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Photosynthetic organisms must balance maximizing productive light absorption and protecting themselves from too much light, which causes damage. Both tasks require pigments—chlorophylls and carotenoids—which absorb light energy and either transfer it to photosystems or disperse it as heat. Wang et al. determined the structure of a fucoxanthin chlorophyll a/c–binding protein (FCP) from a diatom. The structure reveals the arrangement of the specialized photosynthetic pigments in this light-harvesting protein. Fucoxanthin and chlorophyll c absorb the blue-green light that penetrates to deeper water and is not absorbed well by chlorophylls a or b. FCPs are related to the light-harvesting complexes of plants but have more binding sites for carotenoids and fewer for chlorophylls, which may help transfer and disperse light energy. Science , this issue p. eaav0365 Specialized pigments held together by a protein scaffold help diatoms harvest a broad spectrum of light. Diatoms are abundant photosynthetic organisms in aquatic environments and contribute 40% of its primary productivity. An important factor that contributes to the success of diatoms is their fucoxanthin chlorophyll a/c-binding proteins (FCPs), which have exceptional light-harvesting and photoprotection capabilities. Here, we report the crystal structure of an FCP from the marine diatom Phaeodactylum tricornutum , which reveals the binding of seven chlorophylls (Chls) a, two Chls c, seven fucoxanthins (Fxs), and probably one diadinoxanthin within the protein scaffold. Efficient energy transfer pathways can be found between Chl a and c, and each Fx is surrounded by Chls, enabling the energy transfer and quenching via Fx highly efficient. The structure provides a basis for elucidating the mechanisms of blue-green light harvesting, energy transfer, and dissipation in diatoms.

Oxygen-evolving complex of photosystem II (PSII) is a tetra-manganese calcium penta-oxygenic cluster (Mn ₄CaO ₅) catalyzing light-induced water oxidation through several intermediate states (S-states) by a mechanism that is not fully understood. To elucidate the roles of Ca ²⁺ in this cluster and the possible location of water substrates in this process, we crystallized Sr ²⁺-substituted PSII from Thermosynechococcus vulcanus , analyzed its crystal structure at a resolution of 2.1 Å, and compared it with the 1.9 Å structure of native PSII. Our analysis showed that the position of Sr was moved toward the outside of the cubane structure of the Mn ₄CaO ₅-cluster relative to that of Ca ²⁺, resulting in a general elongation of the bond distances between Sr and its surrounding atoms compared with the corresponding distances in the Ca-containing cluster. In particular, we identified an apparent elongation in the bond distance between Sr and one of the two terminal water ligands of Ca ²⁺, W3, whereas that of the Sr-W4 distance was not much changed. This result may contribute to the decrease of oxygen evolution upon Sr ²⁺-substitution, and suggests a weak binding and rather mobile nature of this particular water molecule (W3), which in turn implies the possible involvement of this water molecule as a substrate in the O-O bond formation. In addition, the PsbY subunit, which was absent in the 1.9 Å structure of native PSII, was found in the Sr-PSII structure.

The chloride ion, Cl⁻, is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn₄Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl⁻ with a bromide ion (Br⁻) or an iodide ion (I⁻) in PSII and analyzed the crystal structures of PSII with Br⁻ and I⁻ substitutions. Substitution of Cl⁻ with Br⁻ did not inhibit oxygen evolution, whereas substitution of Cl⁻ with I⁻ completely inhibited oxygen evolution, indicating the efficient replacement of Cl⁻ by I⁻. PSII with Br⁻ and I⁻ substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of the Mn₄Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn₄Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl⁻ anions are required to maintain the coordination structure of the Mn₄Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.

X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal’s domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.

X‐ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation‐sensitive proteins. Here, the development of serial X‐ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X‐ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X‐ray‐induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real‐space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter–scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence‐selective XFH or time‐resolved XFH. In this work, serial X‐ray fluorescence holography is developed and its capabilities are demonstrated by obtaining hologram patterns from the protein crystal Photosystem II before any signs of radiation‐induced damage. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structure of their functional metal clusters.

An experimental system, SPINETT (SACLA Pump-probe INstrumEnt for Tracking Transient dynamics), dedicated for ultrafast pump-probe experiments using X-ray free-electron lasers has been developed. SPINETT consists of a chamber operated under 1 atm helium pressure, two Von Hamos spectrometers, and a large two-dimensional detector having a short work distance. This platform covers complementary X-ray techniques; one can perform time-resolved X-ray absorption spectroscopy, time-resolved X-ray emission spectroscopy, and time-resolved X-ray diffuse scattering. Two types of liquid injectors have been prepared for low-viscosity chemical solutions and for protein microcrystals embedded in a matrix. We performed a test experiment at SPring-8 Angstrom Compact free-electron LAser and demonstrated the capability of SPINETT to obtain the local electronic structure and geometrical information simultaneously.

Photosystem II (PSII) catalyzes light-induced water oxidation through an Si-state cycle, leading to the generation of di-oxygen, protons and electrons. Pump–probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

Photosynthetic water oxidation is catalyzed by the Mn₄CaO₅ cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S₀ to S₄). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S₁, S₂, and S₃ states by x-ray free-electron laser serial crystallography. No insertion of water was found in S₂, but flipping of D1 Glu189 upon transition to S₃ leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn₄CaO₆ cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.

A new approach, time-resolved serial femtosecond crystallography, is used to view the intermediate states of a photosystem complex following illumination, shedding light on proton transfer and O=O bond formation. Bond formation in photosystem II Technical developments, such as X-ray free electron lasers (XFEL), allow for a more detailed view of the structure of the photosystem complexes, making it possible to get a glimpse of the mechanisms of proton transfer and bond formation. Jian-Ren Shen and colleagues use a new approach, time-resolved serial femtosecond crystallography, with X-ray free electron lasers to view the intermediate states formed after two-flash illumination. Upon illumination, the authors see that the disappearance of one water molecule relocates another water molecule towards an oxygen atom, in a manner that may reflect proton transfer. They also gain evidence for the inclusion of a new oxygen atom that would be positioned to form an O=O bond that has been hypothesized but never previously detected. These insights increase our understanding of the mechanism of water oxidation in photosystem II. Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC) 1 , 2 , 3 . The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn 4 CaO 5 cluster organized in an asymmetric, ‘distorted-chair’ form 4 . This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’ 5 structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the Q B /non-haem iron and the Mn 4 CaO 5 cluster. The changes around the Q B /non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn 4 CaO 5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ 4 -oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4 , 5 ). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously 6 , 7 .

Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 angstrom. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 angstrom. From our electron density map, we located all of the metal atoms of the Mn(4)CaO(5) cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn(4)CaO(5) cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.