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.

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.

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.

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.

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.

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.

Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å.

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 Å. 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 Å. 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.

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.

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.

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.

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.

Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by deletion mutagenesis and X-ray crystallography.

PsbM and PsbI are two low molecular weight subunits of photosystem II (PSII), with PsbM being located in the center, and PsbI in the periphery, of the PSII dimer. In order to study the functions of these two subunits from a structural point of view, we crystallized and analyzed the crystal structure of PSII dimers from two mutants lacking either PsbM or PsbI. Our results confirmed the location of these two subunits in the current crystal structure, as well as their absence in the respective mutants. The relative contents of PSII dimers were found to be decreased in both mutants, with a concomitant increase in the amount of PSII monomers, suggesting a destabilization of PSII dimers in both of the mutants. On the other hand, the accumulation level of the overall PSII complexes in the two mutants was similar to that in the wild-type strain. Treatment of purified PSII dimers with lauryldimethylamine N-oxide at an elevated temperature preferentially disintegrated the dimers from the PsbM deletion mutant into monomers and CP43-less monomers, whereas no significant degradation of the dimers was observed from the PsbI deletion mutant. These results indicate that although both PsbM and PsbI are required for the efficient formation and stability of PSII dimers in vivo, they have different roles, namely, PsbM is required directly for the formation of dimers and its absence led to the instability of the dimers accumulated. On the other hand, PsbI is required in the assembly process of PSII dimers in vivo; once the dimers are formed, PsbI was no longer required for its stability.

Location of chloride and its possible functions in oxygen-evolving photosystem II revealed by X-ray crystallography.

The chloride ion, Cl(-), is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn(4)Ca 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 Mn(4)Ca 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 Mn(4)Ca 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 Mn(4)Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.

S1-state model of the O2-evolving complex of photosystem II.

We introduce a quantum mechanics/molecular mechanics model of the oxygen-evolving complex of photosystem II in the S(1) Mn(4)(IV,III,IV,III) state, where Ca(2+) is bridged to manganese centers by the carboxylate moieties of D170 and A344 on the basis of the new X-ray diffraction (XRD) model recently reported at 1.9 Å resolution. The model is also consistent with high-resolution spectroscopic data, including polarized extended X-ray absorption fine structure data of oriented single crystals. Our results provide refined intermetallic distances within the Mn cluster and suggest that the XRD model most likely corresponds to a mixture of oxidation states, including species more reduced than those observed in the catalytic cycle of water splitting.

Structural-functional role of chloride in photosystem II.

Chloride binding in photosystem II (PSII) is essential for photosynthetic water oxidation. However, the functional roles of chloride and possible binding sites, during oxygen evolution, remain controversial. This paper examines the functions of chloride based on its binding site revealed in the X-ray crystal structure of PSII at 1.9 Å resolution. We find that chloride depletion induces formation of a salt bridge between D2-K317 and D1-D61 that could suppress the transfer of protons to the lumen.

Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium.

Ycf12 (Psb30) and PsbZ are two low molecular weight subunits of photosystem II (PSII), with one and two trans-membrane helices, respectively. In order to study the functions of these two subunits from a structural point of view, we constructed deletion mutants lacking either Ycf12 or PsbZ from Thermosynechococcus elongatus, and purified, crystallized and analyzed the structure of PSII dimer from the two mutants. Our results showed that Ycf12 is located in the periphery of PSII, close to PsbK, PsbZ and PsbJ, and corresponded to the unassigned helix X1 reported previously, in agreement with the recent structure at 2.9 A resolution (A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 A resolution: role of quinones, lipids, channels and chloride, Nat. Struct. Mol. Biol. 16 (2009) 334-342). On the other hand, crystals of PsbZ-deleted PSII showed a remarkably different unit cell constants from those of wild-type PSII, indicating a role of PsbZ in the interactions between PSII dimers within the crystal. This is the first example for a different arrangement of PSII dimers within the cyanobacterial PSII crystals. PSII dimers had a lower oxygen-evolving activity from both mutants than that from the wild type. In consistent with this, the relative content of PSII in the thylakoid membranes was lower in the two mutants than that in the wild type. These results suggested that deletion of both subunits affected the PSII activity, thereby destabilized PSII, leading to a decrease in the PSII content in vivo. While PsbZ was present in PSII purified from the Ycf12-deletion mutant, Ycf12 was present in crude PSII but absent in the finally purified PSII from the PsbZ-deletion mutant, indicating a preferential, stabilizing role of PsbZ for the binding of Ycf12 to PSII. These results were discussed in terms of the PSII crystal structure currently available.

Distribution of the cationic state over the chlorophyll pair of the photosystem II reaction center.

The reaction center chlorophylls a (Chla) of photosystem II (PSII) are composed of six Chla molecules including the special pair Chla P(D1)/P(D2) harbored by the D1/D2 heterodimer. They serve as the ultimate electron abstractors for water oxidation in the oxygen-evolving Mn(4)CaO(5) cluster. Using the PSII crystal structure analyzed at 1.9 Šresolution, the redox potentials of P(D1)/P(D2) for one-electron oxidation (E(m)) were calculated by considering all PSII subunits and the protonation pattern of all titratable residues. The E(m)(Chla) values were calculated to be 1015-1132 mV for P(D1) and 1141-1201 mV for P(D2), depending on the protonation state of the Mn(4)CaO(5) cluster. The results showed that E(m)(P(D1)) was lower than E(m)(P(D2)), favoring localization of the charge of the cationic state more on P(D1). The P(D1)(•+)/P(D2)(•+) charge ratio determined by the large-scale QM/MM calculations with the explicit PSII protein environment yielded a P(D1)(•+)/P(D2)(•+) ratio of ~80/~20, which was found to be due to the asymmetry in electrostatic characters of several conserved D1/D2 residue pairs that cause the E(m)(P(D1))/E(m)(P(D2)) difference, e.g., D1-Asn181/D2-Arg180, D1-Asn298/D2-Arg294, D1-Asp61/D2-His61, D1-Glu189/D2-Phe188, and D1-Asp170/D2-Phe169. The larger P(D1)(•+) population than P(D2)(•+) appears to be an inevitable fate of the intact PSII that possesses water oxidation activity.

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.

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.

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga.

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222(1) with unit cell constants of a=146.8 A, b=176.9 A, and c=353.7 A, and the other one had a space group of P2(1)2(1)2(1) with unit cell constants of a=209.2 A, b=237.5 A, and c=299.8 A. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 A resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 A resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.