Capturing structural intermediates in an animal-like cryptochrome photoreceptor by time-resolved crystallography.

Animal-like cryptochromes are photoreceptors that control circadian rhythm and signaling in many eukaryotes. Transient photoreduction of the cryptochrome flavin chromophore initiated signaling via a poorly understood mechanism. By serial femtosecond crystallography (SFX), we show that the photoreduction mechanism of cryptochrome involves three loci [carboxyl-terminal region, a transient protonation pathway, and flavin adenine dinucleotide (FAD)-binding site] acting in unison to accomplish three effects: radical pair stabilization, protonation of FAD radical, and formation of the signaling state.

Crystallization and initial X-ray crystallographic analysis of a de novo-designed protein with left-handed βαβ units.

A newly designed protein featuring a rare left-handed βαβ motif has successfully been crystallized and characterized by preliminary X-ray diffraction. The computational design was conducted using a combination of Rosetta BluePrintBDR, ProteinMPNN and AlphaFold2, generating eight candidates based on predicted stability and folding accuracy. The final construct was expressed, purified and crystallized in space group P2.

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.

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.

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.

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.

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

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

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

The high-spin S state was investigated with photosystem II (PSII) from spinach, , and . In extrinsic protein-depleted PSII, high-spin electron paramagnetic resonance (EPR) signals were not detected in either species, whereas all species showed ∼ 5 signals in the presence of a high concentration of Ca instead of the multiline signal. In the intact and PsbP/Q-depleted PSII from spinach, the = 4.

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 MnCaO cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S to S). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S, S, and S states by x-ray free-electron laser serial crystallography. No insertion of water was found in S, but flipping of D1 Glu upon transition to S leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane MnCaO cluster with an oxyl/oxo bridge.

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.

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.

β-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 β-carotene (Bcr) in intact PSII-CC followed by charge separation (CS) at the reaction center (RC) with different excitation wavelengths were compared.

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

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.

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 (S-S) 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.

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. (., , , 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.

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.

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.

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 Å resolution (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 Å from the nitrogen Nδ of the imidazole ring of D2-His336.

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.

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.

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.

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.

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.