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Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.

DOI： 10.1016/j.bbabio.2021.148471

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Union of Crystallography (IUCr)  

Photosystem II (PSII) catalyzes light-induced water oxidation through an S<italic>
_i
</italic>-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.

DOI： 10.1107/s2052252521002177

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Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

DOI： 10.1038/s42003-021-01919-3

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Iron-stress induced protein A (IsiA) is a chlorophyll-binding membrane-spanning protein in photosynthetic prokaryote cyanobacteria, and is associated with photosystem I (PSI) trimer cores, but its structural and functional significance in light harvesting remains unclear. Here we report a 2.7-Å resolution cryo-electron microscopic structure of a supercomplex between PSI core trimer and IsiA from a thermophilic cyanobacterium Thermosynechococcus vulcanus. The structure showed that 18 IsiA subunits form a closed ring surrounding a PSI trimer core. Detailed arrangement of pigments within the supercomplex, as well as molecular interactions between PSI and IsiA and among IsiAs, were resolved. Time-resolved fluorescence spectra of the PSI-IsiA supercomplex showed clear excitation-energy transfer from IsiA to PSI, strongly indicating that IsiA functions as an energy donor, but not an energy quencher, in the supercomplex. These structural and spectroscopic findings provide important insights into the excitation-energy-transfer and subunit assembly mechanisms in the PSI-IsiA supercomplex.

DOI： 10.1038/s42003-020-0949-6

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Microcrystals of photosystem II (PSII) have recently been used to investigate the intermediate structures of the water oxidizing complex during water oxidation by serial femtosecond crystallography using X-ray free electron lasers. To clarify the water oxidation mechanism, it is crucial to know whether the reaction proceeds properly in the microcrystals. In this work, we monitored the water oxidation reaction in a single PSII microcrystal using Fourier transform infrared (FTIR) microspectroscopy with the transmission method. Flash-induced micro-FTIR difference spectra of S-state transitions in a PSII microcrystal showed features virtually identical to the corresponding spectra previously obtained using the attenuated total reflection method for multiple microcrystals, representing the reactions near the crystal surface, as well as the spectra in solution. This observation indicates that the reaction processes of water oxidation proceed with relatively high efficiencies retaining native intermediate structures in the entire inside of a PSII microcrystal.

DOI： 10.1021/acs.jpcb.9b10154

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S-0 to S-4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S-1, S-2, and S-3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S-2, but flipping of D1 Glu(189) upon transition to S-3 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.

DOI： 10.1126/science.aax6998

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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.

DOI： 10.1074/jbc.RA118.004304

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

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.

DOI： 10.1021/acs.jpclett.8b00638

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn4CaO5. VTVH MCD spectra taken in the S2 state provide a clear g = 2, S = 1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749 nm, negative (−) at 773 nm and (+) at 808 nm are assigned as 4A → 2E spin-flips within the d3 configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S3 exhibits a more intense (−) MCD peak at 764 nm and has a stronger MCD saturation characteristic. This S3 MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S2 EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.

DOI： 10.1016/j.bbabio.2017.10.004

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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)(1-3). The structure of PSII has been analysed at 1.9 angstrom resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 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 angstrom using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 angstrom 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 angstrom 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 mu 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 angstrom between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously(6,7.)

DOI： 10.1038/nature21400

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Proton matrix ENDOR spectra were measured for Ca2+-depleted and Sr2+-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Theymosynechococcus vulcanus in the S-2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca2+-containing and Sr2+-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca2+-containing and Sr2+-substituted are similar in the S-2 state, in agreement with the similarity of the crystal structure of both Ca2+-containing and Sr2+-substituted PSII in the Si state. Nevertheless, slightly different hyperfine separations were found between Ca2+-containing and Sr2+-substituted PSII because of modifications of the water protons ligating to the Sr2+ ion. Importantly, Ca2+ depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca2+ and Y, directly. With respect to the crystal structure and the functions of Ca2+ in oxygen evolution, it was concluded that the roles of Ca2+ and Sr2+ involve the maintenance of the hydrogen bond network near the Ca2+ site and electron transfer pathway to the manganese cluster.

DOI： 10.1074/jbc.M115.675496

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DOI： 10.1016/j.bbabio.2014.11.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 angstrom resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well defined protein environment(1). However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation(2), and slight differences were found in the Mn-Mn distances determined by XRD1, EXAFS(3-7) and theoretical studies(8-14). Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanus in the S-1 state at a resolution of 1.95 angstroms using femtosecond X-ray pulses of the SPring-8 angstrom compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn-Mn distances that are shorter by 0.1-0.2 angstroms. The valences of each manganese atom were tentatively assigned as Mn1D(III), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn-ligand distances and analysis of the Jahn-Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation.

DOI： 10.1038/nature13991

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Visible/UV absorption in PS II core complexes is dominated by the chl-a absorptions, which extend to ~ 700 nm. A broad 700-730 nm PS II core complex absorption in spinach has been assigned [1] to a charge transfer excitation between ChlD1 and ChlD2. Emission from this state, which peaks at 780 nm, has been seen [2] for both plant and cyanobacterial samples. We show that Thermosynechococcus vulcanus PS II core complexes have parallel absorbance in the 700-730 nm region and similar photochemical behaviour to that seen in spinach. This establishes the low energy charge transfer state as intrinsic to the native PS II reaction centre. High-sensitivity MCD measurements made in the 700-1700 nm region reveal additional electronic excitations at ~ 770 nm and ~ 1550 nm. The temperature and field dependence of MCD spectra establish that the system peaking near 1550 nm is a heme-to-Fe(III) charge transfer excitation. These transitions have not previously been observed for cyt b559 or cyt c550. The distinctive characteristics of the MCD signals seen at 770 nm allow us to assign absorption in this region to a dz2 → dx2 - y2 transition of Mn(III) in the Ca-Mn4O5 cluster of the oxygen evolving centre. Current measurements were performed in the S1 state. Detailed analyses of this spectral region, especially in higher S states, promise to provide a new window on models of water oxidation.

DOI： 10.1016/j.bbabio.2014.11.003

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