Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1101/2022.05.12.491653

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Other Link： https://link.springer.com/article/10.1007/s11120-023-01049-3/fulltext.html

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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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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s11120-021-00880-w

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

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 [2Fe-2S] 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 angstrom resolution. 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 Cys(53)ValAsnCys(56) 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.

DOI： 10.1016/j.bbabio.2019.148084

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

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S2 LS and S2 HS is pH dependent, with a pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5 and pKa ≈ 7.5 (n ≈ 1) for Mn4SrO5. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of high pH, NH3-S2 LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3TyrZ [rad] state, consistent with EPR data showing minor or no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2 HS was capable of advancing to S3 at low temperature (198 K). This is an experimental demonstration that the S2 LS is formed first and advances to S3 via the S2 HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2 LS to S2 HS transition which allow the S2 HS to S3 transition to occur below 200 K. This work also provides a protocol for generating S3 in concentrated samples without the need for saturating flashes.

DOI： 10.1016/j.bbabio.2018.02.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Photosynthesis converts solar energy into chemical energy. Photosystem II (PSII) oxidizes water to produce oxygen, electrons and protons under solar light irradiation. This light-driven water oxidation initiates a series of reactions in photosynthesis. Basic photoelectrochemical studies on PSII are directed toward the enzymatic applications of PSII for sustainable production of electricity or solar fuels. To maximize the photoelectrochemical catalytic activity of PSII on electrode substrates, interfacial designs between PSII and electrode substrates are important. Herein, we report bio-inorganic photoanodes of PSII and ferricyanide-intercalated layered double hydroxide (LDH) for visible-light-driven water oxidation. PSII is simply drop-cast onto a ferricyanide-intercalated cobalt–aluminum LDH and then shows a turnover frequency of 0.5 ± 0.1 s−1 and a turnover number of 920 ± 40 for 1 h at pH 6.5 at +0.5 V vs. NHE under visible light irradiation. Photoelectrochemical experiments using a PSII inhibitor or a bio-engineered PSII suggest that interfacial electron transfer from the plastoquinone QA site of PSII to ferricyanide may play an important role in improving the photo-electrocatalytic activity and stability of PSII. Our studies will open up new possibilities in fundamental or advanced photoelectrochemical studies of PSII.

DOI： 10.1016/j.electacta.2018.01.133

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

We have demonstrated in vitro motility assay of microtubules on a kinesin-coated substrate by using adenosine triphosphate (ATP) generated from microsphere gels containing thylakoid membranes. Upon photoirradiation, the gels generated ATP, which was utilized for performing gliding motions of microtubules. This work may facilitate the development of a localized ATP generation system in the in vitro motility assay, which consequently would widen the applications of biomolecular motors in nanotechnology.

DOI： 10.1246/cl.160903

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc.  

Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/ Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E12, of Tll0287 was 255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E12 increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies.

DOI： 10.1074/jbc.M116.746263

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Two mutants, D1-H198Q and D1-H198A, have been previously constructed in Thermosynechococcus elongatus with the aim at modifying the redox potential of the P-680(center dot+)/P-680 couple by changing the axial ligand of P-D1, one the two chlorophylls of the Photosystem II primary electron donor [Sugiura et al., Biochim. Biophys. Acta 1777 (2008) 331-342]. However, after the publication of this work it was pointed out to us by Dr. Eberhard Schlodder (Technische Universitat Berlin) that in both mutants the pheophytin band shift which is observed upon the reduction of Q(A) was centered at 544 nm instead of 547 nm, clearly showing that the D1 protein corresponded to PsbA1 whereas the mutants were supposedly constructed in the psbA(3) gene so that the conclusions in our previous paper were wrong. O-2 evolving mutants have been therefore reconstructed and their analyze shows that they are now correct mutants which are suitable for further studies. Indeed, the D1-H198Q mutation downshifted by approximate to 3 nm the P-680(center dot+)/P-680 difference absorption spectrum in the Soret region and increased the redox potential of the P-680(center dot+)/P-680 couple and the D1-H198A mutation decreased the redox potential of the P-680(center dot+)/P-680 couple all these effects being comparable to those which were observed in Synechocystis sp. PCC 6803 [Diner et al., Biochemistry 40 (2001) 9265-9281 and Merry et al. Biochemistry 37 (1998) 17,439-17,447]. We apologize for having presented wrong data and wrong conclusions in our earlier publication. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2016.09.012

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DOI： 10.1016/j.bbabio.2015.03.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S-0 to S-4) before O-2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (Y-z). Cl is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S = 5/2) and low spin (S = 1/2) forms of the Systate. The S-3-state was also found to be heterogeneous with: i) a S = 3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2Yz)' split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S = 5/2) form of S-2 shows a marked heterogeneity with a g = 4.3 form generated at low temperature that converts to a relaxed form at g = 4.9 at higher temperatures. The high spin g = 4.9 form can then progress to the EPR detectable form of S-3 at temperatures as low as 180 K whereas the low spin (S = 1/2) S-2-state can only advance to the S-3 state at temperatures >= 235 K. Both of the two S-2 configurations and the two S-3 configurations are each shown to be in equilibrium at >= 235K but not at 198 K. Since both S-2 configurations are formed at 198 K, they likely arise from two specific populations of Si. The existence of heterogeneous populations in Si, Sy and S-3 states may be related to the structural flexibility associated with the positioning of the oxygen O-5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S-2-state(s) and the S-3-state(s).

DOI： 10.1016/j.bbabio.2015.03.006

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DOI： 10.1016/j.bbabio.2014.11.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Cytb(559) in Photosystem II is a heterodimeric b-type cytochrome. The subunits, PsbE and PsbF, consist each in a membrane alpha-helix. Roles for Cytb559 remain elusive. In Thermosynechococcus elongatus, taking advantage of the robustness of the PSII variant with PsbA3 as the D1 subunit (WT*3), 4 mutants were designed hoping to get mutants nevertheless the obligatory phototrophy of this cyanobacterium. In two of them, an axial histidine ligand of the haem-iron was substituted for either a methionine, PsbE/H23M, which could be potentially a ligand or for an alanine, PsbE/H23A, which cannot. In the other mutants, PsbE/Y19F and PsbE/T26P, the environment around PsbE/H23 was expected to be modified. From EPR, MALDI-TOF and O-2 evolution activity measurements, the following results were obtained: Whereas the PsbE/H23M and PsbE/H23A mutants assemble only an apo-Cytb(559) the steady-state level of active PSII was comparable to that in WT*3. The lack of the haem or, in PsbE/T26P, conversion of the high-potential into a lower potential form, slowed-down the recovery rate of the O-2 activity after high-light illumination but did not affect the photoinhibition rate. This resulted in the following order for the steady-state level of active PSII centers under high-light conditions: PsbE/H23M approximate to PsbE/H23A << PsbE/Y19F <= PsbE/T26P <= WT*3. These data show i) that the haem has no structural role provided that PsbE and PsbF are present, ii) a lack of correlation between the rate of photoinhibition and the E-m of the haem and iii) that the holo-Cytb(559) favors the recovery of a functional enzyme upon photoinhibition. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2014.11.009

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The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium The rmosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbAl and PsbA3, 31 between PsbAl and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr(z) were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino add sequences identified the residues Cys144 and Pro173 found in PsbAl and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr(z)/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the alpha-helix bearing Tyr(z) and between the two alpha-helices bearing Tyr(z) and its hydrogen-bonded partner, Dl/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X-and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr(z) by P-680(+center dot) was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn4CaO5 cluster in the S-2 and S-3 states could weaken the strength of the H-bond interaction between Tyr(z)(center dot) and Dl/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr(z). (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2014.08.008

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DOI： 10.1016/j.bbabio.2014.08.008

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Cyanobacteria have several psbA genes encoding PsbA, the D1 reaction center protein of the photosystem II (PSII) complex which bears, with PsbD, the D2 protein, most of the cofactors involved in electron-transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the three possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In this review, we briefly summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting PSII in T. elongatus.

DOI： 10.1007/s11164-014-1828-x

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DOI： 10.1016/j.bbabio.2013.12.011

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DOI： 10.1016/j.bbabio.2013.09.009

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S236_6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The electron density map of the 3D crystal of Photosystem II from Thermosynechococcus vulcanus with a 1.9 angstrom 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 angstrom from the nitrogen N delta of the imidazole ring of D2-His336. By MALDI-TOF/MS upon tryptic digestion, it is shown that similar to 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.

DOI： 10.1021/bi401213m

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PsbV2 is a c-type cytochrome present in a very low abundance in the thermophilic cyanobacterium Thermosynechococcus elongatus. We purified this cytochrome and solved its crystal structure at a resolution of 1.5 Å. The protein existed as a dimer in the crystal, and has an overall structure similar to other c-type cytochromes like Cytc6 and Cytc550, for example. However, the 5th and 6th heme iron axial ligands were found to be His51 and Cys101, respectively, in contrast to the more common bis-His or His/Met ligands found in most cytochromes. Although a few other c-type cytochromes were suggested to have this axial coordination, this is the first crystal structure reported for a c-type heme with this unusual His/Cys axial coordination. Previous spectroscopic characterizations of PsbV2 are discussed in relation to its structural properties. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2013.08.023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

PsbV2 is a c-type cytochrome present in a very low abundance in the thermophilic cyanobacterium Thermosynechococcus elongatus. We purified this cytochrome and solved its crystal structure at a resolution of 1.5 angstrom. The protein existed as a dimer in the crystal, and has an overall structure similar to other c-type cytochromes like Cytc(6) and Cytc(550), for example. However, the 5th and 6th heme iron axial ligands were found to be His51 and Cys101, respectively, in contrast to the more common bis-His or His/Met ligands found in most cytochromes. Although a few other c-type cytochromes were suggested to have this axial coordination, this is the first crystal structure reported for a c-type heme with this unusual His/Cys axial coordination. Previous spectroscopic characterizations of PsbV2 are discussed in relation to its structural properties. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2013.08.023

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DOI： 10.1016/j.bbabio.2013.06.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Charge recombination in the light-induced radical pair S(n)Tyr(Z)(center dot)Q(A)(-center dot) in Photosystem II (PSII) from Thermosynechococcus elongatus has been studied at cryogenic temperatures by time-resolved EPR for different configurations of PSII that are expected to affect the driving force of the reaction (oxidation states S-0, S-1 or S-2 of the Mn4CaO5 cluster; PsbA1, PsbA2, or PsbA3 as D1 protein). The kinetics were independent of temperature in the studied range from 4.2 to 50 K and were not affected by exchange of H2O for D2O, consistent with single-step electron tunneling over the distance of similar to 32 angstrom without any repopulation through Boltzmann equilibration of intermediates lying higher in energy. In PsbA1-PSII, the charge recombinations in the radical pairs S(n)Tyr(Z)(center dot)Q(A)(-center dot) (k(et) = 3.4 x 10(-3) s(-1) for S-1) were slower than in PsbA3-PSII despite an expected lower driving force owing to a downshifted E-m(Q(A)/Q(A)(-center dot)) in PsbA1-PSII. Conversely, the reaction was slower in the presence of S-2 than in the presence of SI, despite an expected larger driving force due to an upshifted E-m(Tyr(Z)(center dot)/Tyr(Z)) in S-2. These observations indicate that the charge recombination occurs in the Marcus inverted region. Assuming that the driving force of the reaction (-Delta G(0) approximate to 1.2 eV at room temperature for S-1) does not vary strongly with temperature, the data indicate an optimal electron transfer rate (for a hypothetical -Delta G(0) = lambda) substantially faster than would be predicted from extrapolation of room temperature intraprotein ET rates over shorter distances. Possible origins of this deviation are discussed, including a possible enhancement of the electronic coupling of Tyr(Z)(center dot) and Q(A)(-center dot). by aromatic cofactors located in between. Observed similar S(1)Tyrz(center dot)Q(A)(-center dot) charge recombinations in PsbA2-PSII and PsbA3-PSII predict that E-m(Q(A)/Q(A)(-center dot)) in PsbA2-PSII is similar to that in PsbA3-PSII.

DOI： 10.1021/jp400337j

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S147_6

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DOI： 10.1016/j.bbabio.2012.06.006

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DOI： 10.1016/j.bbabio.2012.01.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Water oxidation by plants and cyanobacteria is performed via a light-driven cycle of five intermediates called S states (S-0-S-4) at the water oxidizing center (WOC) in photosystem II (PSII). The information about misses, i.e., the probabilities that the S-state transitions failed to advance, is crucial for detailed analysis of various spectroscopic data in investigations of the water oxidation mechanism. In this study, we have determined the miss probabilities of the individual S-state transitions using light induced Fourier transform infrared (FTIR) difference spectroscopy. The extent of S-state transitions in the WOC upon each saturating flash was monitored by detecting the flow of electrons from the WOC to ferricyanide, an exogenous electron acceptor, using the CN stretching bands of ferricyanide and ferrocyanide. Simulation of the oscillation pattern of the flash-number dependence of the signal amplitude provided the miss probabilities for the S-0 -> S-1, S-1 -> S-2, S-2 -> S-3, and S-3 -> S-0 transitions (alpha(0)-alpha(3), respectively) without any assumption about fitting parameters. The results for PSII preparations from Thermosynechococcus elongatus and spinach showed a general tendency of misses in the order, alpha(0) <= alpha(1) < alpha(2) < alpha(3), indicating that a more oxidized WOC has a higher miss probability. A very similar result observed for the Y-D-less mutant (D2-Y160F) of T. elongatus confirmed that Y-D does not affect the estimated misses. It was further shown that NO3- treatment specifically increased alpha(3), consistent with inactivation of the S-3 state reported previously. These results demonstrate the usefulness of this FTIR method for estimating individual miss probabilities in the S-state cycle in elucidation of the molecular mechanism of photosynthetic water oxidation.

DOI： 10.1021/bi300708a

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DOI： 10.1016/j.bbabio.2012.02.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn4CaO5 cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S-i states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S-3 -> S-0 transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn4CaO5 cluster that leads to O-2 formation. Early proton movement was also detected during the S-2 -> S-3 transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn4CaO5 cluster in the S-2 and S-3 states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S-0 -> S-1 transition, which could contribute to the stabilization of the S-1 state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.

DOI： 10.1021/bi300294n

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

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr(Z)(center dot) in the (S(3)Tyr(Z)(center dot))' is slightly modified; (ii) the split EPR signal arising from Tyr(Z)(center dot) in the (S(2)Tyr(Z)(center dot))' state induced by near-infrared illumination at 4.2 K of the S(3)Tyr(Z) state is significantly modified; and (iii) the slow phases of P-680(+). reduction by Tyr(Z) are slowed down from the hundreds of mu s time range to the ms time range, whereas both the S(1)Tyr(Z)(center dot) -> S(2)Tyr(Z) and the S(3)Tyr(Z)(center dot) -> 3 S(0)Tyr(Z) + O-2 transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr(Z) phenol and its environment, likely the Tyr-O center dot center dot center dot H center dot center dot center dot N epsilon-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr(Z) being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the alpha-helix bearing Tyr(Z) and between the two alpha-helices bearing Tyr(Z) and its hydrogen-bonded partner, His-190, are responsible for these changes.

DOI： 10.1074/jbc.M112.340323

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S63_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

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 Mn4CaO5 cluster. Using the PSII crystal structure analyzed at 1.9 angstrom resolution, the redox potentials of P-D1/center dot P-D2 for one-electron oxidation (E-m) were calculated by considering all PSII subunits and the protonation pattern of all titratable residues. The Em(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 Mn4CaO5 cluster. The results showed that Em(P-D1) was lower than E-m(P-D2) P-D2 favoring localization of the charge of the cationic state more on P-D1. The P-D1(center dot+)/P-D2(center dot+) charge ratio determined by the large-scale QM/MM calculations with the explicit PS II protein environment yielded a P-D1(center dot+)/P-D2(center dot+) ratio of similar to 80/similar to 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., Dl -Asn181/D2-Argl 80, D1-Asn298/D2-Arg294, D1-Asp61/D2-His61, D1-Glu189/D2-Phe188, and D1-Asp170/D2-Phe169. The larger P-D1(center dot+) population than P-D2(center dot+) appears to be an inevitable fate of the intact PSII that possesses water oxidation activity.

DOI： 10.1021/ja203947k

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DOI： 10.1021/bi200313p.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The role of Ca2+ in the Photosystem II water oxidation mechanism has been investigated in the higher redox states of the enzyme. The involvement of Ca2+ in structuring the environment of the tyrosine Y-Z in the S-3 state has been investigated by studying the effect of a Ca/Sr exchange by pulse-EPR spectroscopy. It is shown that Ca and Yz are involved in a common hydrogen bond network in S-3. The comparison of the temperature dependence of the rate of the water oxidation reaction shows that the slowdown of the S3YZ center dot -> S-0 transition in Sr-PSII mainly arises from a decrease of the entropic part of the Delta G(#) of the reaction. This suggests that Ca/Sr exchange perturbs the distribution of the conformational microstates and thereby hinders the overall water splitting process.

DOI： 10.1039/c1ee01408k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC APPLIED SPECTROSCOPY  

We have constructed a 1064 nm deep near-infrared (NIR) excited multichannel Raman microspectrometer using an InP/InGaAsP multichannel detector. This microspectrometer achieves high sensitivity suitable for in vivo measurements of single living cells with lateral resolution of 0.7 mu m and depth resolution of 3.1 mu m. It has been applied to the structural analysis of living cyanobacterial cells, well-known model organisms for photosynthesis research, which are too photolabile to be measured with visible laser excitation. High signal-to-noise ratio (SIN) Raman spectra have been obtained from carotenoid, chlorophyll a, and phycocyanin in a single living cyanobacterial cell with no appreciable interference from autofluorescence or photodamage. Sub-micrometer mapping of Raman intensities provides clear distribution images of the three pigments inside the cell.

DOI： 10.1366/10-06196

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The electronic structures of the native Mn4OxCa cluster and the biosynthetically substituted Mn4OxSr cluster of the oxygen evolving complex (OEC) of photosystem II (PSII) core complexes isolated from Thermosynechococcus elongatus, poised in the S-2 state, were studied by X- and Q-band CW-EPR and by pulsed Q-band Mn-55-ENDOR spectroscopy. Both wild type and tyrosine D less mutants grown photoautotrophically in either CaCl2 or SrCl2 containing media were measured. The obtained CW-EPR spectra of the S-2 state displayed the characteristic, clearly noticeable differences in the hyperfine pattern of the multiline EPR signal [Boussac et al. J. Biol. Chem. 2004, 279, 22809-22819]. In sharp contrast, the manganese (Mn-55) ENDOR spectra of the Ca and Sr forms of the OEC were remarkably similar. Multifrequency simulations of the X- and Q-band CW-EPR and Mn-55-pulsed ENDOR spectra using the Spin Hamiltonian formalism were performed to investigate this surprising result. It is shown that (i) all four manganese ions contribute to the Mn-55-ENDOR spectra; (ii) only small changes are seen in the fitted isotropic hyperfine values for the Ca2+ and Sr2+ containing OEC, suggesting that there is no change in the overall spin distribution (electronic coupling scheme) upon Ca2+/Sr2+ substitution; (iii) the changes in the CW-EPR hyperfine pattern can be explained by a small decrease in the anisotropy of at least two hyperfine tensors. It is proposed that modifications at the Ca2+ site may modulate the fine structure tensor of the Mn-III ion. DFT calculations support the above conclusions. Our data analysis also provides strong support for the notion that in the S-2 state the coordination of the Mn-III ion is square-pyramidal (5-coordinate) or octahedral (6-coordinate) with tetragonal elongation. In addition, it is shown that only one of the currently published OEC models, the Siegbahn structure [Siegbahn, P. E. M. Acc. Chem. Res. 2009, 42, 1871-1880, Pantazis, D. A. et al. Phys. Chem. Chem. Phys. 2009, 11, 6788-6798], is consistent with all data presented here. These results provide important information for the structure of the OEC and the water-splitting mechanism. In particular, the 5-coordinate Mn-III is a potential site for substrate 'water' (H2O, OH-) binding. Its location within the cuboidal structural unit, as opposed to the external 'dangler' position, may have important consequences for the mechanism of O-O bond formation.

DOI： 10.1021/ja110145v

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The main cofactors involved in Photosystem II (PSII) oxygen evolution activity are borne by two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is predominantly encoded by either the psbA(1) or the psbA(3) gene, the expression of which depends on the environmental conditions. In this work, the Q(B) site properties in PsbA1-PSII and PsbA3-PSII were probed through the binding properties of DCMU, a urea-type herbicide, and bromoxynil, a phenolic-type herbicide. This was done by using helium temperature EPR spectroscopy and by monitoring the time-resolved changes of the redox state of Q(A) by absorption spectroscopy in PSII purified from a His(6)-tagged WT strain expressing PsbA1 or from a His(6)-tagged strain in which both the psbA(1) and psbA(2) genes have been deleted and which therefore only express PsbA3. It is shown that, in both PsbA1-PSII and PsbA3-PSII, bromoxynil does not bind to PSII when Q(B) is in its semiquinone state which indicates a much lower affinity for PSII when Q(A) is in its semiquinone state than when it is in its oxidized state. This is consistent with the midpoint potential of Q(A)(center dot-)/Q(A) being more negative in the presence of bromoxynil than in its absence [Krieger-Liszkay and Rutherford, Biochemistry 37 (1998) 17339-17344]. The addition in the dark of DCMU, but not that of bromoxynil, to PSII with a secondary electron acceptor in the Q(B)(center dot-) state induces the oxidation of the non-heme iron in a fraction of PsbA3-PSII but not in PsbA1-PSII. These results are explained as follows: i) bromoxynil has a lower affinity for PSII with the non-heme iron oxidized than DCMU therefore, ii) the midpoint potential of the Fe(II)/Fe(III) couple is lower with DCMU bound than with bromoxynil bound in PsbA3-PSII; and iii) the midpoint potential of the Fe(II)/Fe(III) couple is higher in PsbA1-PSII than in PsbA3-PSII. The observation of DCMU-induced oxidation of the non-heme iron leads us to propose that Q(2), an electron acceptor identified by Joliot and Joliot [FEBS Lett 134 (1981) 155-158], is the non-heme iron. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2010.10.004

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S96_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S10_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S9_6

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

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA(1) or the psbA(3) gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491-1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b(559) (Cyt b(559)) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/Delta PsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b(559) properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/Delta PsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b(559) likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.

DOI： 10.1074/jbc.M110.136945

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA(3) gene compared to that deduced from the psbA(1) gene points a difference of 21 residues. In this work. PSII isolated from a wild type T. elongatus strain expressing PsbA(1) or from a strain in which both the psbA(1) and psbA(2) genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo(D1) is increased by 17 mV from -522 mV in PsbA1-PSII to -505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S(2)QA(-center dot). charge recombination and the C equivalent to N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q(B) pocket. The temperature dependences of the S(2)Q(A)(-center dot) charge recombination show that the strength of the H-bond to Pheo(D1) is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q(A) (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P-680(+center dot) and Y-z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P-680/P-680(+center dot) couple (and hence that of P-1(680)center dot/P-680(+)center dot) is tuned as well when shifting from PsbAl to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2010.03.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA(3) gene (WT*). The Delta Psb30 mutant appears more susceptible to photodamage, has a cytochrome b(559) that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the Delta Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b(559) into the low potential form. Structural reasons for such effects are discussed. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2010.03.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The primary electron acceptor pheophytin (PheO(D1)) plays a crucial role in regulation of forward and backward electron transfer in photosystem II (PSII). It is known that some cyanobacteria control the Pheo(D1) potential in high-light acclimation by exchanging the DI proteins from different copies of the psbA genes. To clarify file mechanism of the potential control of PheODh we studied the hydrogen bond interactions of Pheo(D1) in the neutral and anionic States using light-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra of Pheol:)l upon its photoreduction were obtained using three different PSII preparations, PSII core complexes from Thermosynehococcus elongatus possessing PsbA1 as a D1 subunit (PSII-PsbA1), those with PsbA3 (PSII-PsbA3), and PSII membranes from spinach. The DI-Gln130 side chain, which is hydrogen bonded to the 13(1)-keto C=O group of Pheo(D1) PSII membranes from spinach. The D1-Gln130 in PSII-PsbA3 and spinach PSIL The spectrum of PSII-PsbA1 exhibited 131-keto C=O bands at 1682 and 1605 cm(-1) in neutral Pheol), and its anion, respectively, while the corresponding bands were observed at frequencies lower by 1-3 and 18-19cm(-1), respectively, in the latter two preparations. This larger frequency shift in PhcoD(1)(-) than Plleol), by the change of the hydrogen bond donor Was well reproduced by density functional theory (DFT) calculations for the Pheo models hydrogen bonded with acetamide and acetic acid. The DFT calculations also exhibited a higher redox potential for Pheo reduction in the model with acetic acid than that with acetamide, consistent with previous observations for the D1-Gln130GIu mutant of Synechocystis. It is thus concluded that a stronger hydrogen bond effect oil the Pheo(-) anion than the neutral Pheo causes the shift in the redox potential, which IS utilized In the photoprotection mechanism of PSII.

DOI： 10.1021/bi9018829

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA(3) gene over the psbA(1) gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at <10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q(x) band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the PheO(D1) Q(x) band induced by Q(A)(-) (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light When green light was used there was little difference between the two variants. With far red light the stable (t(1/2)>50 ms) W yield was similar to 95% in D1:3, and similar to 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q(x) band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13(1)-keto of PheOD1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P(D1), in particular Ser153Ala. Photoinduced Q(A)(-) formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant A reduced efficiency for the oxidation of side-pathway donors in the D1:I variant could be explained by a variation in the location and/or redox potential of P(+). (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2009.07.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

By using a nondestructive, ultrasensitive, fluorescence kinetic technique, we measure in situ the photochemical energy conversion efficiency and electron transfer kinetics on the acceptor side of histidine-tagged photosystem II core complexes tethered to gold surfaces. Atomic force microscopy images coupled with Rutherford backscattering spectroscopy measurements further allow us to assess the quality, number of layers, and surface density of the reaction center films. Based on these measurements, we calculate that the theoretical photo-electronic current density available for an ideal monolayer of core complexes is 43 mu A cm(-2) at a photon flux density of 2000 mu mol quanta m(-2) s(-1) between 365 and 750 nm. While this current density is approximately two orders of magnitude lower than the best organic photovoltaic cells (for an equivalent area), it provides an indication for future improvement strategies. The efficiency could be improved by increasing the optical cross section, by tuning the electron transfer physics between the core complexes and the metal surface, and by developing a multilayer structure, thereby making biomimetic photoelectron devices for hydrogen generation and chemical sensing more viable.

DOI： 10.1002/cssc.200900255

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S67_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S68_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S134_1

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Language：English   Publishing type：Research paper (international conference proceedings)  

DOI： 10.1063/1.3482818

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The redox potential of the primary plastoquinone electron acceptor Q(A), E-m(Q(A)/Q(A)(-)), in an oxygen-evolging photosystem (PS) 11 complex from a thermophilic cyanobacterium Thermosynechococcus elongatus was determined to be - 140 +/- 2 mV vs. SHE by thin-layer cell spectroclectrochemistry for the first time. The E-m(Q(A)/Q(A)(-)) value obtained here together with the recently determined redox potential of pheophytin (Phe) a [Kato et at. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 17365-17370] yields -330 to -370 mV for the free energy change by electron transfer from Phe a(-) to Q(A) and provides a renewed picture for the energetics oil the electron acceptor side in PS II.

DOI： 10.1021/bi901691j

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Thin-layer cell spectroelectrochemistry, featuring rigorous potential control and rapid redox equilibration within the cell, was used to measure the redox potential E(m)(Phe a/Phe a(-)) of pheophytin (Phe) a, the primary electron acceptor in an oxygen-evolving photosystem (PS) II core complex from a thermophilic cyanobacterium Thermosynechococcus elongatus. Interferences from dissolved O(2) and water reductions were minimized by airtight sealing of the sample cell added with dithionite and mercury plating on the gold minigrid working electrode surface, respectively. The result obtained at a physiological pH of 6.5 was E(m)( Phe a/Phe a(-)) = -505 +/- 6 mV vs. SHE, which is by approximate to 100 mV more positive than the values measured approximate to 30 years ago at nonphysiological pH and widely accepted thereafter in the field of photosynthesis research. Using the P680* - Phe a free energy difference, as estimated from kinetic analyses by previous authors, the present result would locate the E(m)(P680/P680(+)) value, which is one of the key parameters but still resists direct measurements, at approximately +1,210 mV. In view of these pieces of information, a renewed diagram is proposed for the energetics in PS II.

DOI： 10.1073/pnas.0905388106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The non-heme iron is located between the quinone electron acceptors, Q(A) and Q(B), in photosystem II (PSII), and together with its bicarbonate ligand, it regulates the electron and proton transfer reactions of quinone acceptors. In this study, we have investigated the structural coupling of a nearby Tyr residue with the non-heme iron center using Fourier transform infrared (FTIR) spectroscopy. Light-induced FC(2+)/Fe(3+) FTIR difference spectra of PSII core complexes from unlabeled and [4-(13)C]Tyr-labeled Thermosynechococcus elongatus revealed that the CO stretching (nu CO) bands of a Tyr side chain are located at 1253 and 1241 cm(-1) in the Fe(2+) and Fe(3+) states, respectively. Upon deuteration, both nu CO bands were upshifted by 11-12 cm(-1). Taking into account the criteria for determining the hydrogen bond structure of a Tyr side chain from infrared bands reported previously [Takahashi, R., and Noguchi, T. (2007) J. Phys. Chem. B 111, 13833-13844] and the results of DFT calculations of model complexes of p-cresol hydrogen-bonded with bicarbonate, we interpreted the observed nu CO bands and their deuteration effects as indicating that one Tyr side chain with a hydrogen bond donor-acceptor form is strongly coupled to the non-heme iron. From the X-ray structures of PSII core complexes, it is proposed that either D1-Y246 or D2-Y244 provides a hydrogen bond to the oxygen of the bicarbonate ligand but the other Tyr does not directly interact with bicarbonate. The Tyr residue coupled to the non-heme iron may play a key role in the regulatory function of the iron-bicarbonate center by stabilizing the bicarbonate ligand and forming a rigid hydrogen bond network around the non-heme ion.

DOI： 10.1021/bi901195e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Oxygen evolution by Photosystem II (PSII) is catalyzed by a Mn4Ca cluster. Thus far, from the crystallographic three-dimensional (3D) structures, seven amino, acid residues have been identified as possible ligands of the Mn4Ca cluster. Among them, there is only one histidine, His332, which belongs to the D1 polypeptide. The relationships of the D1-His332 amino acid with kinetics and thermodynamic properties of the Mn4Ca cluster in the S-2- and S-3-states of the catalytic cycle were investigated in purified PSII from Thermosynechococcus elongatus. This was done by examining site-directed D1-His332Gln and D1-His332Ser mutants by a variety of spectroscopic techniques such as time-resolved UV-visible absorption change spectroscopy, cw- and pulse-EPR, thermoluminescence, and measurement of substrate water exchange. Both mutants grew photo-autotrophically and active PSII could be purified. On the basis of the parameters assessed in this work, the D1-His332(Gln, Ser) mutations had no effect in the S-2-state. Electron spin-echo envelope modulation (ESEEM) spectroscopy also showed that possible interactions between the nuclear spin of the nitrogen(s) of D1-His332 with the electronic spin S = 1/2 of the Mn4Ca cluster in the S-2-state were not detectable and that the D1-His332Ser mutation did not affect the detected hyperfine couplings. In contrast, the following changes were observed in the S-3-state of the D1-His332 mutants: (1) The redox potential of the S-3/S-2 couple was slightly increased by <= 20 meV, (2) The S-3-EPR spectrum was slightly modified, (3) The D1-His332Gln mutation resulted in it similar to 3 fold decrease of the slow (tightly bound) exchange rate and it similar to 2 fold increase of the fast exchange rate of the water substrate molecules. All these results suggest that the D1-His332 would be more involved in S-3 than in S-2. This could be one element of the conformational changes put forward in the S-2 to S-3 transition.

DOI： 10.1021/bi901067b

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In photosynthetic water oxidation performed in the water oxidizing center (WOC) of photosystem II (PSII), two water molecules are converted into one oxygen molecule and four protons through a light-driven cycle of intermediates called S states (S-0-S-4). To understand the molecular mechanism of water oxidation and the chemical nature of substrate intermediates, it is essential to determine the stoichiometry of proton release from substrate water at individual S-state transitions. In this study, we have monitored proton release during water oxidation by means of isotope-edited Fourier transform infrared (FTIR) spectroscopy. FTIR difference spectra upon successive flash illumination were measured using PSII core complexes from a thermophilic cyanobacterium Thermosynechococcus elongatus, which were suspended in a high concentration (200 mM) Mes buffer at pH 6.0. The spectra involved, in addition to protein bands, the bands of the Mes buffer that trapped virtually all protons from the WOC. Mes-only signals were extracted by subtracting the spectra measured in deuterated-Mes (Mes-d(12)). The flash-number dependence of the intensity increase of the isotope-edited Mes signal showed a clear period-four oscillation. By simulating the oscillation with different assumptions about miss factors, the proton release pattern was estimated to be 0.8-1.0:0.2-0.3:0.9-1.2:1.5-1.6 for the S-0 -> S-1 -> S-2 -> S-3 -> S-0 transitions. The effect of H/D exchange on the COOH region of proteins in FTIR difference spectra of the S-state cycle showed that protonation/deprotonation of carboxylic groups contributed little to the observed proton release pattern. Together with the present and previous FTIR results suggesting no involvement of also His and Cys side groups, it was concluded that proton release from substrate water takes place with a 1:01:2 stoichiometry, which is perturbed by partial protonation/deprotonation of side groups probably of Arg, Lys, or Tyr located nearby the WOC.

DOI： 10.1021/ja901696m

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The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biological systems remains limited, likely because its characterization relies on the controlled but experimentally challenging modifications of the free energy changes associated with either the electron or proton transfer. We have performed such a study here in Photosystem II. The driving force for electron transfer from Tyr(z) to P(680)(*+) has been decreased by similar to 80 meV by mutating the axial ligand of P(680), and that for proton transfer upon oxidation of Tyr(z) by substituting a 3-fluorotyrosine (3F-Tyr(z)) for Tyrz. In Mn-depleted Photosystem 11, the dependence upon pH of the oxidation rates of Tyrz and 3F-Tyrz were found to be similar. However, in the pH range where the phenolic hydroxyl of Tyrz is involved in a H-bond with a proton acceptor, the activation energy of the oxidation of 3F-Tyrz is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr (Seyedsayamdost et al. J. Am. Chem. Soc. 2006, 128,1569-1579). Thus, when the phenol of Y(z) acts as a H-bond donor, its oxidation by P680(*+) is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidation-induced proton transfer from the phenolic hydroxyl of Tyrz has been proposed to occur concertedly with the electron transfer to P680(*+). This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in determining the coupling between proton and electron transfer.

DOI： 10.1021/ja808604h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Despite crystallographic structures now available and intensive work in the past decades, little is known about the higher redox states of the catalytic cycle of Photosystem II, the enzyme responsible for the presence of O-2 on Earth and at the beginning of the process that has produced both the biomass and the fossil fuels. In one of the highest oxidation states, the S-3-state, only signals at S-values higher than 4 have been detected so far at the X-band. In this work, we report for the first time the complete X-band EPR spectrum for the S-3-state of Photosystem II. Simutations show that, for a spin state S = 1, as was previously suggested for S-3, it is not possible to account for all the features observed. A satisfactory simulated spectrum was obtained for a spin state S = 3 with zero-field splitting parameters D = 0.175 cm(-1) and E/D = 0.275. The detection of the full. EPR signal for S-3 opens the door for new investigations and a better understanding of the catalytic cycle of Photosystern II.

DOI： 10.1021/ja900680t

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S60_5

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S98_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Redox-active tyrosine (Tyr) D is indirectly involved in controlling the primary electron transfer in PSII The presence of the oxidized TyrD renders P680(+) more oxidizing by localizing the charge more on P(D1) and thus facilitates trapping of the excitation energy in PSII. We also conclude that the mechanism of the primary charge separation and stabilization is altered upon Q(A) reduction. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2008.09.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Ycf12 (Psb30) is a small hydrophobic subunit of photosystem II (PS II) complexes found in the cyanobacterium, Thermosynechococcus elongatus. However, earlier intense proteomic analysis on the PS II complexes from the cyanobacterium, Synechocystis 6803, could not detect Psb30. In this work, we generated a mutant of Synechocystis 6803 in which a hexa-histidine tag was fused to the C-terminus of Synechocystis Psb30. The mutant accumulated fully functional PS II complexes. Purification of Psb30 by metal affinity chromatography from thylakoid extracts resulted in co-purification of an oxygen-evolving PS II complex with normal subunit composition. This result indicates that Psb30 is expressed and stably associated with the PS II complex in Synechocystis. The histidine-tagged Psb30 in the purified PS II complex was not detected by staining or anti-polyhistidine antibodies. We also generated a mutant in which ycf12 was disrupted. The mutant grew photosynthetically and showed no significant phenotype under moderate growth conditions. Purified PS II complexes from the disruptant showed an oxygen-evolving activity comparable to wild type under low irradiance. However, it showed a remarkably lower activity than wild type under high irradiance. Thus Psb30 is required for the efficient function of PS II complexes, particularly under high irradiance conditions.

DOI： 10.1007/s11120-008-9340-z

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In order to investigate oxygen binding and hydrophobic cavities in photosystem II (PSII), we have introduced xenon under pressure into crystals of PSII isolated from Thermosynechococcus elongatus and used X-ray anomalous diffraction analyses to identify the xenon sites in the complex. Under the conditions employed, 25 Xe-binding sites were identified in each monomer of the dimeric PSII complex. The majority of these were distributed within the membrane spanning portion of the complex with no obvious correlation with the previously proposed oxygen channels. One binding site was located close to the haem of cytochrome b559 in a position analogous to a Xe-binding site of myoglobin. The only Xe-binding site not associated with the intrinsic subunits of PSII was within the hydrophobic core of the PsbO protein.

DOI： 10.1007/s11120-008-9366-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Photosynthetic water oxidation takes place in the water-oxidizing center (WOC) of photosystem II (PSII). To clarify the mechanism of water oxidation, detecting water molecules in the WOC and monitoring their reactions at the molecular level are essential. In this study, we have for the first time detected the DOD bending vibrations of functional D(2)O molecules during the S-state cycle of the WOC by means of Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra upon S-state transitions were measured using the PSII core complexes from Thermosynechococcus elongatus moderately deuterated with D(2)(16)O and D(2)(18)O. D(2)(16)O-minus-D(2)(18)O double difference spectra at individual S-state transitions exhibited six to eight peaks arising from the D(16)OD/D(18)OD bending vibrations in the 1250-1150 cm(-1) region. This observation indicates that at least two water molecules, not in any deprotonated forms, participate in the reaction at each S-state transition throughout the cycle. Most of the peaks exhibited clear counter peaks with opposite signs at different transitions, reflecting a series of reactions of water molecules at the catalytic site. In contrast, negative bands at similar to 1240 cm(-1) in the S(2) -> S(3), S(3) -> S(0), and possibly S(0) -> S(1) transitions, for which no clear counter peaks were found in other transitions, can be interpreted as insertion of substrate water into the WOC from a water cluster in the proteins. The characteristics of the weakly D-bonded OD stretching bands were consistent with the insertion of substrate from internal water molecules in the S(2) -> S(3) and S(3) -> S(0) transitions. The results of this study show that FTIR detection of the DOD bending vibrations is a powerful method for investigating the molecular mechanism of photosynthetic water oxidation as well as other enzymatic reactions involving functional water molecules.

DOI： 10.1021/bi801580e

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We monitored illuminated-minus-dark absorption difference spectra in the range of 450-1100 nm induced by continuous illumination at 8 K of photosystem II (PSII) core complexes from Thermosynechococcus elongatus. The photo-induced oxidation of the side-path donors Cytb(559), beta-carotene and chlorophyll Z, as well as the concomitant stable (t(1/2) > 1 s) reduction of the first plastoquinone electron acceptor, Q(A) (monitored by the well-known 'C550' shift), were quantified as a function of the absorbed photons per PSII. The Q(A) photo-induced reduction data can be described by three distinct quantum efficiency distributions: (i) a very high efficiency of similar to 0.5-1, (ii) a middle efficiency with a very large range of similar to 0.014-0.2, and (iii) a low efficiency of similar to 0.002. Each of the observed side-path donors exhibited similar quantum efficiency distributions, which supports a branched pathway model for side-path oxidation where beta-carotene is the immediate electron donor to the photo-oxidized chlorophyll (P680(+)). The yields of the observed side-path donors account quantitatively for the wide middle efficiency range of photo-induced Q(A) reduction, but not for the PSII fractions that exhibit the highest and lowest efficiencies. The high-efficiency component may be due to Tyr(Z) oxidation.

DOI： 10.1007/s11120-008-9330-1

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This report describes a protocol to incorporate isotopically labelled aromatic amino acids into the proteins of the thermophilic cyanobacterium Thermosynechoccus elongatus. By using the EPR signal of the two redox active tyrosines of Photosystem II, Tyr(D)(center dot) and Tyr(Z)(center dot), as spectroscopic probes it is shown that labelled tyrosines can be incorporated with a high yield in this cyanobacterium. The production of a fully (13)C- or (2)H-labelled enzyme is also described.

DOI： 10.1007/s11120-008-9305-2

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

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(center dot) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximate to 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.

DOI： 10.1074/jbc.M710583200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The influence of the histidine axial ligand to the P-D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P-680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA(1) and psbA(2) genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT*) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA(3) gene. The O-2-evolving activity of His-tagged PSII isolated from WT* was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA(1) is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT*. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P680, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechoeystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His 198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-92811. We conclude that the nature of the axial ligand to PD1 is not an important determinant of the redox and spectroscopic properties Of P680 in T elongatus. (C) 2008 Elsevier B.V All rights reserved.

DOI： 10.1016/j.bbabio.2008.01.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC  

The active site for water oxidation in photosystem II (PSII) consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S(0) to S(4)) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn(4) cluster interacting with TyrZ(center dot), can be trapped by illumination of the S(0), S(1) and S(2) states at cryogenic temperatures. The EPR signals reported were attributed to S(0)TyrZ(center dot), S(1)TyrZ(center dot) and S(2)TyrZ(center dot), respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn(4) cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD(center dot), PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ(center dot) interacting with the Mn cluster in S(0), S(1), S(2) and also probably the S(3) states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 angstrom units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.

DOI： 10.1098/rstb.2007.2216

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Bicarbonate is known to be required for the maximum activity of photosystem II. Although it is well established that bicarbonate is bound to the northeme iron to regulate the quinone reactions, the effect of bicarbonate on oxygen evolution is still controversial, and its binding site and exact physiological roles remain to be clarified. In this study, the structural coupling of bicarbonate to the oxygen-evolving center (OEC) was studied using Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra during the S-state cycle of OEC were recorded using the PSII core complexes from Thermosynechococcus elongatus in the presence of either unlabeled bicarbonate or (13)C-bicarbonate. The H(12)CO(3)-minus-H(13)CO(3)-double difference spectra showed prominent bicarbonate bands at the first flash, whereas no appreciable bands were detected at the second to fourth flashes. The bicarbonate bands at the, first flash were virtually identical to those from the nonheme iron, which was preoxidized by ferricyanide and photoreduced by a single flash, recorded using Mn-depleted PSII complexes. Using the bicarbonate bands of the nonheme iron as an internal standard, it was concluded that no bicarbonate band arising from OEC exists in the S-state FTIR spectra. This conclusion indicates that bicarbonate is not affected by the structural changes in OEC upon the four S-state transitions. It is thus strongly suggested that bicarbonate is neither a ligand to the Mn cluster nor a cofactor closely coupled to OEC, although the possibility cannot be fully excluded that nonexchangeable bicarbonate exists in OEC as a constituent of the Mn-cluster core. The data also provide strong evidence that bicarbonate does not function as a substrate or a catalytic intermediate. Bicarbonate may play major roles in the photoassembly process of the Mn cluster and in the stabilization of OEC by a rather indirect interaction.

DOI： 10.1021/bi702241t

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S166_6

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S167_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S167_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Bromide anomalous X-ray diffraction analyses have been used to locate chloride binding sites in the vicinity of the water splitting/oxygen evolving centre (OEC) of Photosystem II. Three-dimensional crystals of PSII from Thermosynechococcus elongatus were grown from (i) isolated PSII crystals infiltrated with bromide or (ii) PSII obtained from cells cultured in a medium in which the chloride content was totally replaced by bromide. In either case, the anomalous diffraction yielded the same result, the existence of two bromide binding sites in the vicinity of the OEC. Neither are in the first coordination sphere of the Mn and Ca ions which form the catalytic centre of the OEC, being about 6 to 7 angstrom from the metal-cluster. Site 1 is located close to the side chain nitrogen of D2-K317 and the backbone nitrogen of D1-Glu333 while Site 2 is adjacent to backbone nitrogens of CP43-Glu354 and D1-Asn338. Their positioning close to postulated hydrophilic channels may suggest a role in proton removal from, or substrate access to, the OEC.

DOI： 10.1039/b810067p

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S166_4

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DOI： 10.2142/biophys.48.S18_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The redox-active tyrosine Y-D (D2-Tyr160) in photosystem II (PSII) serves as a side-path electron donor to P680. When YD is oxidized, a proton is released from phenolic OH, and a neutral radical Y-D(center dot) is formed. A hydrogen bond network around YD must be deeply involved in the mechanism of the YD reaction. In this study, we have detected water molecules structurally coupled to YD by means of Fourier transform infrared (FTIR) spectroscopy. Light-induced Y-D(center dot)/Y-D FTIR difference spectrum of a hydrated film of the PSII core complexes from Thermosynechococcus elongatus showed major signals at 3636(-)/3617(+) and 3594(+)/3585(-) cm(-1) in the weakly hydrogen bonded OH stretching region. These peaks downshifted by 11-12 cm(-1) upon (H2O)-O-18 substitution and almost disappeared upon H/D exchange, and hence, they were definitely assigned to the water OH vibrations. Small intramolecular couplings of 3-6 cm(-1) estimated from the OH frequencies of residual HOD species in a deuterated film indicate that these OH signals, arise from two different water molecules that have significantly asymmetric hydrogen bond structures. Similar OH signals were observed in PSII-enriched membranes from spinach, suggesting that two water molecules commonly exist near YD irrespective of biological species. These water molecules are coupled to YD most probably through a hydrogen bond network or one of them possibly interacts directly with YD, and thus, they may play crucial roles in the YD reaction by forming a proton-transfer pathway and tuning the redox potential Of Y-D.

DOI： 10.1021/bi701752d

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of similar to 5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex. (c) 2007 Elsevier B. All rights reserved.

DOI： 10.1016/j.bbabio.2007.08.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Chloroplasts take up cytosolic nitrite during nitrate assimilation. In this study we identified a nitrite transporter located in the chloroplasts of higher plants. The transporter, CsNitr1- L, a member of the proton- dependent oligopeptide transporter ( POT) family, was detected during light- induced chloroplast development in de- etiolating cucumber seedlings. We detected a CsNitr1- L - green fluorescent protein ( GFP) fusion protein in the chloroplasts of leaf cells and found that an immunoreactive 51 kDa protein was present in the isolated inner envelope membrane of chloroplasts. CsNitr1- L has an isoform, CsNitr1- S, with an identical 484 amino acid core sequence; however, in CsNitr1- S the 120 amino acid N- terminal extension is missing. Saccharomyces cerevisiae cells expressing CsNitr1- S absorbed nitrite from an acidic medium at a slower rate than mock- transformed control cells, and accumulated nitrite to only one- sixth the concentration of the control cells, suggesting that CsNitr1- S enhances the efflux of nitrite from the cell. Insertion of T- DNA in a single CsNitr1- L homolog ( At1g68570) in Arabidopsis resulted in nitrite accumulation in leaves to more than five times the concentration found in the wild type. These results show that it is possible that both CsNitr1- L and CsNitr1- S encode efflux- type nitrite transporters, but with different subcellular localizations. CsNitr1- L may possibly load cytosolic nitrite into chloroplast stroma in the chloroplast envelope during nitrate assimilation. The presence of genes homologous to CsNitr1- L in the genomes of Arabidopsis and rice indicates that facilitated nitrite transport is of general physiological importance in plant nutrition.

DOI： 10.1093/pcp/pcm073

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The structure and the electronic properties of P680 and its radical cation in photosystem II (PSII) were studied by means of Fourier transform infrared spectroscopy (FTIR). Light-induced P680(+)/P680 FTIR difference spectra in the mid- and near-IR regions were measured using PSII membranes from spinach, core complexes from Thermosynechococcus elongatus, and reaction center (RC) complexes (D1-D2-Cytb559) from spinach. The spectral features of the former two preparations were very similar, indicating that the structures of P680 and its radical cation are virtually identical between membranes and cores and between plants and cyanobacteria. In sharp contrast, the spectrum of the RC complexes exhibited significantly different features. A positive doublet at similar to 1724 and similar to 1710 cm(-1) due to the 13(1)-keto CO stretches of P680(+) in the membrane and core preparations were changed to a prominent single peak at 1712 cm(-1) in the RC complexes. This observation was interpreted to indicate that a positive charge on P680(+) was extensively delocalized over the chlorophyll dimer in RC, whereas it was mostly localized on one chlorophyll molecule (70-80%) in intact P680. The significant change in the electronic structure of P680(+) in RC was supported by a dramatic change in the characteristics of a broad intervalence band in the near-IR region and relatively large shifts of chlorin ring bands. It is proposed that the extensive charge delocalization in P680(+) mainly causes the decrease in the redox potential of P680(+)/P680 in isolated RC complexes. This potential decrease explains the well-known phenomenon that Y(Z) is not oxidized by P680(+) in RC complexes.

DOI： 10.1021/bi700157n

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S-0-S-4) in the water oxidation process that also involves a tyrosine radical intermediate (Tyr(Z)(center dot)). An S(2)Tyr(Z)(center dot) state in which the Mn4Ca cluster and Tyr(Z)(center dot) are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from Tyr(D)(center dot). In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of Tyr(Z)(center dot) in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The Tyr(Z)(center dot) g(x) value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two g(x) values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, Tyr(D)(center dot), was examined in various electrostatic environments. The Tyr(D)(center dot) g(x) value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to Tyr(D)(center dot) was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr(center dot) g(x) values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.

DOI： 10.1021/bi062084f

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S193_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S193_3

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S194_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A Ca2+ ion is an indispensable element in the oxygen-evolving Mn cluster in photosystem II (PSII). To investigate the structural relevance of Ca2+ to the Mn cluster, the effects of Sr2+ substitution for Ca2+ on the structures and reactions of ligands to the Mn cluster during the S-state cycle were investigated using flash-induced Fourier transform infrared ( FTIR) difference spectroscopy. FTIR difference spectra representing the four S-state transitions, S-1 -> S-2, S-2 -> S-3, S-3 -> S-0, and S-0 -> S-1, were recorded by applying four consecutive flashes either to PSII core complexes from Thermosynechococcus elongatus or to PSII-enriched membranes from spinach. The spectra were also recorded using biosynthetically Sr(2+)substituted PSII core complexes from T. elongatus and biochemically Sr2+-substituted PSII membranes from spinach. Several common spectral changes upon Sr2+ substitution were observed in the COO(-)stretching region of the flash-induced spectra for both preparations, which were best expressed in Ca2+-minus-Sr2+ double difference spectra. The significant intensity changes in the symmetric COO- peaks at similar to 1364 and similar to 1418 cm(-1) at the first flash were reversed as opposite intensity changes at the third flash, and the slight shift of the similar to 1446 cm-1 peak at the second flash corresponded to the similar but opposite shift at the fourth flash. Analyses of these changes suggest that there are at least three carboxylate ligands whose structures are significantly perturbed by Ca2+/Sr2+ exchange. They are (1) the carboxylate ligand having a bridging or unidentate structure in the S2 and S3 states and perturbed in the S-1 -> S-2 and S-3 -> S-0 transitions, (2) that with a chelating or bridging structure in the S1 and S0 states and perturbed also in the S-1 -> S-2 and S-3 -> S-0 transitions, and (3) that with a chelating structure in the S-3 and S-0 states and changes in the S-2 -> S-3 and S-0 -> S-1 transitions. Taking into account the recent FTIR studies using site-directed mutagenesis and/or isotope substitution [Chu et al. (2004) Biochemistry 43,3152- 3116; Kimura et al. (2005) J. Biol. Chem. 280, 2078-2083; Strickler et al. (2006) Biochemistry 45, 8801-8811], it was concluded that these carboxylate groups do not originate from either D1-Ala344 (C-terminus) or D1-Glu189, which are located near the Ca2+ ion in the X-ray crystallographic model of the Mn cluster. It was thus proposed that if the X-ray model is correct, the above carboxylate groups sensitive to Sr2+ substitution are ligands to the Mn ions strongly coupled to the Ca2+ ion rather than direct ligands to Ca2+.

DOI： 10.1021/bi061232z

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Loading of cytosolic nitrite into the chloroplast in nitrate assimilation of rice was modified by introducing gene for chloroplastic nitrite transport from cucumber. Rice (Oryza sativa L. japonica cv. Nipponbare) was transformed with a cDNA (CsNitr1-L) that encodes a nitrite transporter of cucumber under the control of the CaMV35S promoter. CsNitr1-L transgenic rice grown by hydroponics with nitrate supplied as the sole nitrogen source grew as well as plants grown with ammonia. In contrast the growth of the untransformed or mock-transformed control rice was retarded on nitrate-containing medium by about 60% compared to plants grown with ammonia. Nitrite was not beneficial to the growth of the untransformed or mock-transformed control rice but sustained that of CsNitr1-L transgenic rice. Low steady-state concentration of nitrite in leaves as well as delayed non-photochemical quenching of chlorophyll fluorescence in CsNitr1-L transgenic rice seems indicative of higher nitrite use in chloroplasts.

DOI： 10.5511/plantbiotechnology.23.47

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S385_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S385_2

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S385_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The Mn4Ca complex that is involved in water oxidation in PSII is affected by near-infrared (NIR) light in certain redox states and these phenomena can be monitored by electron paramagnetic resonance (EPR) at low temperature. Here we report the action spectra of the NIR effects in the S-2 and S-3 states in PSII from plants and the thermophilic cyanobacterium Thermosynechococcus elongatus. The action spectra obtained are very similar in both S states, indicating the presence of the same photoactive form of the Mn4Ca complex in both states. Since the chemical nature of the photoactive species is not known, an unequivocal interpretation of this result cannot be made; however, it appears to be more easily reconciled with the view that the redox state of the Mn4Ca cluster does not change from the S-2 to the S-3 transition, at least in those centers sensitive to NIR light. The temperature dependence of the NIR effect and the action spectra for S-2 indicate the presence of structural heterogeneity in the Mn4Ca cluster.

DOI： 10.1093/pcp/pci088

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Mass transport of the bulk of the analyte to the electrode and through the bioactive layer can be significantly improved by use of the nanoelectrode array and defined arrangement of protein film. This phenomenon has been studied by (i) atomic-force microscopy, (ii) electrochemical measurements of PSII activity, and (iii) digital simulations for an oriented monolayer of histidine-tagged photosystem II (PSII) immobilized on nitrilotriacetic acid (NTA)-modified gold electrodes. The output signal of the electrochemical biosensor is controlled by (i) mass transport from the bioactive layer to electrode and (ii) mass transport between the bulk of the analyte and the electrode. Mass transport through the bioactive layer was electrochemically studied for PSII self-assembled on gold screen-printed electrodes. A densely packed monolayer of PSII has a significant shielding effect toward the diffusion of redox mediator duroquinone (DQ). Mass transport to the planar electrode surface was improved by co-immobilization of bovine-serum albumin (BSA) as spacer biomolecule in the monolayer of PSII. Correlation between the electrochemical properties and surface arrangement of the resulting protein films was clearly observable and confirmed the improved mass-transport properties of structured enzyme monolayers. On the basis of this observation, the application of a bottom-up approach for improvement of electrode performance was proposed and digitally simulated for an infinite array of electrodes ranging in diameter from 50 nm to 5 mu m. The nanoelectrode array, with the optimum time window selected for measurements, enables enhancement of mass transport between the bulk of the analyte and the macroelectrode by a factor of up to 50 in comparison with "classical" planar electrodes. Use of a time window enables minimization of crosstalk between individual electrodes in the array. The measurements require methods which suppress the double-layer capacity.

DOI： 10.1007/s00216-005-3149-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

pH dependence of the efficiencies of the flash-induced S-state transitions in the oxygen-evolving center (OEC) was studied by means of Fourier transform infrared (FTIR) difference spectroscopy using photosystem II (PSII) core complexes from the thermophilic cyanabactrium Thermosynechoccocus elongatus. The PSII core complexes dark-adapted at different pHs in the presence of ferricyanide as an electron acceptor were excited by four consecutive saturating laser flashes, and FTIR difference spectra induced by each flash were recorded in the region of 1800-1200 cm(-1). Each difference spectrum was fitted with a linear combination of standard spectra measured at pH 6.0, which represent the spectra upon individual S-state transitions, and the transition efficiencies were estimated from the fitting parameters. It was found that the S(1) --> S(2) transition probability is independent of pH throughout the pH region of 3.5-9.5, while the S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transition probabilities decrease at acidic pH with pK values of 3.6 +/- 0.2, 4.2 +/- 0.3, and 4.7 +/- 0.5, respectively. These findings, i.e., the pH-independent S(1) - S2 transition probability and the pK values for the inhibition in the acidic range of the other three transitions, were in good agreement with recent results obtained by electron paramagnetic resonance measurements for PSII-enriched membranes of spinach [Bernat, G., Morvaridi, F., Feyziyev, Y., and Styring, S. (2002) Biochemistry 41, 5830-5843]. On the basis of this correspondence for quite different types of PSII preparations exhibiting marked difference in the pH dependence of the apparent proton release pattern, it is concluded that the inhibition of the S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transitions in the acidic region is an inherent property of the OEC. This feature probably reflects proton release from substrate water in these three transitions. On the other hand, all of the S-state transitions remained generally efficient up to pH 9.5 in the alkaline region, except for a slight decrease of the S(3) --> S(0) transition probability above pH 8 (pK - 10). This observation partly differs from the tendency reported for spinach preparations, suggesting that a mechanism different from that in the acidic region is responsible for the transition efficiencies in the alkaline region.

DOI： 10.1021/bi0483312

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S272_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S271_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S272_2

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

Cytochrome c(550) is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c(550) we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and -30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c(550) is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c(550) was also constructed. It showed a lowered affinity for Cl- and Ca2+ as reported earlier for the cytochrome c(550)-less Synechocystis 6803 mutant, but it showed a shorter lived S(2)Q(B)(-) state, rather than a stabilized S-2 state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss ( or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c(550).

DOI： 10.1074/jbc.M408206200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Site-directed mutagenesis in the photosystem II (PSII) oxygen-evolving enzyme was achieved in the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII from this species is the focus of attention because its robustness makes it suitable for enzymological and biophysical studies. PSII, which lacks the redox-active tyrosine Tyro, was engineered by substituting a phenylalanine for tyrosine 160 of the D2 protein. An aim of this work was to engineer a mutant for spectroscopy, in particular, for EPR, on the active enzyme. The Tyr(D)(.) EPR signal was monitored in whole cells (i) to control the expression level of the two genes (psbD(1) and psbD(2)) encoding D2 and (ii) to assess the success of the mutagenesis. Both psbD(1) and psbD(2) could be expressed, and recombination occurred between them. The D2-Y160F mutation was introduced into psbD(1) after psbD(2) was deleted and a His-tag was attached to the CP43 protein. The effects of the Y160F mutation were characterized in cells, thylakoids, and isolated PSII. The efficiency of enzyme function under the conditions tested was unaffected. The distribution and lifetime of the redox states (S-n states) of the enzyme cycle were modified, with more So in the dark and no rapid decay phase of S-3. Although not previously reported, these effects were expected because Tyr(D)(.) is able to oxidize S-0 and Tyr(D) is able to reduce S-2 and S-3. Slight changes in the difference spectra in the visible and infrared recorded upon the formation and reduction of the chlorophyll cation P-680(+) and kinetic measurements of P-680(+) reduction indicated minor structural perturbations, perhaps in the hydrogen-bonding network linking Tyr(D) and P-680, rather than electrostatic changes associated with the loss of a charge from Tyr(D)(.)(H+). We show here that this fully active preparation can provide spectra from the Mn4CaO4 complex and associated radical species uncontaminated by Tyr(D)(.).

DOI： 10.1021/bi048732h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

Electrochemical synthesis of nickel-nitrilotriacetic acid (Ni-NTA) chelators, for subsequent immobilization of (HiS)(6)-tagged proteins (Photosystem II (PSII) as model molecule), on Au or Au-graphite electrodes is compared to chemical synthesis. Results show: (i) higher Ni-NTA surface density, (ii) shorter treatment time (1-12 min vs. 16 h normally needed for self-assembled monolayer (SAM)), (iii) possibility of addressing the chelator to only one Au electrode, in a sensor mu-array. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bioelechem.2003.10.024

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

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr2+-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr2+- and Ca2+-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O-2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O-2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr2+-containing PSII has other kinetics modifications: 1) it has an increased stability of the S-3 redox state; 2) it shows an increase in the rate of electron donation from Tyr(D), the redox-active tyrosine of the D-2 protein, to the oxygen-evolving complex in the S-3-state forming S-2; 3) the rate of oxidation of the S-0-state to the S-1-state by Tyr(D)(.) is increased; and 4) the release of O-2 is slowed down to an extent similar to that seen for the slow-down of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr2+-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and Tyr(Z) as well as structural effects.

DOI： 10.1074/jbc.M401677200

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S173_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S172_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S172_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Two methods for monolayer immobilization of photosystem II (PSII) isolated from thermophilic cyanobacteria Synechococcus elongatus and prospects for its use as a biosensor for detection of herbicides are reported: (i) immobilization based on recombinant (HiS)(6)-tagged PSII coupled with nickel-nitrilotriacetic acid (Ni-NTA) chelator monolayer on An electrode. (ii) immobilization based on the natural PSII coupled with the protein A-anti-DI antibody modified Au electrode. Here, Cysteamine (CYS)-self-assembled monolayer (SAM)-(NTA)-PSII monolayers were compared with traditional bovine serum albumin (BSA)-glutaraldehyde (GA)-PSII crosslinked get matrix and better performances of the derived electro-chemical biosensors were pointed out. Better diffusion of inhibitors and mediators resulted in improved sensitivity, velocity of the response, and lower 150 for herbicides.

DOI： 10.1081/AL-120037593

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

The iron-containing superoxide dismutase (FeSOD) from the thermophilic cyanobacterium Thermosynechococcus elongatus has been isolated. The protein crystallizes readily and we have determined the structure to 1.6 Angstrom resolution. This is the first structural characterization of an FeSOD isolated from a cyanobacterium and one of the highest resolution FeSOD structures determined to date. The activity of the T. elongatus FeSOD has been measured both at 25 degreesC and 50 degreesC and it has been spectroscopically characterized. The T. elongatus FeSOD EPR spectra at pH 5.1, 7.5 and 10.0 are similar. This indicates that no change in the geometry of the Fe-III site occurs over a wide range of pH. This is in contrast to the other FeSODs described in the literature.

DOI： 10.1007/s00775-003-0469-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

First, the crystal structure of cytochrome c-550 (the psbV1 gene product) from the thermophilic cyanobacterium Thermosynechococcus elongatus has been determined to a resolution of 1.8 Angstrom. A comparison of the T elongatus cytochrome c-550 structure to its counterparts from mesophilic organisms, Synechocystis 6803 and Arthrospira maxima, suggests that increased numbers of hydrogen bonds may play a role in the structural basis of thermostability. The cytochrome c-550 in T elongatus also differs from that in Synechocystis 6803 and Arthrospira maxima in its lack of dimerization and the presence of a trigonal planar molecule, possibly bicarbonate, tightly bound to the heme propionate oxygen atoms. Cytochromes c-550 from T elongatus, Synechocystis 6803 and Arthrospira maxima exhibit different EPR spectra. A correlation has been done between the heme-axial ligands geometries and the rhombicity calculated from the EPR spectra. This correlation indicates that binding of cytochrome c-550 to Photosystem 11 is accompanied by structural changes in the heme vicinity. Second, the psbV2 gene product has been found and purified. The UV-visible, EPR and Raman spectra are reported. From the spectroscopic data and from a theoretical structural model based on the cytochrome c-550 structure it is proposed that the 6th ligand of the heme-iron is the Tyr86.

DOI： 10.1093/pcp/pcg084

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform N-15 and C-13 isotopic labeling of photosystem II (PS II). The difference spectra upon first- to fourth-flash illumination were obtained with hydrated (for the 1800-1200 cm(-1) region) or deuterated (for the 3500-3100 cm(-1) region) films of unlabeled, N-15-labeled, and C-13-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon N-15 and C-13 labeling provided the assignments of major peaks in the regions of 3450-3250 and 1700-1630 cm(-1) to the NH stretches and amide I modes of polypeptide backbones, respectively, and the assignments of some of the peaks in the 1600-1500 cm(-1) region to the amide II modes of backbones. Other prominent peaks in the latter region and most of the peaks in the 1450-1300 cm(-1) region exhibited large downshifts upon C-13 labeling but were unchanged by N-15 labeling, and hence assigned to the asymmetric and symmetric COO- stretching vibrations, respectively, of carboxylate groups in Glu, Asp, or the C-terminus. Peak positions corresponded well with each other among the first- to fourth-flash spectra, and most of the bands in the first- and/or second-flash spectra appeared with opposite signs of intensity in the third- and/or fourth-flash spectra. This observation indicates that the protein movements in the S-1-->S-2 and/or S-2-->S-3 transitions are mostly reversed in the S-3-->S-0 and/or S-0-S-1 transitions, representing a catalytic role of the protein moieties of the water-oxidizing complex. Drastic structural changes in carboxylate groups over the S-state cycle suggest that the Asp and/or Glu side chains play important roles in the reaction mechanism of photosynthetic water oxidation.

DOI： 10.1021/bi0341612

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus with a histidine tag on the CP43 protein of PSII Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential (E'(m)) of + 390 mV in about half of the centers and + 275 mV in the other half. The high-potential form, whose E'(m) is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The Em of the lowpotential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E'(m) when bound to the PSII core (-80 mV at pH 6.0) than after its extraction from the complex (-240 mV at pH 6.0). The E'(m) of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.

DOI： 10.1007/s00775-002-0406-7

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S202_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Photosynthetic water oxidation is performed via the light-driven S-state cycle in the water-oxidizing complex (WOC) of photosystem II (PS II). To understand its molecular mechanism, monitoring the reaction of substrate water in each S-state transition is essential. We have for the first time detected the reactions of water molecules in WOC throughout the S-state cycle by observing the OH vibrations of water using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. Moderately hydrated (or deuterated) PS II core films from Synechococcus elongatus were used to obtain the FTIR difference spectra upon the first, second, third, and fourth flash illumination, representing the structural changes in the S-1 --> S-2, S-2 --> S-3, S-3 --> S-0, and S-0 --> S-1 transitions, respectively. In the weakly H-bonded OH region, bands appeared at 3617/3588 cm(-1) as a differential signal in the first-flash spectrum and at 3634, 3621, and 3612 cm(-1) with negative intensities in the second-, third-, and fourth-flash spectra, respectively. These bands shifted down by similar to940 cm(-1) upon deuteration and by similar to10 cm(-1) upon (HO)-O-18 substitution, indicating that they arise from the OH stretches of water including the substrate and its intermediates. Stronuly D-bonded OD bands of water were also identified as broad features in the range of 2600-2200 cm(-1) by taking the double difference between the spectra of (D2O)-O-16- and (D2O)-O-18-deuterated films. In addition, broad continuum features that probably arise from the large proton polarizability of H-bonds were observed around 3000, 2700, 2550, and 2600 cm(-1) in the first-, second-, third-, and fourth-flash spectra, respectively, of the hydrated PS II film, revealing changes in the H-bond network of the protein. The negative OH intensities upon the second to fourth flashes might be related to proton release from substrate water. The results presented here showed that FTIR detection of water OH(D) bands can be a powerful method for investigating the mechanism of photosynthetic water oxidation.

DOI： 10.1021/bi020603i

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Monolayers of genetically modified proteins with an hexahistidine tag, (HiS)(6), were obtained by using a Ni-NTA chelator synthesized on gold-sputtered surfaces (via sulphide bonds), or on gold and graphite (via sililating agents) working electrodes of screen-printed devices. Two kinds of proteins were produced and purified for this study:
(a) a recombinant antibody, derived from the 'single-chain Fv' (scFv) format, and
(b) a photosystem 11 (PSII) core complex isolated from the mutant strain CP43-H of the thermophilic cyanobacterium Synechococcus elongatus.
An scFv previously isolated from a synthetic 'phage display' library was further engineered with an alkaline phosphatase activity genetically added between the carboxy-terminal of the scFvs and the (HiS)6 to allow direct measurement of immobilisation.
Renewable specific binding of (HiS)6 proteins to gold and graphite surfaces and fast and sensitive electrochemical or optical detection of analytes were obtained. Additionally, "on chip" protein preconcentration was conveniently achieved for biosensing purposes, starting from crude unpurified extracts and avoiding protein purification steps. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0928-4931(02)00177-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Differently hydrated films of photosystem II (PSII) core complexes from Synechococcus elongatus were prepared in a humidity-controlled infrared cell. The relative humidity was changed by a simple method of placing a different ratio of glycerol/water solution in the sealed cell. The extent of hydration of the PSII film was lowered as the glycerol ratio increased. FTIR difference spectra of the water oxidizing complex upon the first to sixth flashes were measured at 10 degreesC using these hydrated PSII films. The FTIR spectra (1800-1200 cm(-1)) of the PSII films hydrated using 20% and 40% glycerol/water showed basically the same features as those of the core sample in solution [Noguchi, T., and Sugiura, A (2001) Biochemistry 40, 1497-1502], and the prominent peaks exhibited clear period four oscillation patterns. These observations indicate that the S-state cycle properly functions in these hydrated samples. In the PSII films less hydrated, however, the efficiencies of S-state transitions decreased as the extent of hydration was lowered. This tendency was more significant in the S-2 --> S-3 and S-3 --> S-0 transitions than in the S-1 --> S-2 and S-0 --> S-1 transitions, indicating that the reactions or movements of water molecules are more strongly coupled with the former two transitions than the latter two. The implication of this observation was discussed in light of the water oxidizing mechanism especially in respect to the steps of substrate incorporation and proton release. Furthermore, in the OH stretching region (3800-3000 cm(-1)) of the first-flash spectrum, a differential signal was observed at 3618/3585 cm(-1), which was previously found in the S-2/S-1 spectrum of a frozen sample at 250 K and assigned to the water vibrations [Noguchi, T., and Sugiura, M. (2000) Biochemistry 39, 10943-10949]. The fact that the signal appeared even in rather dehydrated PSII films at a physiological temperature (10 degreesC) supported the idea that this water is located in the close vicinity of the Mn cluster and directly involved in the water oxidizing reaction. The results also showed that moderate hydration of the PSII sample made the whole OH region measurable, escaping from absorption saturation by bulk water, and thus will be a useful technique to monitor the water reactions during the S-state cycle using FTIR spectroscopy.

DOI： 10.1021/bi011954k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns-mus time scale by P680(+) and reduced rapidly (As to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+ with t1/2 approximate to 190 ns at pH 8.5 in approximately half of the centers. This rate is approximate to 10(5) times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t(1/2) = 2-10 mus) donates much faster to P680(+) than does TyrD (t(1/2) > 150 mus). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+ on the chlorophyll (P-D2) that is located on the D2 side of the reaction center.

DOI： 10.1073/pnas.251382598

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Fourier transform infrared (FTIR) difference spectra of all flash-induced S-state transitions of the oxygen-evolving complex were measured using photosystem II (PSII) core complexes of Synechococcus elongatus. The PSII core sample was given eight successive flashes with 1 s intervals at 10 °C, and FTIR difference spectra upon individual flashes were measured. The obtained difference spectra upon the first to fourth flashes showed considerably different spectral features from each other, whereas the fifth, sixth, seventh, and eighth flash spectra were similar to the first, second, third, and fourth flash spectra, respectively. The intensities at the wave numbers of prominent peaks of the first and second flash spectra showed clear period four oscillation patterns. These oscillation patterns were well fitted with the Kok model with 13% misses. These results indicate that the first, second, third, and fourth flash spectra represent the difference spectra upon the S1 → S2, S2 → S3, S3 → S0, and S0 → S1 transitions, respectively. In these spectra, prominent bands were observed in the symmetric (1300-1450 cm-1) and asymmetric (1500-1600 cm-1) stretching regions of carboxylate groups and in the amide I region (1600-1700 cm-1). Comparison of the band features suggests that the drastic coordination changes of carboxylate groups and the protein conformational changes in the S1 → S2 and S2 → S3 transitions are reversed in the S3 → S0 and S0 → S1 transitions. The flash-induced FTIR measurements during the S-state cycle will be a promising method to investigate the detailed molecular mechanism of photosynthetic oxygen evolution.

DOI： 10.1021/bi0023807

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DOI： 10.2142/biophys.41.S72_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The Mn-4-cluster and the cytochrome c(550) in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S-0-state (spin 1/2) and the S-2-state (spin 1/2 and IR-induced spin 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S-3-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the Ss-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at a = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N,, and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn-4-cluster in a fraction of centers, while the g = 5.20 and g 1.51 signals are tentatively attributed to a high-spin state of the Mn-4-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn-4-cluster. Biochemical modifications (Sr2+/Ca2+ substitution, acetate and NH3 treatments) were also investigated. In Sr2+-reconstituted PSII, in addition to the expected modified S-2 multiline signal, a signal at g = 5.2 was present instead of the g approximate to 4 signal seen in plant PSII, In NH3-treated samples, in addition to the expected modified S-2-multiline signal, a g approximate to 4 signal was detected in a small proportion of the reaction centers. This is of note since g approximate to 4 spectra arising from the Mn-4-cluster in the S-2 state have not yet been published in cyanobacterial PSII, The detection of modified S-3-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH3-treated PSII indicate that NH3 is still bound in the S-3-state. The acetate-treated PSII behaves essentially as in plant PSII, A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc(550) was perpendicular to the plane of the membrane.

DOI： 10.1021/bi001159r

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Language：English   Publishing type：Research paper (scientific journal)  

The vibrations of a water molecule in the water-oxidizing complex (WOC) of photosystem II were detected for the first time using Fourier transform infrared (FTIR) spectroscopy. In a flash-induced FTIR difference spectrum upon the S1-to-S2 transition, a pair of positive and negative bands was observed at 3618 and 3585 cm-1, respectively, and both bands exhibited downshifts by 12 cm-1 upon replacement of H216O by H218O. Upon D2O substitution, the bands largely shifted down to 2681 and 2652 cm-1. These observations indicate that the bands at 3618 and 3585 cm-1&gt
arise from the O-H stretching vibrations of a water molecule, probably substrate water, coupled to the Mn cluster in the S2 and S1 states, respectively. The band frequencies indicate that the O-H group forms a weak H-bond and this H-bonding becomes weaker upon S2 formation. Intramolecular coupling with the other O-H vibration of this water molecule was studied by a decoupling experiment using a H2O/D2O (1:1) mixture. The downshifts by decoupling were estimated to be 4 and 12 cm-1 for the 3618 (S2) and 3585 cm-1 (S1) bands, both of which were much smaller than 52 cm-1 of water in vapor, indicating that the observed water has a considerably asymmetric structure
i.e., one of the O-H groups is weakly and the other is strongly H-bonded. The smaller coupling in the S2 than the S1 state means that this H-bonding asymmetry becomes more prominent upon S2 formation. Such a structural change may facilitate the proton release reaction that takes place in the later step by lowering the potential barrier. The present study showed that FTIR detection of the O-H vibrations is a useful and promising method to directly monitor the chemical reactions of substrate water and clarify the molecular mechanism of photosynthetic water oxidation.

DOI： 10.1021/bi001040i

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni2+-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mu mol (mg Chl)(-1) h(-1) at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B-2-, B-1- and Q-bands arising from S(2)Q(B)(-), S(3)Q(B)(-) and S(2)Q(A)(-) charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

A His-tagged PSII core complex was purified from recombinant Chlamydomonas reinhardtii D2-H thylakoids by single-step Ni2+-affinity column chromatography and its properties were partially characterized in terms of their PSII functions and chemical compositions. The PSII core complex that has a His-tag extension at the C-terminus of the D2 protein evolved oxygen at a high rate of 2,400 mu mol (mg Chl)(-1) h(-1) at the optimum pH of 6.5 with ferricyanide and 2,6-dichlorobenzoquinone as electron accepters in the presence of Ca2+ as an essential cofactor, and approximately 90% of the activity was blocked by 10 mu M DCMU. The core complex exhibited the thermoluminescence Q-band but not the B-band regardless of the presence or absence of DCMU, although both bands were observed in the His-tagged thylakoids. The core complex was free from PSI and contained one Y-D, Tyr 160 of the D2 protein, four Mn atoms, two cytochrome b-559, about 46 Chl a molecules, and probably one QA, the primary acceptor quinone of PSII. It was inferred from these results that His-tagging at the C-terminus of the D2 protein does not affect the functional and structural integrity of the PSII core complex, and that the 'His-tag strategy' is highly useful for biochemical, physicochemical, and structural studies of Chlamydomonas PSII.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have developed a simple and rapid procedure to isolate an oxygen-evolving photosystem II (PS II) core complex from Chlamydomonas reinhardtii. A His-tag made of sis consecutive histidine residues was genetically attached at the carboxy terminus of D2 protein to create a metal binding site on the PS Il supramolecular complex, The recombinant cells producing the His-tagged variant of D2 protein grew photo-autotrophically as well as the wild-type cells. Characterization of the oxygen evolution and the thermoluminescence properties revealed that the His-tagging did not affect the functional integrity of the PS II reaction center. A PS Il core complex was isolated from the detergent-solubilized thylakoids of the recombinant cells in 4 h by a single one-step Ni2+ affinity column chromatography. This preparation consists of D1, D2, CP43, CP47, 33 kDa, and a few low molecular weight proteins, and retains a high rate of oxygen-evolving activity (=1000 mu mol/mg Chl/h), (C) 1998 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(98)00328-7

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

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

A cDNA library constructed from poly(A)+ RNA of tobacco BY2 cells treated with 2,4-dichlorophenoxyacetic acid was screened by using a synthetic oligonucleotide corresponding to the heme binding region of avocado CYP71A1. A cloned 2-kb cDNA designated as cTBP contained an open reading frame of 1593 bp encoding a protein of molecular size of 58916. The deduced amino acid sequence included a cysteine residue corresponding to fifth ligand of heme-Fe at 497th. The coding sequence was expressed under the control of tac promoter and rrnB terminator in Escherichia coli to yield 7 to 10 nmol P450 equivalent per litre of the culture in the presence of 6-aminolevulinic acid. The modified coding sequences in which NH2-terminal residues 2-25 were replaced by the NH2-terminal 18 amino acid residues of microsomal bovine CYP17 were also expressed under the control of ADH promoter and terminator in Saccharomyses cerevisiae to yield 29 and 30 pmol of P450 equivalent/mg protein in the microsomal fraction, respectively. On co-expression of each of the modified coding sequences and yeast NADPH-cytochrome P-450 oxidoreductase gene, the yeast microsomes exhibited 7-ethoxycoumarin O-deethylase activity. Based on these results, tobacco cTBP was found to encode a novel P450-1ike species with a monooxygenese activity related to xenobiotic metabolism.

DOI： 10.1016/0167-4781(96)00107-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：DIAGNOSIS PRESS LTD  

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Language：Japanese  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER  

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J-GLOBAL

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CiNii Books

J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：The Society of Japanese Women Scientists  

Photosynthetic organisms such as plants and cyanobacteria convert light energy to chemical energy efficiently by photosynthetic electron transfer accompanied by water oxidation. Elucidation of their molecular mechanisms must be a very important subject not only for general scientific interest but also for helping to find solutions to energy and environmental problems. However, molecular mechanisms of photosynthesis, especially water oxidation for electron donation and regulation mechanisms of cofactors relating to the electron transfer in Photosystem II, are not yet clear. In this review, we interpret the structure of Photosystem II and the electron transfer system based on the redox potential of cofactors and water oxidation mechanisms. Furthermore, we introduce application research for energy reproduction by using Photosystem II.

DOI： 10.5939/sjws.12002

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J-GLOBAL

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J-GLOBAL

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Language：English   Publisher：AMER CHEMICAL SOC  

DOI： 10.1021/ja202794m

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=185062

Language：English   Publisher：Japanese Society of Plant Physiologists  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184925

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CiNii Books

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都大学  

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Venue：Trois-Rivières, Québec, Canada  

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Venue：岡山国際交流センター  

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Venue：岡山大学  

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Venue：大阪大学  

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Venue：札幌コンベンションセンター  

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Venue：東北大学  

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Venue：岡山大学  

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Venue：London, England  

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Venue：愛媛大学  

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Venue：名古屋大学  

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Venue：札幌コンベンションセンター  

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Venue：福岡国際会議場  

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Venue：高知大学  

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Venue：兵庫県立大学  

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Venue：東京工業大学  

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Venue：アスティ徳島  

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Venue：熊本大学  

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Venue：熊本大学  

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Venue：日本大学理工学部船橋キャンパス  

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Venue：京都大学  

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Venue：筑波大学  

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Venue：筑波大学  

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Venue：筑波大学  

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Venue：札幌コンベンションセンター  

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Venue：京都  

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Venue：愛媛大学  

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Venue：関西大学  

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Venue：東京大学  

「若手の会シンポジウム」

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Venue：広島大学  

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