a proper light excitation condition is adding a generous safety margin in the sample area to be excited and examining the structural changes by the TR-SFX experiment under a suitable size and power of the pump laser illumination. In the case of enzymes such as PSII that requires multi-flash excitations for the latter S states, however, the application of this method is not straightforward under the continuous sample flow condition, because the sample area that can be used for the excitation and X-ray diffraction data collection must be well aligned spatially and temporally, and a larger separation between two consecutive flashes may make the illuminated sample to escape from the area that can be irradiated by the XFEL pulses. Thus, higher Si states may not be captured by the TR-SFX method if a too large separation is used between consecutive flashes.
In the present study, we show a method to determine an optimal light illumination condition for successful TR-SFX to analyze the structures of the intermediate Si-states of PSII. By altering the flash interval distance with a maximum delay time and examining the structural changes that occurs during the S1-to-S2 transition, a boundary of the excitation region was accurately determined. Based on the light illumination conditions determined, we analyzed the PSII structure in the S2 state at a 2.4-Å resolution. Structural changes were found in the OEC, the O1- and O4-channels, and the QB-binding site, providing important insights into the proton transfer and substrate water delivery during the water oxidation reaction.
Determination of a boundary of the excitation region in TR-SFX
We performed TR-SFX as described previously7,18. In this approach, PSII microcrystals were mixed with a grease matrix and ejected from a micro-extrusion injector. The flow of the PSII microcrystals were excited by a single flash to advance the S-state to S2. Fig. 2a shows a scheme representing the interaction between the pump excitation region and the XFEL pulse in the TR-SFX experiment with a delay time (Δt) of 10 ms at the pump and XFEL repetition rates of 30 Hz. It should be noted that, although the pump beam was focused on the sample stream with a top-hat shape (Ø250 µm)19, the effective excitation region extended upstream due to the pump-light scattering on the stream. Thus, the excitation region is schematically depicted by a triangle in the figure, indicating a gradual decrease in the pump photon density along the sample stream. When a flow rate is not fast enough for the sample exchange, the pump excitation region interacts with the next pump-XFEL pulse pair (Fig. 2b). To avoid such erroneous light-contamination, we here designed a test experiment with Δt of minus 50 ns, where XFEL pulses were delivered to the microcrystals about 100 ms (99.99995 ms) after the pump illumination (Fig. 2c). On this negative Δt condition, if the flow rate is slow, the microcrystals would be illuminated partially by the preceding flash, resulting in “one-flash” illumination (Fig. 2d). However, at a sufficiently fast flow rate, the microcrystals at the position of the XFEL shot will escape from the preceding flash illumination (Fig. 2e), resulting in a “dark dataset”. Accordingly, we can check light-contamination, including an effect of pump light scattering on the sample stream, under the given experimental conditions (pump-illumination size and intensity, sample flow rate, etc).
Four diffraction datasets were collected at different flow rates of 4.9 μl/m, 7.3 μl/m, 8.5 μl/m and 9.8 μl/m (corresponding to 2.0 times, 3.0 times, 3.5 times, and 4.0 times of that for the dark datasets), respectively. In addition to these four datasets, we collected two independent dark datasets from different preparations (Dark1 for the light-illuminated, flow rate 4.9 μl/m and 7.3 μl/m experiments, and Dark2 for the light-illuminated, flow rate 8.5 μl/m and 9.8 μl/m experiments) at a flow rate of 2.5 μl/m, and a light dataset with Δt of 10 ms at a flow rate of 9.8 μl/m. All datasets were processed at 2.35 to 2.40-Å resolutions (Table 1).
We evaluated the boundary of the excitation region as follows (Methods). Isomorphous difference Fourier maps between the “light”-illuminated and dark datasets were calculated with the phases obtained by the refinement, which showed a negative peak covering W665, the second water molecule from O4 in the O4-channel (Fig. 1c, d). Since a similar negative peak has been observed in the previous studies, indicating that W665 becomes highly disordered during the S1-to-S2 transition7-9, we take the height of the Fourier difference peak of W665 as an indicator for the light-induced structural changes. The Fobs (-50 ns, “light” at 4.9 μl/m) minus Fobs (dark) difference Fourier map showed a sharp peak height of -6.4σ at the position of W665 (Fig. 3a). This peak height is lower than that observed with a delay time of 10 ms after the excitation flash (Fig. 3e), but apparently higher than the maximum noise level (Fig. 3f), suggesting that at this flow rate, the microcrystals at the target position of the XFEL shot has been excited by the preceding flash illumination. Thus, the flow rate of 4.9 μl/m is too slow to avoid the light-contamination by the preceding flash in the excitation region. The average height of the light-minus-dark Fourier difference peak of W665 in two non-crystallographic symmetry-related PSII monomers was reduced when the flow rate was increased (Fig. 3b-d), and reached to a level not visible in the difference map contoured at ±4.0σ at the flow rate of 9.8 μl/m (Fig. 3d), which is also well below the maximum noise level (Fig. 3f). This indicates that at the flow rate of 9.8 μl/m, no apparent light-minus-dark Fourier difference peak was observed. Therefore, we concluded that the flow rate of 9.8 μl/m gives rise to no light-contamination at the position of the XFEL shot. Therefore, we collected the light-illuminated dataset with a Δt of 10 ms after the flash illumination at this flow rate to analyze the PSII structure in the S2 state.
Structural determination of PSII in the S2 state
The Fobs (10 ms, light, 9.8 μl/m) minus Fobs (dark) isomorphous difference Fourier map showed a strong signal with a peak height above -11.3σ at the position of W665, suggesting that the microcrystals were successfully excited to progress to the S2 state (Fig. 3e, f). Many peaks can be seen in the Fourier difference map when we decrease the contour level below ±3σ; thus we consider that the average noise level is at around ±3σ, and peaks above ±3σ may represent real structural changes induced by one-flash illumination. Most of the peaks that are related to the light-induced structural changes were above ±4.5σ and distributed around the electron transfer chain (the OEC, bicarbonate (BCT), non-heme iron, and QB), and were observed similarly in both PSII monomers. However, some weaker peaks at around ±4σ were found at one side of the monomer-monomer interface (Fig. 3g). These weaker peaks may not be related with the light-induced structural changes, and possibly induced by a relatively low isomorphism between the two datasets. These may arise from differences in the batches of samples used, since it was difficult to control the sample purification conditions and sample states, such as dehydration of the crystals, to an entirely uniform one. Nevertheless, we were able to distinguish the essential, light-induced structural changes during the catalytic reaction from the ones associated with the differences in the samples employed based on their peak intensities.
We refined the 10 ms light dataset as a mixture model consisting of 70% S2 state and 30% S1 state for the Mn4CaO5-cluster and the nearby residues (Method). This gives rise to equivalent values of the temperature factors between the two equivalent atoms in the multiple model, and this population of S2 and S1-states after one-flash illumination is similar to the efficiencies of the Si-state transition estimated by the Fourier transform infrared spectroscophy7, 20. The OEC structures in the S1 and S2 states determined in the present study are similar to those reported in the previous studies8,9. The OEC structure in the S2 state was in the open-cubane form, in which the right side of the O5 is open, giving rise to the five-coordinate trigonal bipyramidal coordination of Mn1. All changes in the Mn-Mn and Mn-Ca distances during the S1-to-S2 transition were less than the error range of the coordinates at the current resolution (Fig. 4). However, the changes found in the isomorphous difference Fourier map, such as the shorting of Mn3-Mn4 and elongations of Mn1-Mn3, Mn3-Ca, and Mn4-Ca, were consistent with the previous study with the diffraction data collected at 100 K for the room temperature-trapped S2-state8 (Fig. 4 and Table 2). In association with the movements of these manganese atoms, some ligand residues of the OEC (D1-E189, E333, D342, A344, and CP43-E354) also moved slightly (Fig. 5b).
Structural changes in the O4 and O1 channels
Among the four channels, the O4-channel has been suggested to function as the pathway of proton release during the S0–to-S1 transition based on theoretical calculations21. On the other hand, other groups argued that it serves as the source of substrate water by a “pivot” or “carousel” mechanism in the transition of S2-to-S322-24. As described above, upon transition to the S2 state, W665 in the O4-channel became highly disordered. This is accompanied by slight shifts of the nearby residues D1-D61 and CP43-E354 toward the position of W665, resulting in a narrowing of the space that has been occupied by W665 (Figs. 3e and 5b). A water cluster (W546, W548, W612, W606, and W806) leading to the lumenal surface in the O4-channel also shifted their positions during the S1-to-S2 transition, and the shift of W548 induced structural changes of its H-bond partners R334 and N335 (Fig. 5a). These changes in the O4-channel were similar to the previous study analyzed at the cryogenic temperature8. However, another water molecule, W757, connected to W548 and W606 in this channel, was found to become disordered in the S1-to-S2 transition at the cryogenic temperature8, but this water molecule was not detected in both the S1 and S2 structures in the present study, presumably due to its peripheral location and thus weaker association within the channel, resulting in a higher mobility at room temperature at which the TR-SFX experiments were conducted in the present study.
Another noticeable change observed near the OEC was a negative peak of -7.4σ covering W601, the H-bond donor to O1 and one of the members of a diamond-shaped water cluster in the O1-channel (Fig. 5b). This indicates that W601 became disordered in the S1-to-S2 transition. This change was not found in our previous study performed at the cryogenic temperature8. Another SFX study at ambient temperature by Kern et al. reported the shifts of three water molecules W601, W547, and W536 (W26, W27, and W30)9 of the diamond-shaped water cluster. In contrast, our previous fixed-target SFX study at cryogenic temperature reported W571, a water molecule found at a cryogenic temperature only, became disordered instead of W6018. These differences indicate a high mobility of the water molecules in the O1-channel during the S1-to-S2 transition. Such an increase of the mobility during the S1-to-S2 transition at room temperature may be necessary to allow incorporation of the substrate water molecule(s) into the OEC during the S2-to-S3 transition, which in turn suggests that the O1-channel may be responsible for the substrate water to enter into the reaction site.
QB and the non-heme iron site
After one flash illumination, the second bound quinone electron acceptor QB undergoes reduction and protonation to form a stable plastosemiquinone intermediate QBH•. The isomorphous difference Fourier map showed a positive peak covering the QB head and a pair of positive and negative peaks over the QB tail, suggesting its slight movement during the S1-to-S2 transition (Fig. 5c). The B-factor of the QB head was decreased from 121 Å2 in the S1-state to 103 Å2 in the S2-state, and the H-bond length between the head of QB and S264 was also changed from 2.92 Å in the S1-state to 2.77 Å in the S2-state, indicating a tighter binding of QBH• to S264. Thus, the first protonation of QB may occur at this site. This notion is consistent with the theoretical calculation that the first proton transfer from S264 to QB is an energetically downhill process, whereas it is an uphill one from H215 to QB25. Several pairs of positive and negative peaks were also found around the H-bond network formed by the bicarbonate (BCT), D1-Y246, D1-E244, D2-K264, and D2-E242, and BCT moved 0.39 Å away from the non-heme iron (Fig. 5c). These changes may be related to the reduction of the non-heme iron or proton uptake after one flash illumination, which in turn suggests that these residues form part of the proton inlet channel for the protonation of QB̶.