担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Three psbA genes (psbA1, psbA2, and psbA3) encoding the D1 subunit of photosystem II (PSII) are present in the thermophilic cyanobacterium Thermosynechococcus elongatus and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, leading to the narrowing of the channel, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, hence an enhancement of oxygen evolution in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.

DOI： 10.1016/j.jbc.2022.102668

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：eLife Sciences Publications, Ltd  

Photosystem I (PSI) is a multi-subunit pigment-protein complex that functions in light-harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO₂ fixation in photosynthetic organisms. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the sites of low-energy Chls are still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primordial cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure shows the absence of some subunits commonly found in other cyanobacteria, confirming the primordial nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 is missing in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. These findings provide insights into not only the identity of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls in oxyphototrophs.

DOI： 10.7554/eLife.73990

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その他リンク： https://cdn.elifesciences.org/articles/73990/elife-73990-v1.xml

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Heterocysts are formed in filamentous heterocystous cyanobacteria under nitrogen-starvation conditions, and possess a very low amount of photosystem II (PSII) complexes than vegetative cells. Molecular, morphological, and biochemical characterizations of heterocysts have been investigated; however, excitation-energy dynamics in heterocysts are still unknown. In this study, we examined excitation-energy-relaxation processes of pigment-protein complexes in heterocysts isolated from the cyanobacterium Anabaena sp. PCC 7120. Thylakoid membranes from the heterocysts showed no oxygen-evolving activity under our experimental conditions and no thermoluminescence-glow curve originating from charge recombination of S2QA-. Two dimensional blue-native/SDS-PAGE analysis exhibits tetrameric, dimeric, and monomeric PSI complexes but almost no dimeric and monomeric PSII complexes in the heterocyst thylakoids. The steady-state fluorescence spectrum of the heterocyst thylakoids at 77 K displays both characteristic PSI fluorescence and unusual PSII fluorescence different from the fluorescence of PSII dimer and monomer complexes. Time-resolved fluorescence (TRF) spectra at 77 K, followed by fluorescence decay-associated spectra, showed different PSII and PSI fluorescence bands between heterocysts and vegetative thylakoids. Based on these findings, we discuss the excitation-energy-transfer mechanism in the heterocysts.

DOI： 10.1016/j.bbabio.2021.148509

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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出版者・発行元：Cold Spring Harbor Laboratory  

<title>Abstract</title>Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO₂ fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium <italic>Gloeobacter violaceus</italic> PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the <italic>Gloeobacter</italic> PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in <italic>Gloeobacter</italic>. Since <italic>Gloeobacter</italic> is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.

DOI： 10.1101/2021.09.29.462462

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jpcb.9b10154

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1126/science.aax6998

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA118.004304

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/nature13991

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