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Featured researches published by Ryo Nagao.


Proceedings of the National Academy of Sciences of the United States of America | 2010

Redox potential of pheophytin a in photosystem II of two cyanobacteria having the different special pair chlorophylls

Suleyman I. Allakhverdiev; Tatsuya Tomo; Yuichiro Shimada; Hayato Kindo; Ryo Nagao; Vyacheslav V. Klimov; Mamoru Mimuro

Water oxidation by photosystem (PS) II in oxygenic photosynthetic organisms is a major source of energy on the earth, leading to the production of a stable reductant. Mechanisms generating a high oxidation potential for water oxidation have been a major focus of photosynthesis research. This potential has not been estimated directly but has been measured by the redox potential of the primary electron acceptor, pheophytin (Phe) a. However, the reported values for Phe a are still controversial. Here, we measured the redox potential of Phe a under physiological conditions (pH 7.0; 25 °C) in two cyanobacteria with different special pair chlorophylls (Chls): Synechocystis sp. PCC 6803, whose special pair for PS II consists of Chl a, and Acaryochloris marina MBIC 11017, whose special pair for PS II consists of Chl d. We obtained redox potentials of −536 ± 8 mV for Synechocystis sp. PCC 6803 and −478 ± 24 mV for A. marina on PS II complexes in the presence of 1.0 M betaine. The difference in the redox potential of Phe a between the two species closely corresponded with the difference in the light energy absorbed by Chl a versus Chl d. We estimated the potentials of the special pair of PS II to be 1.20 V and 1.18 V for Synechocystis sp. PCC 6803 (P680) and A. marina (P713), respectively. This clearly indicates conservation in the properties of water-oxidation systems in oxygenic photosynthetic organisms, irrespective of the special-pair chlorophylls.


Photosynthesis Research | 2013

Comparison of oligomeric states and polypeptide compositions of fucoxanthin chlorophyll a/c-binding protein complexes among various diatom species

Ryo Nagao; Shuji Takahashi; Takehiro Suzuki; Naoshi Dohmae; Katsuyoshi Nakazato; Tatsuya Tomo

Fucoxanthin chlorophyll a/c-binding protein (FCP) is a unique light-harvesting apparatus in diatoms. Several biochemical characteristics of FCP oligomer and trimer from different diatom species have been reported previously. However, the integration of information about molecular organizations and polypeptides of FCP through a comparison among diatoms has not been published. In this study, we used two-dimensional clear-native/SDS-PAGE to compare the oligomeric states and polypeptide compositions of FCP complexes from four diatoms: Chaetocerosgracilis, Thalassiosirapseudonana, Cyclotellameneghiniana, and Phaeodactylumtricornutum. FCP oligomer was found in C. gracilis, T. pseudonana, and C. meneghiniana, but not in P. tricornutum. The oligomerization varied among the three diatoms, although a predominant subunit having similar molecular weight was recovered in each FCP oligomer. These results suggest that the predominant subunit is involved in the formation of high FCP oligomerization in each diatom. In contrast, FCP trimer was found in all the diatoms. The trimerizations were quite similar, whereas the polypeptide compositions were markedly different. On the basis of this information and that from mass spectrometric analyses, the gene products in each FCP complex were identified in T. pseudonana and P. tricornutum. Based on these results, we discuss the role of FCP oligomer and trimer from the four diatoms.


Journal of Biological Chemistry | 2010

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom, Chaetoceros gracilis

Ryo Nagao; Akira Moriguchi; Tatsuya Tomo; Ayako Niikura; Saori Nakajima; Takehiro Suzuki; Akinori Okumura; Masako Iwai; Jian Ren Shen; Masahiko Ikeuchi; Isao Enami

Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ′, PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ′, and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl− and Ca2+ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl− and Ca2+ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.


Biochemistry | 2014

Fourier transform infrared detection of a polarizable proton trapped between photooxidized tyrosine YZ and a coupled histidine in photosystem II: relevance to the proton transfer mechanism of water oxidation.

Shin Nakamura; Ryo Nagao; Ryouta Takahashi; Takumi Noguchi

The redox-active tyrosine YZ (D1-Tyr161) in photosystem II (PSII) functions as an immediate electron acceptor of the Mn4Ca cluster, which is the catalytic center of photosynthetic water oxidation. YZ is also located in the hydrogen bond network that connects the Mn4Ca cluster to the lumen and hence is possibly related to the proton transfer process during water oxidation. To understand the role of YZ in the water oxidation mechanism, we have studied the hydrogen bonding interactions of YZ and its photooxidized neutral radical YZ(•) together with the interaction of the coupled His residue, D1-His190, using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The YZ(•)-minus-YZ FTIR difference spectrum of Mn-depleted PSII core complexes exhibited a broad positive feature around 2800 cm(-1), which was absent in the corresponding spectrum of another redox-active tyrosine YD (D2-Tyr160). Analyses by (15)N and H/D substitutions, examination of the pH dependence, and density functional theory and quantum mechanics/molecular mechanics (QM/MM) calculations showed that this band arises from the N-H stretching vibration of the protonated cation of D1-His190 forming a charge-assisted strong hydrogen bond with YZ(•). This result provides strong evidence that the proton released from YZ upon its oxidation is trapped in D1-His190 and a positive charge remains on this His. The broad feature of the ~2800 cm(-1) band reflects a large proton polarizability in the hydrogen bond between YZ(•) and HisH(+). QM/MM calculations further showed that upon YZ oxidation the hydrogen bond network is rearranged and one water molecule moves toward D1-His190. From these data, a novel proton transfer mechanism via YZ(•)-HisH(+) is proposed, in which hopping of the polarizable proton of HisH(+) to this water triggers the transfer of the proton from substrate water to the luminal side. This proton transfer mechanism could be functional in the S2 → S3 transition, which requires proton release before electron transfer because of an excess positive charge on the Mn4Ca cluster.


Plant and Cell Physiology | 2010

Topological Analysis of the Extrinsic PsbO, PsbP and PsbQ Proteins in a Green Algal PSII Complex by Cross-Linking with a Water-Soluble Carbodiimide

Ryo Nagao; Takehiro Suzuki; Akinori Okumura; Ayako Niikura; Masako Iwai; Naoshi Dohmae; Tatsuya Tomo; Jian Ren Shen; Masahiko Ikeuchi; Isao Enami

The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the alpha subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.


FEBS Letters | 2010

Species-dependence of the redox potential of the primary quinone electron acceptor QA in photosystem II verified by spectroelectrochemistry

Tadao Shibamoto; Yuki Kato; Ryo Nagao; Tatsuya Tomo; Tadashi Watanabe

The redox potentials E m(QA/ Q A − ) of the primary quinone electron acceptor QA in oxygen‐evolving photosystem II complexes of three species were determined by spectroelectrochemistry. The E m(QA/ Q A − ) values were experimentally found to be −162 ± 3 mV for a higher plant spinach, −171 ± 3 mV for a green alga Chlamydomonas reinhardtii and −104 ± 4 mV vs. SHE for a red alga Cyanidioschyzon merolae. On the basis of possible deviations for the experimental values, as estimated to differ by 9–29 mV from each true value, plausible causes for such remarkable species‐dependence of E m(QA/ Q A − ) are discussed, mainly by invoking the effects of extrinsic subunits on the delicate structural environment around QA.


Journal of Physical Chemistry B | 2014

Light-harvesting ability of the fucoxanthin chlorophyll a/c-binding protein associated with photosystem II from the Diatom Chaetoceros gracilis as revealed by picosecond time-resolved fluorescence spectroscopy.

Ryo Nagao; Makio Yokono; Ayaka Teshigahara; Seiji Akimoto; Tatsuya Tomo

The fucoxanthin chlorophyll a/c-binding protein (FCP) is a unique antenna complex possessed by diatoms. Although FCP complexes have been isolated from various diatoms, there is no direct evidence for the existence of FCP associated with photosystem II (FCPII). Here, we report the isolation and spectroscopic characterization of FCPII complex from the diatom Chaetoceros gracilis. The FCPII complex was purified using sucrose centrifugation and anion-exchange chromatography. Clear-native PAGE and SDS-PAGE analyses revealed that the FCPII complex was composed of FCP-A oligomer and FCP-B/C trimer. Time-resolved fluorescence spectra of the FCPII complex were measured at 77 K. The characteristic lifetimes and fluorescence components were determined using global fitting analysis, followed by the construction of fluorescence decay-associated spectra (FDAS). FDAS exhibited fluorescence rises and decays, reflecting excitation energy transfer, with the time constants of 150 ps, 800 ps, and 2.9 ns. The long time constants are most likely attributed to the intercomplex excitation energy transfer between FCP-A oligomer and FCP-B/C trimer in the FCPII complex. The 5.6 ns FDAS likely originates from the final energy traps. In contrast, the FDAS exhibited no quenching component with any time constant. These results indicate that the FCPII complex is efficient in light harvesting and excitation energy transfer.


Journal of Physical Chemistry B | 2013

High excitation energy quenching in fucoxanthin chlorophyll a/c-binding protein complexes from the diatom Chaetoceros gracilis.

Ryo Nagao; Makio Yokono; Seiji Akimoto; Tatsuya Tomo

The fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) is responsible for excellent light-harvesting strategies that enable survival in fluctuating light conditions. Here, we report the light-harvesting and quenching states of two FCP complexes, FCP-A and FCP-B/C, isolated from the diatom Chaetoceros gracilis. Pigment analysis revealed that FCP-A is enriched in Chl c, whereas FCP-B/C is enriched in diadinoxanthin, reflecting differences in low-temperature steady-state absorption and fluorescence spectra of each FCP complex. Time-resolved fluorescence spectra were measured at 77 K, and the characteristic lifetimes were determined using global fitting analysis of the spectra. Tens of picosecond (ps) components revealed energy transfer to low-energy Chl a from Chls a and c, whereas the other components showed only fluorescence decay components with no concomitant rise components. The normalized amplitudes of hundreds of picosecond components were relatively 30% in the total fluorescence, whereas those of longest-lived components were 60%. The hundreds of picosecond components were assigned as excitation energy quenching, whereas the longest-lived components were assigned as fluorescence from the final energy traps. These results suggest that 30% of FCP complex forming quenching state and the other 60% of FCP complex forming light-harvesting state exist heterogeneously in each FCP fraction under continuous low-light condition.


Biochimica et Biophysica Acta | 2012

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom, Chaetoceros gracilis.

Ryo Nagao; Tatsuya Tomo; Eri Noguchi; Takehiro Suzuki; Akinori Okumura; Rei Narikawa; Isao Enami; Masahiko Ikeuchi

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c-binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75kDa) and one serine protease (156kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside-solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94kDa metalloproteases were mostly in the FCP-A fraction along with the 156kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.


Biochimica et Biophysica Acta | 2014

Identification of the basic amino acid residues on the PsbP protein involved in the electrostatic interaction with photosystem II.

Taishi Nishimura; Chihiro Uno; Kunio Ido; Ryo Nagao; Takumi Noguchi; Fumihiko Sato; Kentaro Ifuku

The PsbP protein is an extrinsic subunit of photosystem II (PSII) that is essential for photoautotrophic growth in higher plants. Several crystal structures of PsbP have been reported, but the binding topology of PsbP in PSII has not yet been clarified. In this study, we report that the basic pocket of PsbP, which consists of conserved Arg48, Lys143, and Lys160, is important for the electrostatic interaction with the PSII complex. Our release-reconstitution experiment showed that the binding affinities of PsbP-R48A, -K143A, and -K160A mutated proteins to PSII were lower than that of PsbP-WT, and triple mutations of these residues greatly diminished the binding affinity to PSII. Even when maximum possible binding had occurred, the R48A, K143A, and K160A proteins showed a reduced ability to restore the rate of oxygen evolution at low chloride concentrations. Fourier transform infrared resonance (FTIR) difference spectroscopy results were consistent with the above finding, and suggested that these mutated proteins were not able to induce the normal conformational change around the Mn cluster during S1 to S2 transition. Finally, chemical cross-linking experiments suggested that the interaction between the N-terminus of PsbP with PsbE was inhibited by these mutations. These data suggest that the basic pocket of PsbP is important for proper association and interaction with PSII. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

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Tatsuya Tomo

Tokyo University of Science

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Isao Enami

Tokyo University of Science

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