The Blinking of Small-Angle X-ray Scattering Reveals the Degradation Process of Protein Crystals at Microsecond Timescale.

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.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

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.

Estimation of the relative contributions to the electronic energy transfer rates based on Förster theory: The case of C-phycocyanin chromophores.

In the present study, we provide a reformulation of the theory originally proposed by Förster which allows for simple and convenient formulas useful to estimate the relative contributions of transition dipole moments of a donor and acceptor (chemical factors), their orientation factors (intermolecular structural factors), intermolecular center-to-center distances (intermolecular structural factors), spectral overlaps of absorption and emission spectra (photophysical factors), and refractive index (material factor) to the excitation energy transfer (EET) rate constant. To benchmark their validity, we focused on the EET occurring in C-phycocyanin (C-PC) chromophores. To this aim, we resorted to quantum chemistry calculations to get optimized molecular structures of the C-PC chromophores within the density functional theory (DFT) framework. The absorption and emission spectra, as well as transition dipole moments, were computed by using the time-dependent DFT (TDDFT). Our method was applied to several types of C-PCs showing that the EET rates are determined by an interplay of their specific physical, chemical, and geometrical features. These results show that our formulas can become a useful tool for a reliable estimation of the relative contributions of the factors regulating the EET transfer rate.

Dynamic interactions in the l-lactate oxidase active site facilitate substrate binding at pH4.5.

The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

Capturing structural changes of the S1 to S2 transition of photosystem II using time-resolved serial femtosecond crystallography.

Photosystem II (PSII) catalyzes light-induced water oxidation through an S i -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.

Formation of the High-Spin S2 State Related to the Extrinsic Proteins in the Oxygen Evolving Complex of Photosystem II.

The high-spin S2 state was investigated with photosystem II (PSII) from spinach, Thermosynechococcus vulcanus, and Cyanidioschyzon merolae. In extrinsic protein-depleted PSII, high-spin electron paramagnetic resonance (EPR) signals were not detected in either species, whereas all species showed g ∼ 5 signals in the presence of a high concentration of Ca2+ instead of the multiline signal. In the intact and PsbP/Q-depleted PSII from spinach, the g = 4.1 EPR signal was detected. These results show that formation of the high-spin S2 state of the manganese cluster is regulated by the extrinsic proteins through a charge located near the Mn4 atom in the Mn4CaO5 cluster but is independent of the intrinsic proteins. The shift to the g ∼ 5 state is caused by tilting of the z-axis in the Mn4 coordinates through hydrogen bonds or external divalent cations. The structural modification may allow insertion of an oxygen atom during the S2-to-S3 transition.

An oxyl/oxo mechanism for oxygen-oxygen coupling in PSII revealed by an x-ray free-electron laser.

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 versatile experimental system for tracking ultrafast chemical reactions with X-ray free-electron lasers.

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.

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties.

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = -440 mV, is lower than that of Fd1, Em = -372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Šresolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV-Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.

ß-Carotene Probes the Energy Transfer Pathway in the Photosystem II Core Complex.

The dynamics of the intact photosystem II core complex (PSII-CC) has been investigated extensively to elucidate its excellent photofunction. However, it is significantly difficult to observe the primary photosynthetic processes in PSII-CC because a vast number of chlorophylls (Chl) in the core complex show similar spectral features. In the present work, the dynamics of the energy transfer (ET) from ß-carotene (Bcr) in intact PSII-CC followed by charge separation (CS) at the reaction center (RC) with different excitation wavelengths were compared. Upon excitation at 510 nm, which selectively excites Bcr (Bcr651) inside of the D1-D2 RC, the pheophytin anion absorption band appeared within 9.6 ps. On the other hand, upon excitation at 490 nm, mainly exciting unspecified Bcr in the antenna complex, the anion band appeared after 20 ps. These excitation wavelength dependence experiments revealed a new ET pathway of PSII-CC, which indicates that the initial CS of PSII-CC is limited by ET to the RC.

Structural basis for blue-green light harvesting and energy dissipation in diatoms.

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.

Thylakoid membrane lipid sulfoquinovosyl-diacylglycerol (SQDG) is required for full functioning of photosystem II in Thermosynechococcus elongatus.

Sulfoquinovosyl-diacylglycerol (SQDG) is one of the four lipids present in the thylakoid membranes. Depletion of SQDG causes different degrees of effects on photosynthetic growth and activities in different organisms. Four SQDG molecules bind to each monomer of photosystem II (PSII), but their role in PSII function has not been characterized in detail, and no PSII structure without SQDG has been reported. We analyzed the activities of PSII from an SQDG-deficient mutant of the cyanobacterium Thermosynechococcus elongatus by various spectroscopic methods, which showed that depletion of SQDG partially impaired the PSII activity by impairing secondary quinone (QB) exchange at the acceptor site. We further solved the crystal structure of the PSII dimer from the SQDG deletion mutant at 2.1 Å resolution and found that all of the four SQDG-binding sites were occupied by other lipids, most likely PG molecules. Replacement of SQDG at a site near the head of QB provides a possible explanation for the QB impairment. The replacement of two SQDGs located at the monomer-monomer interface by other lipids decreased the stability of the PSII dimer, resulting in an increase in the amount of PSII monomer in the mutant. The present results thus suggest that although SQDG binding in all of the PSII-binding sites is necessary to fully maintain the activity and stability of PSII, replacement of SQDG by other lipids can partially compensate for their functions.

Fourier Transform Infrared Analysis of the S-State Cycle of Water Oxidation in the Microcrystals of Photosystem II.

Photosynthetic water oxidation is performed in photosystem II (PSII) through a light-driven cycle of intermediates called S states (S0-S4) at the water oxidizing center. Time-resolved serial femtosecond crystallography (SFX) has recently been applied to the microcrystals of PSII to obtain the structural information on these intermediates. However, it remains unanswered whether the reactions efficiently proceed throughout the S-state cycle retaining the native structures of the intermediates in PSII crystals. We investigated the water oxidation reactions in the PSII microcrystals using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. In comparison with the FTIR spectra in solution, it was shown that all of the metastable intermediates in the microcrystals retained their native structures, and the efficiencies of the S-state transitions remained relatively high, although those of the S2 → S3 and S3 → S0 transitions were slightly lowered possibly due to some restriction of water movement in the crystals.

Understanding Two Different Structures in the Dark Stable State of the Oxygen-Evolving Complex of Photosystem†II: Applicability of the Jahn-Teller Deformation Formula.

Tanaka et al. (J. Am. Chem. Soc., 2017, 139, 1718) recently reported the three-dimensional (3D) structure of the oxygen evolving complex (OEC) of photosystem II (PSII) by X-ray diffraction (XRD) using extremely low X-ray doses of 0.03 and 0.12 MGy. They observed two different 3D structures of the CaMn4O5 cluster with different hydrogen-bonding interactions in the S1 state of OEC keeping the surrounding polypeptide frameworks of PSII the same. Our Jahn-Teller (JT) deformation formula based on large-scale quantum mechanics/molecular mechanics (QM/MM) was applied for these low-dose XRD structures, elucidating important roles of JT effects of the MnIII ion for subtle geometric distortions of the CaMn4O5 cluster in OEC of PSII. The JT deformation formula revealed the similarity between the low-dose XRD and damage-free serial femtosecond X-ray diffraction (SFX) structures of the CaMn4O5 cluster in the dark stable state. The extremely low-dose XRD structures were not damaged by X-ray irradiation. Implications of the present results are discussed in relation to recent SFX results and a blue print for the design of artificial photocatalysts for water oxidation.

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

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). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' 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 QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/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 Mn4CaO5 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 µ4-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.

Dynamics of Excitation Energy Transfer Between the Subunits of Photosystem II Dimer.

Energy transfer dynamics in monomer and dimer of the photosystem II core complex (PSII-CC) was investigated by means of femtosecond transient absorption (TA) spectroscopy. There is no profound difference between the TA dynamics of the monomer and the dimer in the weak excitation intensity condition (≤21 nJ). However, the fast recovery of the ground state bleach was pronounced at higher excitation intensities, and the excitation intensity dependence of the dimer was more significant than that of the monomer. This result indicates efficient exciton-exciton annihilation taking place in the dimer due to energy transfer between the two monomer units. The annihilation dynamics was reproduced by a simple model based on binomial theorem, which indicated that although PSII-CC dimer has two reaction centers, only one charge-separated state remained after annihilation.

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga.

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.

Evidence for an unprecedented histidine hydroxyl modification on D2-His336 in Photosystem II of Thermosynechoccocus vulcanus and Thermosynechoccocus elongatus.

The electron density map of the 3D crystal of Photosystem II from Thermosynechococcus vulcanus with a 1.9 Šresolution (PDB: 3ARC ) exhibits, in the two monomers in the asymmetric unit cell, an, until now, unidentified and uninterpreted strong difference in electron density centered at a distance of around 1.5 Šfrom the nitrogen Nδ of the imidazole ring of D2-His336. By MALDI-TOF/MS upon tryptic digestion, it is shown that ~20-30% of the fragments containing the D2-His336 residue of Photosystem II from both Thermosynechococcus vulcanus and Thermosynechococcus elongatus bear an extra mass of +16 Da. Such an extra mass likely corresponds to an unprecedented post-translational or chemical hydroxyl modification of histidine.

Structure of Sr-substituted photosystem II at 2.1 A resolution and its implications in the mechanism of water oxidation.

Oxygen-evolving complex of photosystem II (PSII) is a tetra-manganese calcium penta-oxygenic cluster (Mn4CaO5) 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 Mn4CaO5-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.

Deformation of chlorin rings in the Photosystem II crystal structure.

The crystal structure of Photosystem II (PSII) analyzed at a resolution of 1.9 Å revealed deformations of chlorin rings in the chlorophylls for the first time. We investigated the degrees of chlorin ring deformation and factors that contributed to them in the PSII crystal structure, using a normal-coordinate structural decomposition procedure. The out-of-plane distortion of the P(D1) chlorin ring can be described predominantly by a large "doming mode" arising from the axial ligand, D1-His198, as well as the chlorophyll side chains and PSII protein environment. In contrast, the deformation of P(D2) was caused by a "saddling mode" arising from the D2-Trp191 ring and the doming mode arising from D2-His197. Large ruffling modes, which were reported to lower the redox potential in heme proteins, were observed in P(D1) and Chl(D1), but not in P(D2) and Chl(D2). Furthermore, as P(D1) possessed the largest doming mode among the reaction center chlorophylls, the corresponding bacteriochlorophyll P(L) possessed the largest doming mode in bacterial photosynthetic reaction centers. However, the majority of the redox potential shift in the protein environment was determined by the electrostatic environment. The difference in the chlorin ring deformation appears to directly refer to the difference in "the local steric protein environment" rather than the redox potential value in PSII.