A distinct double-ring LH1–LH2 photocomplex from an extremophilic phototroph
Kazutoshi Tani; Kenji V. P. Nagashima; Risa Kojima; Masaharu Kondo; Ryo Kanno; Issei Satoh; Mai Kawakami; Naho Hiwatashi; Kazuna Nakata; Sakiko Nagashima; Kazuhito Inoue; Yugo Isawa; Ryoga Morishita; Shinichi Takaichi; Endang R. Purba; Malgorzata Hall; Long-Jiang Yu; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Yukihiro Kimura; Yutaka Nagasawa; Takehisa Dewa; Zheng-Yu Wang-Otomo, Springer Science and Business Media LLC
Nature Communications, 2025年02月06日

Genomic highlights of the phylogenetically unique halophilic purple nonsulfur bacterium, Rhodothalassium salexigens
Michael T. Madigan; Kelly S. Bender; Mary N. Parenteau; Yukihiro Kimura; Zheng-Yu Wang-Otomo; W. Matthew Sattley, Abstract

Rhodothalassium (Rts.) salexigens is a halophilic purple nonsulfur bacterium and the sole species in the genus Rhodothalassium, which is itself the sole genus in the family Rhodothalassiaceae and sole family in the order Rhodothalassiales (class Alphaproteobacteria). The genome of this phylogenetically unique phototroph comprises 3.35 Mb and is highly chimeric, with nearly half of its genes originating from families other than the Rhodothalassiaceae, many of which lack phototrophic species. Photosynthesis genes in Rts. salexigens are not arranged in a typical photosynthesis gene cluster but are scattered across the genome, suggesting an origin from horizontal transfers. Despite an encoded RuBisCO, autotrophy has not been observed in Rts. salexigens, and enzymes that oxidize common inorganic electron donors are not encoded. Phospholipid biosynthesis in Rts. salexigens is restricted, and phosphoglycerolipids are the only phospholipids present in its intracytoplasmic membranes. Rts. salexigens fixes nitrogen using a Mo-containing nitrogenase and uses ammonia despite previous results that indicated it was a glutamate auxotroph. Glycine betaine is the sole osmolyte in Rts. salexigens, and enzymes are encoded that facilitate both its uptake and its biosynthesis from glycine. The genomic data also support chemotactic swimming motility, growth over a range of salinities, and the production of membrane-strengthening hopanoids., Springer Science and Business Media LLC
Extremophiles, 2025年01月25日

A Native LH1–RC–HiPIP Supercomplex from an Extremophilic Phototroph
Kazutoshi Tani; Ryo Kanno; Kenji V. P. Nagashima; Mai Kawakami; Naho Hiwatashi; Kazuna Nakata; Sakiko Nagashima; Kazuhito Inoue; Shinichi Takaichi; Endang R. Purba; Malgorzata Hall; Long-Jiang Yu; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Yukihiro Kimura; Zheng-Yu Wang-Otomo, Springer Science and Business Media LLC
Communications Biology, 2025年01月11日

Insights into the divergence of the photosynthetic LH1 complex obtained from structural analysis of the unusual photocomplexes of Roseospirillum parvum
Xiang-Ping Wang; Guang-Lei Wang; Yuan Fu; Akane Minamino; Mei-Juan Zou; Fei Ma; Bo Xu; Zheng-Yu Wang-Otomo; Yukihiro Kimura; Michael T. Madigan; Jörg Overmann; Long-Jiang Yu, Springer Science and Business Media LLC
Communications Biology, 2024年12月19日

The Thermal-Stable LH1–RC Complex of a Hot Spring Purple Bacterium Powers Photosynthesis with Extremely Low-Energy Near-Infrared Light
Yukihiro Kimura; Ryo Kanno; Kaisei Mori; Yoshiki Matsuda; Ryuta Seto; Shinji Takenaka; Hiroyuki Mino; Tatsunari Ohkubo; Mai Honda; Yuji C. Sasaki; Jun-ichi Kishikawa; Kaoru Mitsuoka; Kazuhiro Mio; Malgorzata Hall; Endang R. Purba; Toshiaki Mochizuki; Akira Mizoguchi; Bruno M. Humbel; Michael T. Madigan; Zheng-Yu Wang-Otomo; Kazutoshi Tani, American Chemical Society (ACS)
Biochemistry, 2024年12月16日

Spectral modulation of B850 bacteriochlorophyll a in light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum by detergents and calcium ions
Yoshitaka Saga; Yuhi Sasamoto; Kazuki Inada; Zheng-Yu Wang-Otomo; Yukihiro Kimura, Elsevier BV
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2024年08月

High-resolution structure and biochemical properties of the LH1–RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum
Kazutoshi Tani; Ryo Kanno; Ayaka Harada; Yuki Kobayashi; Akane Minamino; Shinji Takenaka; Natsuki Nakamura; Xuan-Cheng Ji; Endang R. Purba; Malgorzata Hall; Long-Jiang Yu; Michael T. Madigan; Akira Mizoguchi; Kenji Iwasaki; Bruno M. Humbel; Yukihiro Kimura; Zheng-Yu Wang-Otomo, Abstract

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and β-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/β1- or α1/β3-polypeptides through a different Ca²⁺-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca²⁺-bound LH1 complexes. Two water molecules were identified as additional Ca²⁺-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1–RC. While modest but distinct effects of Ca²⁺ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1–RC complex was observed upon removal of Ca²⁺. The presence of Ca²⁺ in the photocomplex of Alc. vinosum suggests that Ca²⁺-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery., Springer Science and Business Media LLC
Communications Biology, 2024年02月12日

Genomic basis for the unique phenotype of the alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis
Michael T. Madigan; Kelly S. Bender; Sophia A. Sanguedolce; Mary N. Parenteau; Marisa H. Mayer; Yukihiro Kimura; Zheng-Yu Wang-Otomo; W. Matthew Sattley, Although several species of purple sulfur bacteria inhabit soda lakes, Rhodobaca bogoriensis is the first purple nonsulfur bacterium cultured from such highly alkaline environments. Rhodobaca bogoriensis strain LBB1(T) was isolated from Lake Bogoria, a soda lake in the African Rift Valley. The phenotype of Rhodobaca bogoriensis is unique among purple bacteria; the organism is alkaliphilic but not halophilic, produces carotenoids absent from other purple nonsulfur bacteria, and is unable to grow autotrophically or fix molecular nitrogen. Here we analyze the draft genome sequence of Rhodobaca bogoriensis to gain further insight into the biology of this extremophilic purple bacterium. The strain LBB1(T) genome consists of 3.91 Mbp with no plasmids. The genome sequence supports the defining characteristics of strain LBB1(T), including its (1) production of a light-harvesting 1-reaction center (LH1-RC) complex but lack of a peripheral (LH2) complex, (2) ability to synthesize unusual carotenoids, (3) capacity for both phototrophic (anoxic/light) and chemotrophic (oxic/dark) energy metabolisms, (4) utilization of a wide variety of organic compounds (including acetate in the absence of a glyoxylate cycle), (5) ability to oxidize both sulfide and thiosulfate despite lacking the capacity for autotrophic growth, and (6) absence of a functional nitrogen-fixation system for diazotrophic growth. The assortment of properties in Rhodobaca bogoriensis has no precedent among phototrophic purple bacteria, and the results are discussed in relation to the organism's soda lake habitat and evolutionary history., SPRINGER JAPAN KK
EXTREMOPHILES, 2023年08月

Atomic force microscopic analysis of the light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum.
Masayuki Morimoto; Haruna Hirao; Masaharu Kondo; Takehisa Dewa; Yukihiro Kimura; Zheng-Yu Wang-Otomo; Hitoshi Asakawa; Yoshitaka Saga, Structural information on the circular arrangements of repeating pigment-polypeptide subunits in antenna proteins of purple photosynthetic bacteria is a clue to a better understanding of molecular mechanisms for the ring-structure formation and efficient light harvesting of such antennas. Here, we have analyzed the ring structure of light-harvesting complex 2 (LH2) from the thermophilic purple bacterium Thermochromatium tepidum (tepidum-LH2) by atomic force microscopy. The circular arrangement of the tepidum-LH2 subunits was successfully visualized in a lipid bilayer. The average top-to-top distance of the ring structure, which is correlated with the ring size, was 4.8 ± 0.3 nm. This value was close to the top-to-top distance of the octameric LH2 from Phaeospirillum molischianum (molischianum-LH2) by the previous analysis. Gaussian distribution of the angles of the segments consisting of neighboring subunits in the ring structures of tepidum-LH2 yielded a median of 44°, which corresponds to the angle for the octameric circular arrangement (45°). These results indicate that tepidum-LH2 has a ring structure consisting of eight repeating subunits. The coincidence of an octameric ring structure of tepidum-LH2 with that of molischianum-LH2 is consistent with the homology of amino acid sequences of the polypeptides between tepidum-LH2 and molischianum-LH2.
Photosynthesis research, 2023年03月17日

Rhodobacter capsulatus forms a compact crescent-shaped LH1-RC photocomplex.
Kazutoshi Tani; Ryo Kanno; Xuan-Cheng Ji; Itsusei Satoh; Yuki Kobayashi; Malgorzata Hall; Long-Jiang Yu; Yukihiro Kimura; Akira Mizoguchi; Bruno M Humbel; Michael T Madigan; Zheng-Yu Wang-Otomo, Rhodobacter (Rba.) capsulatus has been a favored model for studies of all aspects of bacterial photosynthesis. This purple phototroph contains PufX, a polypeptide crucial for dimerization of the light-harvesting 1-reaction center (LH1-RC) complex, but lacks protein-U, a U-shaped polypeptide in the LH1-RC of its close relative Rba. sphaeroides. Here we present a cryo-EM structure of the Rba. capsulatus LH1-RC purified by DEAE chromatography. The crescent-shaped LH1-RC exhibits a compact structure containing only 10 LH1 αβ-subunits. Four αβ-subunits corresponding to those adjacent to protein-U in Rba. sphaeroides were absent. PufX in Rba. capsulatus exhibits a unique conformation in its N-terminus that self-associates with amino acids in its own transmembrane domain and interacts with nearby polypeptides, preventing it from interacting with proteins in other complexes and forming dimeric structures. These features are discussed in relation to the minimal requirements for the formation of LH1-RC monomers and dimers, the spectroscopic behavior of both the LH1 and RC, and the bioenergetics of energy transfer from LH1 to the RC.
Nature communications, 2023年02月15日

Advances in the Spectroscopic and Structural Characterization of Core Light-Harvesting Complexes from Purple Phototrophic Bacteria
Yukihiro Kimura; Kazutoshi Tani; Michael T. Madigan; Zheng-Yu Wang-Otomo, American Chemical Society (ACS)
The Journal of Physical Chemistry B, 2023年01月03日

Selective Expression of Light-Harvesting Complexes Alters Phospholipid Composition in the Intracytoplasmic Membrane and Core Complex of Purple Phototrophic Bacteria
I. Satoh; K. Gotou; S. Nagatsuma; K. V; P. Nagashima; M. Kobayashi; L.-J. Yu; M. T. Madigan; Y. Kimura; Zheng-Yu Wang-Otomo, Elsevier BV
Biochim. Biophy. Acta, 2023年, ［査読有り］

Asymmetric structure of the native Rhodobacter sphaeroides dimeric LH1–RC complex
Kazutoshi Tani; Ryo Kanno; Riku Kikuchi; Saki Kawamura; Kenji V. P. Nagashima; Malgorzata Hall; Ai Takahashi; Long-Jiang Yu; Yukihiro Kimura; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Zheng-Yu Wang-Otomo, Abstract

Rhodobacter sphaeroides is a model organism in bacterial photosynthesis, and its light-harvesting-reaction center (LH1–RC) complex contains both dimeric and monomeric forms. Here we present cryo-EM structures of the native LH1–RC dimer and an LH1–RC monomer lacking protein-U (ΔU). The native dimer reveals several asymmetric features including the arrangement of its two monomeric components, the structural integrity of protein-U, the overall organization of LH1, and rigidities of the proteins and pigments. PufX plays a critical role in connecting the two monomers in a dimer, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in the other monomer. One protein-U was only partially resolved in the dimeric structure, signaling different degrees of disorder in the two monomers. The ΔU LH1–RC monomer was half-moon-shaped and contained 11 α- and 10 β-polypeptides, indicating a critical role for protein-U in controlling the number of αβ-subunits required for dimer assembly and stabilization. These features are discussed in relation to membrane topology and an assembly model proposed for the native dimeric complex., Springer Science and Business Media LLC
Nature Communications, 2022年12月

An LH1–RC photocomplex from an extremophilic phototroph provides insight into origins of two photosynthesis proteins
Kazutoshi Tani; Ryo Kanno; Keigo Kurosawa; Shinichi Takaichi; Kenji V. P. Nagashima; Malgorzata Hall; Long-Jiang Yu; Yukihiro Kimura; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Zheng-Yu Wang-Otomo, Abstract

Rhodopila globiformis is the most acidophilic of anaerobic purple phototrophs, growing optimally in culture at pH 5. Here we present a cryo-EM structure of the light-harvesting 1–reaction center (LH1–RC) complex from Rhodopila globiformis at 2.24 Å resolution. All purple bacterial cytochrome (Cyt, encoded by the gene pufC) subunit-associated RCs with known structures have their N-termini truncated. By contrast, the Rhodopila globiformis RC contains a full-length tetra-heme Cyt with its N-terminus embedded in the membrane forming an α-helix as the membrane anchor. Comparison of the N-terminal regions of the Cyt with PufX polypeptides widely distributed in Rhodobacter species reveals significant structural similarities, supporting a longstanding hypothesis that PufX is phylogenetically related to the N-terminus of the RC-bound Cyt subunit and that a common ancestor of phototrophic Proteobacteria contained a full-length tetra-heme Cyt subunit that evolved independently through partial deletions of its pufC gene. Eleven copies of a novel γ-like polypeptide were also identified in the bacteriochlorophyll a-containing Rhodopila globiformis LH1 complex; γ-polypeptides have previously been found only in the LH1 of bacteriochlorophyll b-containing species. These features are discussed in relation to their predicted functions of stabilizing the LH1 structure and regulating quinone transport under the warm acidic conditions., Springer Science and Business Media LLC
Communications Biology, 2022年11月07日, ［査読有り］

Salt- and pH-Dependent Thermal Stability of Photocomplexes from Extremophilic Bacteriochlorophyll b-Containing Halorhodospira Species
Yukihiro Kimura; Kazuna Nakata; Shingo Nojima; Shinji Takenaka; Michael T. Madigan; Zheng-Yu Wang-Otomo, Halorhodospira (Hlr.) species are the most halophilic and alkaliphilic of all purple bacteria. Hlr. halochloris exhibits the lowest LH1 Qy transition energy among phototrophic organisms and is the only known triply extremophilic anoxygenic phototroph, displaying a thermophilic, halophilic, and alkaliphilic phenotype. Recently, we reported that electrostatic charges are responsible for the unusual spectroscopic properties of the Hlr. halochloris LH1 complex. In the present work, we examined the effects of salt and pH on the spectroscopic properties and thermal stability of LH1-RCs from Hlr. halochloris compared with its mesophilic counterpart, Hlr. abdelmalekii. Experiments in which the photocomplexes were subjected to different levels of salt or variable pH revealed that the thermal stability of LH1-RCs from both species was largely retained in the presence of high salt concentrations and/or at alkaline pH but was markedly reduced by lowering the salt concentration and/or pH. Based on the amino acid sequences of LH1 polypeptides and their composition of acidic/basic residues and the Hofmeister series for cation/anion species, we discuss the importance of electrostatic charge in stabilizing the Hlr. halochloris LH1-RC complex to allow it to perform photosynthesis in its warm, hypersaline, and alkaline habitat., MDPI AG
Microorganisms, 2022年05月02日

A Ca2+-binding motif underlies the unusual properties of certain photosynthetic bacterial core light-harvesting complexes
Kazutoshi Tani; Kazumi Kobayashi; Naoki Hosogi; Xuan-Cheng Ji; Sakiko Nagashima; Kenji V.P. Nagashima; Airi Izumida; Kazuhito Inoue; Yusuke Tsukatani; Ryo Kanno; Malgorzata Hall; Long-Jiang Yu; Isamu Ishikawa; Yoshihiro Okura; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Yukihiro Kimura; Zheng-Yu Wang-Otomo, Elsevier BV
Journal of Biological Chemistry, 2022年04月

Correction to: Allochromatium tepidum, sp. nov., a hot spring species of purple sulfur bacteria.
Michael T Madigan; Jill N Absher; Joseph E Mayers; Marie Asao; Deborah O Jung; Kelly S Bender; Megan L Kempher; Mackenzie K Hayward; Sophia A Sanguedolce; Abigail C Brown; Shinichi Takaichi; Ken Kurokawa; Atsushi Toyoda; Hiroshi Mori; Yusuke Tsukatani; Zheng-Yu Wang-Otomo; David M Ward; W Matthew Sattley
Archives of microbiology, 2022年03月11日

Identification of metal-sensitive structural changes in the Ca2+-binding photocomplex from Thermochromatium tepidum by isotope-edited vibrational spectroscopy
Yukihiro Kimura; Michie Imanishi; Li Yong; Yuki Yura; Takashi Ohno; Yoshitaka Saga; Michael T Madigan; Zheng-Yu Wang-Otomo, AIP Publishing
The Journal of Chemical Physics, 2022年02月14日

Allochromatium tepidum, sp. nov., a hot spring species of purple sulfur bacteria.
Michael T Madigan; Jill N Absher; Joseph E Mayers; Marie Asao; Deborah O Jung; Kelly S Bender; Megan L Kempher; Mackenzie K Hayward; Sophia A Sanguedolce; Abigail C Brown; Shinichi Takaichi; Ken Kurokawa; Atsushi Toyoda; Hiroshi Mori; Yusuke Tsukatani; Zheng-Yu Wang-Otomo; David M Ward; W Matthew Sattley, We describe a new species of purple sulfur bacteria (Chromatiaceae, anoxygenic phototrophic bacteria) isolated from a microbial mat in the sulfidic geothermal outflow of a hot spring in Rotorua, New Zealand. This phototroph, designated as strain NZ, grew optimally near 45 °C but did not show an absorption maximum at 915 nm for the light-harvesting-reaction center core complex (LH1-RC) characteristic of other thermophilic purple sulfur bacteria. Strain NZ had a similar carotenoid composition as Thermochromatium tepidum, but unlike Tch. tepidum, grew photoheterotrophically on acetate in the absence of sulfide and metabolized thiosulfate. The genome of strain NZ was significantly larger than that of Tch. tepidum but slightly smaller than that of Allochromatium vinosum. Strain NZ was phylogenetically more closely related to mesophilic purple sulfur bacteria of the genus Allochromatium than to Tch. tepidum. This conclusion was reached from phylogenetic analyses of strain NZ genes encoding 16S rRNA and the photosynthetic functional gene pufM, from phylogenetic analyses of entire genomes, and from a phylogenetic tree constructed from the concatenated sequence of 1090 orthologous proteins. Moreover, average nucleotide identities and digital DNA:DNA hybridizations of the strain NZ genome against those of related species of Chromatiaceae supported the phylogenetic analyses. From this collection of properties, we describe strain NZ here as the first thermophilic species of the genus Allochromatium, Allochromatium tepidum NZT, sp. nov.
Archives of microbiology, 2022年01月04日

A previously unrecognized membrane protein in the Rhodobacter sphaeroides LH1-RC photocomplex
Kazutoshi Tani; Kenji V. P. Nagashima; Ryo Kanno; Saki Kawamura; Riku Kikuchi; Malgorzata Hall; Long-Jiang Yu; Yukihiro Kimura; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Zheng-Yu Wang-Otomo, Rhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αβ-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general., Springer Science and Business Media LLC
Nature Communications, 2021年12月

Electrostatic charge controls the lowest LH1 Q(y) transition energy in the triply extremophilic purple phototrophic bacterium, Halorhodospira halochloris
Yukihiro Kimura; Shingo Nojima; Kazuna Nakata; Takuya Yamashita; Xiang-Ping Wang; Shinji Takenaka; Seiji Akimoto; Masayuki Kobayashi; Michael T. Madigan; Zheng-Yu Wang-Otomo; Long-Jiang Yu, Halorhodospira (Hlr.) halochloris is a unique phototrophic purple bacterium because it is a triple extremophile-the organism is thermophilic, alkalophilic, and halophilic. The most striking photosynthetic feature of Hlr. halochloris is that the bacteriochlorophyll (BChl) b-containing core light-harvesting (LH1) complex surrounding its reaction center (RC) exhibits its LH1 Q(y) absorption maximum at 1016 nm, which is the lowest transition energy among phototrophic organisms. Here we report that this extraordinarily red-shifted LH1 Q(y) band of Hlr. halochloris exhibits interconvertible spectral shifts depending on the electrostatic charge distribution around the BChl b molecules. The 1016 nm band of the Hlr. halochloris LH1-RC complex was blue-shifted to 958 nm upon desalting or pH decrease but returned to its original position when supplemented with salts or pH increase. Resonance Raman analysis demonstrated that these interconvertible spectral shifts are not associated with the strength of hydrogen-bonding interactions between BChl b and LH1 polypeptides. Furthermore, circular dichroism signals for the LH1 Q(y) transition of Hlr. halochloris appeared with a positive sign (as in BChl b-containing Blastochloris species) and opposite those of BChl a-containing purple bacteria, possibly due to a combined effect of slight differences in the transition dipole moments between BChl a and BChl b and in the interactions between adjacent BChls in their assembled state. Based on these findings and LH1 amino acid sequences, it is proposed that Hlr. halochloris evolved its unique and tunable light-harvesting system with electrostatic charges in order to carry out photosynthesis and thrive in its punishing hypersaline and alkaline habitat., ELSEVIER
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2021年11月

Photosynthetic Growth and Energy Conversion in an Engineered Phototroph Containing Thermochromatium tepidum Light-Harvesting Complex 1 and the Rhodobacter sphaeroides Reaction Center Complex
Kenji V. P. Nagashima; Sakiko Nagashima; Masaharu Kitashima; Kazuhito Inoue; Michael T. Madigan; Yukihiro Kimura; Zheng-Yu Wang-Otomo, Light-harvesting complex 1 (LH1) of the thermophilic purple sulfur bacterium Thermochromatium tepidum can be expressed in the purple non-sulfur bacterium Rhodobacter sphaeroides and forms a functional RC-LH1 complex with the native Rba. sphaeroides reaction center (Nagashima, K. V. P., et al. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 10906-10911). Although there is a large uphill energy gap between Tch. tepidum LH1 and the Rba. sphaeroides RC in this chimeric complex, it has been shown that light energy can be transferred, consistent with that seen in the native Rba. sphaeroides RC-LH1 complex. In this study, the contribution of this chimeric complex to growth and photosynthetic energy conversion in the hybrid organism was quantified. The mutant synthesizing this chimeric complex was grown phototrophically under 940 nm light-emitting diode (LED) light preferentially absorbed by Tch. tepidum LH1 and showed faster growth at low intensities of this wavelength than both a mutant strain of Rba. sphaeroides lacking LH2 and a mutant lacking all light-harvesting complexes. When grown with 850 nm LED light, the strain containing the native Rba. sphaeroides LH1-RC grew faster than the chimeric strain. Electron transfer from the RC to the membrane-integrated cytochrome bc(1) complex was also estimated by flash-induced absorption changes in heme b. The rate of ubiquinone transport through the LH1 ring structure in the chimeric strain was virtually the same as that in native Rba. sphaeroides. We conclude that Tch. tepidum LH1 can perform the physiological functions of native LH1 in Rba. sphaeroides., AMER CHEMICAL SOC
BIOCHEMISTRY, 2021年09月

Cryo-EM Structure of the Photosynthetic LH1-RC Complex from Rhodospirillum rubrum
Kazutoshi Tani; Ryo Kanno; Xuan-Cheng Ji; Malgorzata Hall; Long-Jiang Yu; Yukihiro Kimura; Michael T. Madigan; Akira Mizoguchi; Bruno M. Humbel; Zheng-Yu Wang-Otomo, Rhodospirillum (Rsp.) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl a(G)) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 angstrom resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 alpha beta-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the Q(A) site in the RC. The geranylgeraniol side chains of BChl a(G) coordinated by LH1 beta-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 alpha-associated BChls a(G). The structure also revealed key protein-protein interactions in both Nand C-terminal regions of the LH1 alpha beta-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex., AMER CHEMICAL SOC
BIOCHEMISTRY, 2021年08月

Exciton Origin of Color-Tuning in Ca2+-Binding Photosynthetic Bacteria
Kou Timpmann; Margus Ratsep; Liina Kangur; Alexandra Lehtmets; Zheng-Yu Wang-Otomo; Arvi Freiberg, Flexible color adaptation to available ecological niches is vital for the photosynthetic organisms to thrive. Hence, most purple bacteria living in the shade of green plants and algae apply bacteriochlorophyll a pigments to harvest near infra-red light around 850-875 nm. Exceptions are some Ca2+-containing species fit to utilize much redder quanta. The physical basis of such anomalous absorbance shift equivalent to similar to 5.5 kT at ambient temperature remains unsettled so far. Here, by applying several sophisticated spectroscopic techniques, we show that the Ca2+ ions bound to the structure of LH1 core light-harvesting pigment-protein complex significantly increase the couplings between the bacteriochlorophyll pigments. We thus establish the Ca-facilitated enhancement of exciton couplings as the main mechanism of the record spectral red-shift. The changes in specific interactions such as pigment-protein hydrogen bonding, although present, turned out to be secondary in this regard. Apart from solving the two-decade-old conundrum, these results complement the list of physical principles applicable for efficient spectral tuning of photo-sensitive molecular nano-systems, native or synthetic., MDPI
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 2021年07月

Crystal structure of a photosynthetic LH1-RC in complex with its electron donor HiPIP
Tomoaki Kawakami; Long-Jiang Yu; Tai Liang; Koudai Okazaki; Michael T. Madigan; Yukihiro Kimura; Zheng-Yu Wang-Otomo, Photosynthetic electron transfers occur through multiple components ranging from small soluble proteins to large integral membrane protein complexes. Co-crystallization of a bacterial photosynthetic electron transfer complex that employs weak hydrophobic interactions was achieved by using high-molar-ratio mixtures of a soluble donor protein (high-potential iron-sulfur protein, HiPIP) with a membrane-embedded acceptor protein (reaction center, RC) at acidic pH. The structure of the co-complex offers a snapshot of a transient bioenergetic event and revealed a molecular basis for thermodynamically unfavorable interprotein electron tunneling. HiPIP binds to the surface of the tetraheme cytochrome subunit in the light-harvesting (LH1) complex-associated RC in close proximity to the low-potential heme-1 group. The binding interface between the two proteins is primarily formed by uncharged residues and is characterized by hydrophobic features. This co-crystal structure provides a model for the detailed study of long-range trans-protein electron tunneling pathways in biological systems. The high potential iron-sulfur (HiPIP) proteins are direct electron donors to the light-harvesting-reaction center complexes (LH1-RC) in photosynthetic beta- and gamma -Proteobacteria. Here, the authors present the 2.9 angstrom crystal structure of the HiPIP-bound LH1-RC complex from the thermophilic purple sulfur bacterium Thermochromatium tepidum and discuss mechanistic implications for the electron transfer pathway., NATURE RESEARCH
NATURE COMMUNICATIONS, 2021年02月

Quinone transport in the closed light-harvesting 1 reaction center complex from the thermophilic purple bacterium Thermochromatium tepidum
Rikako Kishi; Michie Imanishi; Masayuki Kobayashi; Shinji Takenaka; Michael T. Madigan; Zheng-Yu Wang-Otomo; Yukihiro Kimura, Redox-active quinones play essential roles in efficient light energy conversion in type-II reaction centers of purple phototrophic bacteria. In the light-harvesting 1 reaction center (LH1-RC) complex of purple bacteria, Q(B) is converted to Q(B)H(2) upon light-induced reduction and Q(B)H(2) is transported to the quinone pool in the membrane through the LH1 ring. In the purple bacterium Rhodobacter sphaeroides, the C-shaped LH1 ring contains a gap for quinone transport. In contrast, the thermophilic purple bacterium Thermochromatium (Tch.) tepidum has a closed O-shaped LH1 ring that lacks a gap, and hence the mechanism of photosynthetic quinone transport is unclear. Here we detected light-induced Fourier transform infrared (FTIR) signals responsible for changes of Q(B) and its binding site that accompany photosynthetic quinone reduction in Tch. tepidum and characterized Q(B) and Q(B)H(2) marker bands based on their N-15- and C-13-isotopic shifts. Quinone exchanges were monitored using reconstituted photosynthetic membranes comprised of solubilized photosynthetic proteins, membrane lipids, and exogenous ubiquinone (UQ) molecules. In combination with C-13-labeling of the LH1-RC and replacement of native UQ(8) by ubiquinones of different tail lengths, we demonstrated that quinone exchanges occur efficiently within the hydrophobic environment of the lipid membrane and depend on the side chain length of UQ. These results strongly indicate that unlike the process in Rba. sphaeroides, quinone transport in Tch. tepidum occurs through the size-restricted hydrophobic channels in the closed LH1 ring and are consistent with structural studies that have revealed narrow hydrophobic channels in the Tch. tepidum LH1 transmembrane region., ELSEVIER
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2021年01月

Cryo-EM structure of a Ca2+-bound photosynthetic LH1-RC complex containing multiple alpha beta-polypeptides
Kazutoshi Tani; Ryo Kanno; Yuki Makino; Malgorzata Hall; Mizuki Takenouchi; Michie Imanishi; Long-Jiang Yu; Joerg Overmann; Michael T. Madigan; Yukihiro Kimura; Akira Mizoguchi; Bruno M. Humbel; Zheng-Yu Wang-Otomo, The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 angstrom resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the alpha beta -polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the alpha beta -polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of alpha- and beta -polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside. Here the authors report a cryo-EM structure of the light-harvesting-reaction center complex (LH1- RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970, providing insights into the mechanisms that underlie the absorbance properties of both the LH1 and the RC of this spectrally unusual purple bacterium., NATURE RESEARCH
NATURE COMMUNICATIONS, 2020年10月

The two light-harvesting membrane chromoproteins of Thermochromatium tepidum expose distinct robustness against temperature and pressure
Liina Kangur; Margus Ratsep; Kou Timpmann; Zheng-Yu Wang-Otomo; Arvi Freiberg, An increased robustness against high temperature and the much red-shifted near-infrared absorption spectrum of excitons in the LH1-RC core pigment-protein complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted much interest. In the present work, thermal and hydrostatic pressure stability of the peripheral LH2 and core LH1-RC complexes from this bacterium were in parallel investigated by various optical spectroscopy techniques applied over a wide spectral range from farultraviolet to near-infrared. In contrast to expectations, very distinct robustness of the complexes was established, while the sturdiness of LH2 surpassed that of LH1-RC both with respect to temperatures between 288 and 360 K, and pressures between 1 bar and 14 kbar. Subtle structural variances related to the hydrogen bond network are likely responsible for the extra stability of LH2., ELSEVIER
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2020年08月

Lycopene-Family Carotenoids Confer Thermostability on Photocomplexes from a New Thermophilic Purple Bacterium
Ryuta Seto; Shinichi Takaichi; Toshiyuki Kurihara; Rikako Kishi; Mai Honda; Shinji Takenaka; Yusuke Tsukatani; Michael T. Madigan; Zheng-Yu Wang-Otomo; Yukihiro Kimura, Blastochloris tepida is a newly described thermophilic purple bacterium containing bacteriochlorophyll b. Using purified light-harvesting 1 reaction center (LH1-RC) core complexes from Blc. tepida, we compared the biochemical, spectroscopic, and thermal denaturation properties of these complexes with those of its mesophilic counterpart, Blc. viridis. Besides their growth temperature optima, a striking difference between the two species was seen in the carotenoid composition of their LH1-RC complexes. The more thermostable Blc. tepida complex contained more carotenoids with longer conjugation lengths (n > 9), such as lycopenes (n = 11), and had a total carotenoid content significantly higher than that of the Blc. viridis complex, irrespective of the light intensity used for growth. The thermostability of LH1-RCs from both Blc. tepida and Blc. viridis decreased significantly in cells grown in the presence of diphenylamine, a compound that inhibits the formation of highly conjugated carotenoids. In contrast to the thermophilic purple bacterium Thermochromatium tepidum, where Ca2+ is essential for LH1-RC thermostability, Ca2+ neither was present in nor had any effect on the thermostability of the Blc. tepida LH1 RC. These results point to a mechanism that carotenoids with elongated conjugations enhance hydrophobic interactions with proteins in the Blc. tepida LH1-RC, thereby allowing the complexes to withstand thermal denaturation. This conclusion is bolstered by a structural model of the Blc. tepida LH 1-RC and is the first example of photocomplex thermostability being linked to a carotenoid-based mechanism., AMER CHEMICAL SOC
BIOCHEMISTRY, 2020年06月

Direct injection of pigment–protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC
Shinichi Takaichi; Akira Okoshi; Seiu Otomo; Masahiro Misumi; Kintake Sonoike; Jiro Harada, Springer Science and Business Media {LLC}
Photosynthesis Research, 2020年04月20日, ［査読有り］

Blastochloris tepida, sp. nov., a thermophilic species of the bacteriochlorophyll b-containing genus Blastochloris
Michael T. Madigan; Sol M. Resnick; Megan L. Kempher; Alice C. Dohnalkova; Shinichi Takaichi; Zheng-Yu Wang-Otomo; Atsushi Toyoda; Ken Kurokawa; Hiroshi Mori; Yusuke Tsukatani, A new taxon is created for the thermophilic purple nonsulfur bacterium previously designated as Rhodopseudomonas strain GI. Strain GI was isolated from a New Mexico (USA) hot spring microbial mat and grows optimally above 40 degrees C and to a maximum of 47 degrees C. Strain GI is a bacteriochlorophyll b-containing species of purple nonsulfur bacteria and displays a budding morphology, typical of species of the genus Blastochloris. Although resembling the species Blc. viridis in many respects, the absorption spectrum, carotenoid content, and lipid fatty acid profile of strain GI is distinct from that of Blc. viridis strain DSM133(T) and other recognized Blastochloris species. Strain GI forms its own subclade within the Blastochloris clade of purple nonsulfur bacteria based on comparative 16S rRNA gene sequences, and its genome is significantly larger than that of strain-DSM133(T); average nucleotide identity between the genomes of Blc. viridis and strain GI was below 85%. Moreover, concatenated sequence analyses of PufLM and DnaK clearly showed strain GI to be distinct from both Blc. viridis and Blc. sulfoviridis. Because of its unique assortment of properties, it is proposed to classify strain GI as a new species of the genus Blastochloris, as Blc. tepida, sp.n., with strain-GI(T) designated as the type strain (= ATCC TSD-138 = DSM 106918)., SPRINGER
ARCHIVES OF MICROBIOLOGY, 2019年12月

A Dual Role for Ca2+ in Expanding the Spectral Diversity and Stability of Light-Harvesting 1 Reaction Center Photocomplexes of Purple Phototrophic Bacteria
Michie Imanishi; Mizuki Takenouchi; Shinichi Takaich; Shiori Nakagawa; Yoshitaka Saga; Shinji Takenaka; Michael T. Madigan; Joerg Overmann; Zheng-Yu Wang-Otomo; Yukihiro Kimura, The light-harvesting 1 reaction center (LH1-RC) complex in the purple sulfur bacterium Thiorhodovibrio (Trv.) strain 970 cells exhibits its LH1 Q(y) transition at 973 nm, the lowest-energy Q(y) absorption among purple bacteria containing bacteriochlorophyll a (BChl a). Here we characterize the origin of this extremely red-shifted Qy transition. Growth of Try. strain 970 did not occur in cultures free of Ca2+, and elemental analysis of Ca2+-grown cells confirmed that purified Try. strain 970 LH1-RC complexes contained Ca2+. The LH1 Qy band of Try. strain 970 was blue-shifted from 959 to 875 nm upon Ca2+ depletion, but the original spectral properties were restored upon Ca2+ reconstitution, which also occurs with the thermophilic purple bacterium Thermochromatium (Tch.) tepidum. The amino acid sequences of the LH1 alpha- and beta-polypeptides from Try. strain 970 closely resemble those of Tch. tepidum; however, Ca2+ binding in the Try. strain 970 LH1-RC occurred more selectively than in Tch. tepidum LH1-RC and with a reduced affinity. Ultraviolet resonance Raman analysis indicated that the number of hydrogen-bonding interactions between BChl a and LH1 proteins of Try. strain 970 was significantly greater than for Tch. tepidum and that Ca2+ was indispensable for maintaining these bonds. Furthermore, perfusion-induced Fourier transform infrared analyses detected Ca2+-induced conformational changes in the binding site closely related to the unique spectral properties of Try. strain 970. Collectively, our results reveal an ecological strategy employed by Try. strain 970 of integrating Ca2+ into its LH1-RC complex to extend its light-harvesting capacity to regions of the near-infrared spectrum unused by other purple bacteria., AMER CHEMICAL SOC
BIOCHEMISTRY, 2019年06月

Phospholipid distributions in purple phototrophic bacteria and LH1-RC core complexes.
Nagatsuma S; Gotou K; Yamashita T; Yu LJ; Shen JR; Madigan MT; Kimura Y; Wang-Otomo ZY
Biochimica et biophysica acta. Bioenergetics, 2019年06月, ［査読有り］

Novel features of LH1-RC from Thermochromatium tepidum revealed from its atomic resolution structure
Yu Long-Jiang; Suga Michihiro; Wang-Otomo Zheng-Yu; Shen Jian-Ren, Light-harvesting-1 (LH1)-reaction center (RC) super-complex is a membrane protein-pigment complex existing in purple photosynthetic bacteria, where LH1 absorbs light energy and transfers them rapidly and efficiently to RC to initiate the charge separation and electron transfer reactions. The structure of LH1-RC has been reported at relatively low resolutions from several different species of bacteria previously, but was solved at an atomic resolution recently from a thermophilic photosynthetic bacterium Thermochromatium tepidum. This high-resolution structure revealed the detailed organization of the super-complex including a number of unique features that are important for its functioning, such as a more intact RC structure, transporting routes for quinones to replace the bound QB as well as for the in-and-out of the closed LH1 ring, detailed coordinating environment of the Ca2+ ions in LH1 important for the remarkable red shift of the absorption spectrum, as well as for the enhanced thermostability. These results thus greatly advance our understanding on the mechanisms of energy transfer, quinone exchange, the red shift in the LH1-Qy transition and the enhanced thermal stability, in this super-complex.
FEBS JOURNAL, 2018年12月, ［査読有り］

Cooperative Photoprotection by Multicompositional Carotenoids in the LH1 Antenna from a Mutant Strain of Rhodobacter sphaeroides
Yu Jie; Tan Li-Ming; Kawakami Tomoaki; Wang Peng; Fu Li-Min; Wang-Otomo Zheng-Yu; Zhang Jian-Ping
JOURNAL OF PHYSICAL CHEMISTRY B, 2018年08月23日, ［査読有り］

Biochemical and Spectroscopic Characterizations of a Hybrid Light-Harvesting Reaction Center Core Complex
Kimura Yukihiro; Hashimoto Kanako; Akimoto Seiji; Takenouchi Mizuki; Suzuki Kengo; Kishi Rikako; Imanishi Michie; Takenaka Shinji; Madigan Michael T; Nagashima Kenji V. P; Wang-Otomo Zheng-Yu, The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium tepidum exhibits a largely red-shifted LH1 Q(y) absorption at 915 nm due to binding of Ca2+, resulting in an "uphill" energy transfer from LH1 to the reaction center (RC). In a recent study, we developed a heterologous expression system (strain TS2) to construct a functional hybrid LH1-RC with LH1 from Tch. tepidum and the RC from Rhodobacter sphaeroides [Nagashima, K. V. P., et al. (2017) Proc. Natl. Acad. Sci. U.S. A. 114, 10906]. Here, we present detailed characterizations of the hybrid LH1-RC from strain TS2. Effects of metal cations on the phototrophic growth of strain TS2 revealed that Ca2+ is an indispensable element for its growth, which is also true for Tch. tepidum but not for Rba. sphaeroides. The thermal stability of the TS2 LH1-RC was strongly dependent on Ca2+ in a manner similar to that of the native Tch. tepidum, but interactions between the heterologous LH1 and RC became relatively weaker in strain TS2. A Fourier transform infrared analysis demonstrated that the Ca2+-binding site of TS2 LH1 was similar but not identical to that of Tch. tepidum. Steady-state and time-resolved fluorescence measurements revealed that the uphill energy transfer rate from LH1 to the RC was related to the energy gap in an order of Rba. sphaeroides, Tch. tepidum, and strain TS2; however, the quantum yields of LH1 fluorescence did not exhibit such a correlation. On the basis of these findings, we discuss the roles of Ca2+, interactions between LH1 and the RC from different species, and the uphill energy transfer mechanisms., AMER CHEMICAL SOC
BIOCHEMISTRY, 2018年07月31日, ［査読有り］

New Insights into the Mechanism of Uphill Excitation Energy Transfer from Core Antenna to Reaction Center in Purple Photosynthetic Bacteria
Li-Ming Tan; Jie Yu; Tomoaki Kawakami; Masayuki Kobayashi; Peng Wang; Zheng-Yu Wang-Otomo; Jian-Ping Zhang, The uphill excitation energy transfer (EET) from the core antenna (LH1) to the reaction center (RC) of purple photosynthetic bacteria was investigated at room temperature by comparing the native LH1-RC from Thermochromatium (Tch.) tepidum with the hybrid LH1-RC from a mutant strain of Rhodobacter (Rba.) sphaeroides. The latter protein with chimeric Tch-LH1 and Rba-RC exhibits a substantially larger RC-to-LH1 energy difference (ΔE = 630 cm-1) of 3-fold thermal energy (3kBT). The spectroscopic and kinetics results are discussed on the basis of the newly reported high-resolution structures of Tch. tepidum LH1-RC, which allow us to propose the existence of a passage formed by LH1 BChls that facilitates the LH1 → RC EET. The semilogarithmic plot of the EET rate against ΔE was found to be linear over a broad range of ΔE, which consolidates the mechanism of thermal activation as promoted by the spectral overlap between the LH1 fluorescence and the special pair absorption of RC., American Chemical Society
Journal of Physical Chemistry Letters, 2018年06月21日, ［査読有り］

Properties and structure of a low-potential, penta-heme cytochrome c552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum
Jing-Hua Chen; Long-Jiang Yu; Alain Boussac; Zheng-Yu Wang-Otomo; Tingyun Kuang; Jian-Ren Shen, The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron–sulfur protein, cytochrome c′, and one of two low-potential cytochrome c552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum., Springer Netherlands
Photosynthesis Research, 2018年04月24日, ［査読有り］

Structure of photosynthetic LH1-RC supercomplex at 1.9 Å resolution
Long-Jiang Yu; Michihiro Suga; Zheng-Yu Wang-Otomo; Jian-Ren Shen, Light-harvesting complex 1 (LH1) and the reaction centre (RC) form a membrane-protein supercomplex that performs the primary reactions of photosynthesis in purple photosynthetic bacteria. The structure of the LH1-RC complex can provide information on the arrangement of protein subunits and cofactors
however, so far it has been resolved only at a relatively low resolution. Here we report the crystal structure of the calcium-ion-bound LH1-RC supercomplex of Thermochromatium tepidum at a resolution of 1.9 Å. This atomic-resolution structure revealed several new features about the organization of protein subunits and cofactors. We describe the loop regions of RC in their intact states, the interaction of these loop regions with the LH1 subunits, the exchange route for the bound quinone QB with free quinone molecules, the transport of free quinones between the inside and outside of the LH1 ring structure, and the detailed calcium-ion-binding environment. This structure provides a solid basis for the detailed examination of the light reactions that occur during bacterial photosynthesis., Nature Publishing Group
Nature, 2018年04月01日, ［査読有り］

C-terminal cleavage of the LH1 α-polypeptide in the Sr2+-cultured Thermochromatium tepidum
Yukihiro Kimura; Tomoaki Kawakami; Teruhisa Arikawa; Yong Li; Long-Jiang Yu; Takashi Ohno; Michael T. Madigan; Zheng-Yu Wang-Otomo, The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Qy absorption at 915 nm and enhanced thermostability. Ca2+ can be biosynthetically replaced by Sr2+ in growing cultures of Tch. tepidum. However, the resulting Sr2+-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca2+-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca2+- and Sr2+-cultured cells. In this study, we examined the effects of the biosynthetic Sr2+-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr2+-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr2+-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr2+ substitutes for Ca2+ in the LH1 complex. Limited proteolysis of the native Ca2+-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr2+-substituted LH1-RC complexes in their Ca2+-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum., Springer Netherlands
Photosynthesis Research, 2018年03月01日, ［査読有り］

Carotenoid Singlet Fission Reactions in Bacterial Light Harvesting Complexes As Revealed by Triplet Excitation Profiles
Jie Yu; Li-Min Fu; Long-Jiang Yu; Ying Shi; Peng Wang; Zheng-Yu Wang-Otomo; Jian-Ping Zhang, Carotenoids (Cars) in bacterial photosynthesis are known as accessory light harvesters and photoprotectors. Recently, the singlet fission (SF) reaction initiated by Car photoabsorption has been recognized to be an effective excitation deactivation channel disfavoring the light harvesting function. Since the SF reaction and the triplet sensitization reaction underlying photoprotection both yield triplet excited state Cars ((3)Car*), their contribution to the overall (3)Car* photoproduction are difficult to disentangle. To tackle this problem, we resorted to the triplet excitation profiles (TEPs), i.e., the actinic spectra of the overall (3)Car* photoproduction. The TEPs combined with the conventional fluorescence excitation spectra allowed us to extract the neat SF contribution, which can serve as a spectroscopic measure for the SF reactivity. This novel spectroscopic strategy was applied to analyze the light harvesting complexes (LHs) from Tch. tepidum and Rba. sphaeroides 2.4.1. The results unambiguously showed that the SF reaction of Cars proceeds with an intramolecular scheme, even in the case of LH1-RC from Rba. sphaeroides 2.4.1 likely binding a secondary pool of Cars. Regarding the SF-reactivity, the geometric distortion in the conjugated backbone of Cars was shown to be the structural determinant, while the length of the Car conjugation was suggested to be relevant to the effective localization of the geminate triplets to avoid being annihilated. The SF reaction scheme and structure-activity relationship revealed herein will be useful not only in deepening our understanding of the roles of Cars in photosynthesis, but also in enlightening the applications of Cars in artificial light conversion systems., AMER CHEMICAL SOC
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2017年11月, ［査読有り］

Spectrally Selective Spectroscopy of Native Ca-Containing and Ba-Substituted LH1-RC Core Complexes from Thermochromatium tepidum
Margus Ratsep; Kou Timpmann; Tomoaki Kawakami; Zheng-Yu Wang-Otomo; Arvi Freiberg, The LH1-RC core complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted interest of many researchers because of its several unique properties, such as increased robustness against environmental hardships and the much red-shifted near infrared absorption spectrum of the LH1 antenna exciton polarons. The known near-atomic-resolution crystal structure of the complex well supported this attention. Yet several mechanistic aspects of the complex prominence remained to be understood. In this work, samples of the native, Ca2+-containing core complexes were investigated along with those destabilized by Ba2+ substitution, using various spectrally selective steady-state and picosecond time-resolved spectroscopic techniques at physiological and cryogenic temperatures. As a result, the current interpretation of exciton spectra of the complex was significantly clarified. Specifically, by evaluating the homogeneous and inhomogeneous compositions of the spectra, we showed that there is little to no effect of cation substitution on the dynamic or kinetic properties of antenna excitons. Reasons of the extra red shift of absorption/fluorescence spectra observed in the Ca-LH1-RC and not in the Ba-LH1-RC complex should thus be searched in subtle structural differences following the inclusion of different cations into the core complex scaffold., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2017年11月, ［査読有り］

Probing structure-function relationships in early events in photosynthesis using a chimeric photocomplex
Kenji V. P. Nagashima; Mai Sasaki; Kanako Hashimoto; Shinichi Takaichi; Sakiko Nagashima; Long-Jiang Yu; Yuto Abe; Kenta Gotou; Tomoaki Kawakami; Mizuki Takenouchi; Yuuta Shibuya; Akira Yamaguchi; Takashi Ohno; Jian-Ren Shen; Kazuhito Inoue; Michael T. Madigan; Yukihiro Kimura; Zheng-Yu Wang-Otomo, The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per alpha beta-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that alpha-D49, beta-L46, and a deletion at position 43 of the alpha-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, alpha-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex., NATL ACAD SCIENCES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2017年10月, ［査読有り］

Metal Cations Induced alpha beta-BChl a Heterogeneity in LH1 as Revealed by Temperature-Dependent Fluorescence Splitting
Fei Ma; Long-Jiang Yu; Manuel J. Llansola-Portoles; Bruno Robert; Zheng-Yu Wang-Otomo; Rienk van Grondelle, Two spectral forms of the core light-harvesting complex (LH1) of the purple bacterium Thermochromatium (Tch.) tepidum, the native Ca2+-binding and the Ba2+-substituted one, exhibit different fluorescence (FL) emission spectra at low temperature (T). While Ca-LH1 exhibits one emission band, an unusual splitting of the fluorescence is observed for Ba-LH1. These two sub-bands display the same spectral-width dependence according to T, but their intensity evolves differently with T. Based on the crystal structures, we propose that the FL splitting originates from a large alpha beta-BChl a transition energy heterogeneity, approximate to 600 cm(-1), which is much larger compared with other LH1 and LH2 complexes (80-200 cm(-1)). This large heterogeneity is induced by the inhomogeneous Coulomb (and possibly hydrogen-bonding) interactions exerted by Ba2+. The energy levels of the two LH1s were compared using exciton calculations in combination with Redfield theory. To simulate the FL splitting, an electronic transition containing two resonant bands was considered. This work shows how metal cations incorporated into the polypeptide modulate the electronic properties of BChl a aggregates., WILEY-V C H VERLAG GMBH
CHEMPHYSCHEM, 2017年08月, ［査読有り］

Excitonic and Vibrational Coherence in the Excitation Relaxation Process of Two LH1 Complexes as Revealed by Two-Dimensional Electronic Spectroscopy
Fei Ma; Long-Jiang Yu; Ruud Hendrikx; Zheng-Yu Wang-Otomo; Rienk van Grondelle, Ultrafast excitation relaxation within a manifold exciton state and longlived vibrational coherence are two universal characteristics of photosynthetic antenna complexes. In this work, we studied the two-dimensional electronic spectra of two core light-harvesting (LH1) complexes of Thermochromatium (Tch.) tepidum, native Ca2+-LH1 and modified Ba2+-LH1. The role of the vibrational coherence in the exciton relaxation was revealed by comparing the two LH1 with similar structures but different electronic properties and by the evolution of the exciton and vibrational coherence as a function of temperature., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY LETTERS, 2017年06月, ［査読有り］

Dependence of the hydration status of bacterial light-harvesting complex 2 on polyol cosolvents
Ying Shi; Jie Yu; Long-Jiang Yu; Peng Wang; Li-Min Fu; Jian-Ping Zhang; Zheng-Yu Wang-Otomo, Low molecular weight (MW) polyols are organic osmolytes influencing protein structure and activity. We have intended to investigate the effects of low MW polyols on the optical and the excited-state properties of the light-harvesting complex 2 (LH2) isolated from the photosynthetic bacterium Thermochromatium (Tch.) tepidum, a thermophile growing at similar to 50 degrees C. Steady state spectroscopy demonstrated that, on increasing glycerol or sorbitol fractions up to 60% (polyol/water, v/v), the visible absorption of carotenoids (Crts) remained unchanged, while the near infrared Q(y) absorption of bacteriochlorophyll a (BChl) at 800 nm (B800) and 850 nm (B850) varied slightly. Further increasing the fraction of glycerol (but not sorbitol) to 80% (v/v) induced distinct changes of the near infrared absorption and fluorescence spectra. Transient absorption spectroscopy revealed that, following the fast processes of BChl-to-Crt triplet energy transfer, rather weak Q(y) signals of B800 and B850 remained and evolved in phase with the kinetics of triplet excited state Crt ((3)Crt*), which are attributed to the Q(y) band shift as a result of (3)Crt*-BChl interaction. The steady state and the transient spectral responses of the Q(y) bands are found to correlate intimately with the water activity varying against polyol MW and mixing ratio, which are rationalized by the change of the hydration status of the C-and N-termini of LH2. Our results suggest that, with reference to the mesophilic purple bacterium Rhodobacter sphaeroides 2.4.1, Tch. tepidum adopts substantially more robust LH2 hydration against the osmotic effects from the low MW polyols., ROYAL SOC CHEMISTRY
PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 2017年05月, ［査読有り］

Effects of Calcium Ions on the Thermostability and Spectroscopic Properties of the LH1-RC Complex from a New Thermophilic Purple Bacterium Allochromatium tepidum
Yukihiro Kimura; Shuwen Lyu; Akira Okoshi; Koudai Okazaki; Natsuki Nakamura; Akira Ohashi; Takashi Ohno; Manami Kobayashi; Michie Imanishi; Shinichi Takaichi; Michael T. Madigan; Zheng-Yu Wang-Otomo, The light harvesting-reaction center (LH1-RC) complex from a new thermophilic purple sulfur bacterium Allochromatium (Alc.) tepidum was isolated and characterized by spectroscopic and thermodynamic analyses. The purified Alc. tepidum LH1-RC complex showed a high thermostability comparable to that of another thermophilic purple sulfur bacterium Thermochromatium tepidum, and spectroscopic characteristics similar to those of a mesophilic bacterium Alc. vinosum. Approximately 4-5 Ca2+ per LH1-RC were detected by inductively coupled plasma atomic emission spectroscopy and isothermal titration calorimetry. Upon removal of Ca2+, the denaturing temperature of the Alc. tepidum LH1-RC complex dropped accompanied by a blue-shift of the LH1 Q(y) absorption band. The effect of Ca2+ was also observed in the resonance Raman shift of the C3-acetyl vC=O band of bacteriochlorophyll-a, indicating changes in the hydrogen-bonding interactions between the pigment and LH1 polypeptides. Thermodynamic parameters for the Ca2+-binding to the Alc. tepidum LH1-RC complex indicated that this reaction is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. Our data support a hypothesis that Alc. tepidum may be a transitional organism between mesophilic and thermophilic purple bacteria and that Ca2+ is one of the major keys to the thermostability of LH1-RC complexes in purple bacteria., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2017年05月, ［査読有り］

Direct Observation of Energy Detrapping in LH1-RC Complex by Two-Dimensional Electronic Spectroscopy
Fei Ma; Long-Jiang Yu; Ruud Hendrikx; Zheng-Yu Wang-Otomo; Rienk van Grondelle, The purple bacterial core light harvesting antenna-reaction center (LH1-RC) complex is the simplest system able to achieve the entire primary function of photosynthesis. During the past decade, a variety of photosynthetic proteins were studied by a powerful technique, two-dimensional electronic spectroscopy (2DES). However, little attention has been paid to LH1-RC, although its reversible uphill energy transfer, trapping, and backward detrapping processes, represent a crucial step in the early photosynthetic reaction dynamics. Thus, in this work, we employed 2DES to study two LH1-RC complexes of Thermochromatium (Tch.) tepidum. By direct observation of detrapping, the complex reversible process was clearly identified and an overall scheme of the excitation evolution in LH1-RC was obtained., AMER CHEMICAL SOC
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2017年01月, ［査読有り］

Spectroscopic and Thermodynamic Characterization of the Metal-Binding Sites in the LH1-RC Complex from Thermophilic Photosynthetic Bacterium Thermochromatium tepidum
Yukihiro Kimura; Yuki Yura; Yusuke Hayashi; Yong Li; Moe Onoda; Long-Jiang Yu; Zheng-Yu Wang-Otomo; Takashi Ohno, The light-harvesting 1 reaction center (LH1- RC) complex from thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits enhanced thermo-stability and an unusual LH1 Q(gamma) transition, both induced by Ca2+ binding. In this study, metal-binding sites and metal protein interactions in the LH1 RC complexes from wild-type (B915) and biosynthetically Sr2+-substituted (B888) Tch. tepidum were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopies. The ITC measurements revealed stoichiometric ratios of approximately 1:1 for binding of Ca2+, Sr2+, or Ba2+ to the LH1 alpha beta-subunit, indicating the presence of 16 binding sites in both B915 and B888. The AA analysis provided direct evidence for Ca2+ and Sr2+ binding to B915 and B888, respectively, in their purified states. Metal-binding experiments supported that Ca2+ and Sr2+ (or Ba2+) competitively associate with the binding sites in both species. The ATR-FTIR difference spectra upon Ca2+ depletion and Sr2+ substitution demonstrated that dissociation and binding of Ca2+ are predominantly responsible for metal-dependent conformational changes of B915 and B888. The present results are largely compatible with the recent structural evidence that another binding site for Sr2+ (or Ba2+) exists in the vicinity of the Ca2+-binding site, a part of which is shared in both metal-binding sites., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2016年12月, ［査読有り］

Structural Basis for the Unusual Q(y) Red-Shift and Enhanced Thermostability of the LH1 Complex from Thermochromatium tepidum
Long-Jiang Yu; Tomoaki Kawakami; Yukihiro Kimura; Zheng-Yu Wang-Otomo, While the majority of the core light-harvesting complexes (LH1) in purple photosynthetic bacteria exhibit a Q(y) absorption band in the range of 870-890 nm, LH1 from the thermophilic bacterium Thermochromatium tepidum displays the Q(y) band at 915 nm with an enhanced thermostability. These properties are regulated by Ca2+ ions. Substitution of the Ca2+ with other divalent metal ions results in a complex with the Q(y) band blue-shifted to 880-890 nm and a reduced thermostability. Following the recent publication of the structure of the Ca-bound LH1-reaction center (RC) complex [Niwa, S., et al. (2014) Nature SOS, 228], we have determined the crystal structures of the Sr- and Ba-substituted LH1-RC complexes with the LH1 Q(y) band at 888 nm. Sixteen Sr2+ and Ba2+ ions are identified in the LH1 complexes. Both Sr2+ and Ba2+ are located at the same positions, and these are clearly different from, though close to, the Ca2+-binding sites. Conformational rearrangement induced by the substitution is limited to the metal binding sites. Unlike the Ca-LH1-RC complex, only the a-polypeptides are involved in the Sr and Ba coordinations in LH1. The difference in the thermostability between these complexes can be attributed to the different patterns of the network formed by metal binding. The Sr- and Ba-LH1-RC complexes form a single-ring network by the LH1 alpha-polypeptides only, in contrast to the double-ring network composed of both alpha- and beta-polypeptides in the Ca-LH1-RC complex. On the basis of the structural information, a combined effect of hydrogen bonding, structural integrity, and charge distribution is considered to influence the spectral properties of the core antenna complex., AMER CHEMICAL SOC
BIOCHEMISTRY, 2016年11月, ［査読有り］

Challenges facing an understanding of the nature of low-energy excited states in photosynthesis
Jeffrey R. Reimers; Malgorzata Biczysko; Douglas Bruce; David F. Coker; Terry J. Frankcombe; Hideki Hashimoto; Juergen Hauer; Ryszard Jankowiak; Tobias Kramer; Juha Linnanto; Fikret Mamedov; Frank Mueh; Margus Ratsep; Thomas Renger; Stenbjom Styring; Jian Wan; Zhuan Wang; Zheng-Yu Wang-Otomo; Yu-Xiang Weng; Chunhong Yang; Jian-Ping Zhang; Arvi Freiberg; Elmars Krausz, While the majority of the photochemical states and pathways related to the biological capture of solar energy are now well understood and provide paradigms for artificial device design, additional low-energy states have been discovered in many systems with obscure origins and significance. However, as low-energy states are naively expected to be critical to function, these observations pose important challenges. A review of known properties of low energy states covering eight photochemical systems, and options for their interpretation, are presented. A concerted experimental and theoretical research strategy is suggested and outlined, this being aimed at providing a fully comprehensive understanding. (C) 2016 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2016年09月, ［査読有り］

Bacterial Light-Harvesting Complexes Showing Giant Second-Order Nonlinear Optical Response as Revealed by Hyper-Rayleigh Light Scattering
Fei Ma; Long-Jiang Yu; Xiao-Hua Ma; Peng Wang; Zheng-Yu Wang-Otomo; Jian-Ping Zhang, The second-order nonlinear optical, (NLO) properties of light-harvesting complexes (LHs) from, the purple photosynthetic bacteria Thermochromatium (Tch.) tepidum were investigated for the first time by means of hyper-Rayleigh scattering (HRS). The carotenoid (Car) molecules bound to the isolated LH1 and LH2 proteins gave rise to second-harmonic Scattering; however, they showed an opposite effect of the collective contribution,from Car, that is, the first hyperpolarizability (beta) reduced substantially from (10 510 +/- 370) X 10(-30) esu for LH1 to (360 +/- 120) X 10(-30) esu for LH2 Chromatophores of Tch. tepidum also Showed a giant hyperpolarizability,of (11 640 +/- 630) x 10(-30) esu, On the basis of the structural information on :bacterial LHs, it is found that the effective, beta of an LH is governed by the microenvironment and orientational correlation among the Car chromophores, which is concluded to be coherently enhanced for LH1. For LH2, however, additional. destructive effects between different Car molecules may account for the small beta value. This work demonstrates that LH1 and native membranes of purple bacteria can be potent NLO materials and that HRS is a promising spectroscopic means for investigating Structural information of pigment-protein supramolecules., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2016年09月, ［査読有り］

Temperature dependent LH1 -> RC energy transfer in purple bacteria Tch. tepidum with shiftable LH1-Q(y) band: A natural system to investigate thermally activated energy transfer in photosynthesis
Fei Ma; Long-Jiang Yu; Zheng-Yu Wang-Otomo; Rienk van Grondelle, The native LH1-RC complex of the purple bacterium Thermochromatium (Tch.) tepidum has an ultra-red LH1-Q(y) absorption at 915 nm, which can shift to 893 and 882 nm by means of chemical modifications. These unique complexes are a good natural system to investigate the thermally activated energy transfer process, with the donor energies different while the other factors (such as the acceptor energy, special pair at 890 nm, and the distance/relative orientation between the donor and acceptor) remain the same. The native B915-RC, B893-RC and B882-RC complexes, as well as the LH1-RC complex of Rhodobacter (Rba.) sphaeroides were studied by temperature-dependent time-resolved absorption spectroscopy. The energy transfer time constants, k(ET)(-1), are 65, 45, 46 and 45 ps at room temperature while 225, 58, 85, 33 ps at 77 K. for the B915-RC, B893-RC, B882-RC and Rba. sphaeroides LH1-RC, respectively. The dependences of k(ET) on temperature have different trends. The reorganization energies are determined to be 70, 290, 200 and 45 cm(-1), respectively, by fitting k(ET) vs temperature using Marcus equation. The activation energies are 200, 60, 115 and 20 cm(-1), respectively. The influences of the structure (the arrangement of the 32 BChl a molecules) on k(ET) are discussed based on these results, to reveal how the B915-RC complex accomplishes its energy transfer function with a large uphill energy of 290 cm(-1). (C) 2015 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2016年04月, ［査読有り］

The origin of the unusual Q(y) red shift in LH1-RC complexes from purple bacteria Thermochromatium tepidum as revealed by Stark absorption spectroscopy
Fei Ma; Long-Jiang Yu; Zheng-Yu Wang-Otomo; Rienk van Grondelle, Native LH1-RC of photosynthetic purple bacteria Thennochromatium (Tch.) tepidum, B915, has an ultra-red BChl a Q(y) absorption. Two blue-shifted complexes obtained by chemical modification, B893 and B882, have increasing wfull widths at half maximum (FWHM) and decreasing transition dipole oscillator strength. 77K Stark absorption spectroscopy studies were employed for the three complexes, trying to understand the origin of the 915 nm absorption. We found that Tr(Delta alpha) and vertical bar Delta mu vertical bar of both Q(y) and carotenoid (Car) bands are larger than for other purple bacterial LH complexes reported previously. Moreover, the red shifts of the Q(y) bands are associated with (1) increasing Tr(Delta alpha) and vertical bar Delta mu vertical bar of the Q(y) band, (2) the red shift of the Car Stark signal and (3) the increasing vertical bar Delta mu vertical bar of the Car band. Based on the results and the crystal structure, a combined effect of exciton-charge transfer (CT) states mixing, and inhomogeneous narrowing of the BChl a site energy is proposed to be the origin of the 915 nm absorption. CT-exciton state mixing has long been found to be the origin of strong Stark signal in LH1 and special pair, and the more extent of the mixing in Tch. tepidum LH1 is mainly the consequence of the shorter BChl-BChl distances. The less flexible protein structure results in a smaller site energy disorder (inhomogeneous narrowing), which was demonstrated to be able to influence vertical bar Delta mu vertical bar and absorption. (C) 2015 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2015年12月, ［査読有り］

Thermal Adaptability of the Light-Harvesting Complex 2 from Thermochromatium tepidum: Temperature-Dependent Excitation Transfer Dynamics
Ying Shi; Ning-Jiu Zhao; Peng Wang; Li-Min Fu; Long-Jiang Yu; Jian-Ping Zhang; Zheng-Yu Wang-Otomo, The photosynthetic purple bacterium Thermochromatium (Tch.) tepidum is a thermophile that grows at an optimal temperature of similar to 50 degrees C. We have investigated, by means of steady-state and time-resolved optical spectroscopies, the effects of temperature on the near-infrared light absorption and the excitation energy transfer (EET) dynamics of its light-harvesting complex 2 (LH2), for which the mesophilic counterpart of Rhodobacter (Rba.) sphaeroides 2.4.1 (similar to 30 degrees C) was examined in comparison. In a limited range around the physiological temperature (10-55 degrees C), the B800-to-B850 EET process of the Tch. tepidum LH2, but not the Rba. sphaeroides LH2, was found to be characteristically temperature-dependent, mainly because of a temperature-tunable spectral overlap. At 55 degrees C, the LH2 complex from Tch. tepidum maintained efficient near-infrared light harvesting and B800-to-B850 EET dynamics, whereas this EET process was disrupted in the case of Rba. sphaeroides 2.4.1 owing to the structural distortion of the LH2 complex. Our results reveal a remarkable thermal adaptability of the light-harvesting function of Tch. tepidum, which could enhance our understanding of the survival strategy of this thermophile in response to environmental challenges., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2015年11月, ［査読有り］

Characterization of the quinones in purple sulfur bacterium Thermochromatium tepidum
Yuuka Kimura; Tomoaki Kawakami; Long-Jiang Yu; Miku Yoshimura; Masayuki Kobayashi; Zheng-Yu Wang-Otomo, Abstract Quinone distributions in the thermophilic purple sulfur bacterium Thermochromatium tepidum have been investigated at different levels of the photosynthetic apparatus. Here we show that, on average, the intracytoplasmic membrane contains 18 ubiquinones (UQ) and 4 menaquinones (MQ) per reaction center (RC). About one-third of the quinones are retained in the light-harvesting-reaction center core complex (LH1-RC) with a similar ratio of UQ to MQ. The numbers of quinones essentially remains unchanged during crystallization of the LH1-RC. There are 1-2 UQ and 1 MQ associated with the RC-only complex in the purified solution sample. Our results suggest that a large proportion of the quinones are confined to the core complex and at least five UQs remain invisible in the current LH1-RC crystal structure., Elsevier
FEBS Letters, 2015年07月03日, ［査読有り］

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c' from the thermophilic purple sulfur bacterium Thermochromatium tepidum
Yukihiro Kimura; Sachiko Kasuga; Masashi Unno; Takashi Furusawa; Shinsuke Osoegawa; Yuko Sasaki; Takashi Ohno; Zheng-Yu Wang-Otomo, A soluble cytochrome (Cyt) c' from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c' and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c' by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c'. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c', indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c'. The control Cyt c' and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c' exhibited characteristic bands at 1,635 and 1,625 cm(-1), ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c'., SPRINGER
PHOTOSYNTHESIS RESEARCH, 2015年04月, ［査読有り］

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum
Anne-Marie Carey; Kirsty Hacking; Nichola Picken; Suvi Honkanen; Sharon Kelly; Dariusz M. Niedzwiedzki; Robert E. Blankenship; Yuuki Shimizu; Zheng-Yu Wang-Otomo; Richard J. Cogdell, This study systematically investigated the different types of LH2 produced by Allochromatium (Ala) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and 0800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both alpha and beta polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila. (C) 2014 Published by Elsevier B.V., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2014年11月, ［査読有り］

Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 angstrom
Satomi Niwa; Long-Jiang Yu; Kazuki Takeda; Yu Hirano; Tomoaki Kawakami; Zheng-Yu Wang-Otomo; Kunio Miki, The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca2+-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 alpha beta-subunit that form a completely closed structure. The Ca2+ ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure., NATURE PUBLISHING GROUP
NATURE, 2014年04月, ［査読有り］

Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å
Satomi Niwa; Long-Jiang Yu; Kazuki Takeda; Yu Hirano; Tomoaki Kawakami; Zheng-Yu Wang-Otomo; Kunio Miki, The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca2+-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 alpha beta-subunit that form a completely closed structure. The Ca2+ ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure., NATURE PUBLISHING GROUP
Nature, 2014年04月, ［査読有り］

1P255 紅色硫黄細菌由来光捕集1反応中心複合体における金属-タンパク質間相互作用の熱力学的解析(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))
Kimura Yukihiro; Hayashi Yusuke; Otomo Seiu; Ohno Takashi, 一般社団法人 日本生物物理学会
生物物理, 2014年

1P253 同位体標識された好熱性紅色光合成細菌における膜タンパク質耐熱化の分子機構解析(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))
Yura Yuki; Kimura Yukihiro; Otomo Seiu; Ohno Takashi, 一般社団法人 日本生物物理学会
生物物理, 2014年

ATR-FTIR Detection of Metal-Sensitive Structural Changes in the Light-Harvesting 1 Reaction Center Complex from the Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum
Yong Li; Yukihiro Kimura; Teruhisa Arikawa; Zheng-Yu Wang-Otomo; Takashi Ohno, Thermochromatium tepidum grows at the highest temperature among purple bacteria, and the light-harvesting 1 reaction center (LIU-RC) complex enhances the thermal stability by utilizing Ca2+, although the molecular mechanism has yet to be resolved. Here, we applied perfusion-induced attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy to highly purified LH1-RC complexes from Tch. tepidum and detected for the first time metal-sensitive fine structural changes involved in the enhanced thermal stability of this complex. The Tch. tepidum LH1-RC complex exhibited Sr2/Ca2+ ATR FTIR difference bands that reflect 1800 1760 1660 1560 changes in the polypeptide backbones and amino acid residues upon the replacement of native Ca2+ with Sr2+. The difference bands also appeared in the following Ca27Sr(2+) difference spectra with almost identical intensities but inverse signs, demonstrating that the structural changes induced by the metal exchange are fully reversible. In contrast, these ATR FTIR signals were faintly detected in the mesophilic counterpart Allochromatium vinosum. A comparative analysis using LH1 complexes lacking the RCs strongly indicated that the metal-sensitive bands originate from polypeptide backbones and amino acid residues near the putative Ca2+-binding site at the C-terminal region of the Tch. tepidum LH1 complexes. Structural changes induced by Sr2+ and Ba2+ substitutions were essentially identical. However, Cd2+ substitution exhibited unique structural modifications, which may be responsible for the severely deteriorated thermal stability of Cd2+-substituted complexes. Possible assignments for the present ATR FTIR signals and their relation with the molecular mechanism of enhancing the thermal stability of Tch. tepidum LH1-RC proteins are discussed on the basis of the recent structural information on the Ca2+- binding site., AMER CHEMICAL SOC
BIOCHEMISTRY, 2013年12月, ［査読有り］

Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum
Long-Jiang Yu; Masaki Unno; Yukihiro Kimura; Kasumi Yanagimoto; Hirozo Oh-oka; Zheng-Yu Wang-Otomo, The cytochrome (Cyt) c-554 in thermophilic green photosynthetic bacterium Chlorobaculum tepidum serves as an intermediate electron carrier, transferring electrons to the membrane-bound Cyt c (z) from various enzymes involved in the oxidations of sulfide, thiosulfate, and sulfite compounds. Spectroscopically, this protein exhibits an asymmetric alpha-absorption band for the reduced form and particularly large paramagnetic H-1 NMR shifts for the heme methyl groups with an unusual shift pattern in the oxidized form. The crystal structure of the Cyt c-554 has been determined at high resolution. The overall fold consists of four alpha-helices and is characterized by a remarkably long and flexible loop between the alpha 3 and alpha 4 helices. The axial ligand methionine has S-chirality at the sulfur atom with its (CH3)-H-epsilon group pointing toward the heme pyrrole ring I. This configuration corresponds to an orientation of the lone-pair orbital of the sulfur atom directed at the pyrrole ring II and explains the lowest-field H-1 NMR shift arising from the 18(1) heme methyl protons. Differing from most other class I Cyts c, no hydrogen bond was formed between the methionine sulfur atom and polypeptide chain. Lack of this hydrogen bond may account for the observed large paramagnetic H-1 NMR shifts of the heme methyl protons. The surface-exposed heme pyrrole ring II edge is in a relatively hydrophobic environment surrounded by several electronically neutral residues. This portion is considered as an electron transfer gateway. The structure of the Cyt c-554 is compared with those of other Cyts c, and possible interactions of this protein with its electron transport partners are discussed., SPRINGER
PHOTOSYNTHESIS RESEARCH, 2013年12月, ［査読有り］

Strontium ions are functionally replaceable with calcium ions in the light-harvesting 1 reaction center core complex from thermophilic purple sulfur bacterium thermochromatium tepidum
Yukihiro Kimura; Yuta Inada; Longjiang Yu; Takashi Ohno; Zhengyu Wang, Thermochromatium tepidum (TTP) is a themophilic purple sulfur photosynthetic bacterium of which light-harvesting 1 reaction center (LH1RC) complexes exhibit an unusual LH1 Qy absorption at 915 nm and thermal resistance relative to those of mesophilic counterparts. Recently, we demonstrated that these unique properties were regulated by an inorganic cofactor, Ca2+. Wild-type TTP cells grew anaerobically at 50 °C in a culture medium containing a small amount of CaCl2. When Ca2+ was removed from the medium or replaced with other metal cations, the photosynthetic growth was largely suppressed, however, only Sr2+ was biofunctionally replaceable with Ca2+. The resulting Sr2+-substituted TTP (Sr2+-TTP) cells showed different spectral properties compared with the native ones in the LH1 Qy region. The LH1RC complex purified from Sr2+-TTP cells exhibited its Qy maximum at 888 nm (B888). Depletion of Sr2+ from the B888 species induced a blue-shift of the LH1 Qy peak and decreased the thermal stability. Upon the Sr2+-reconstitution, the LH1-RC recovered its thermal stability and Qy peak position depending on the Sr2+-concentration with an approximately 104 M−1 of binding constant. This is the first evidence for a functional LH1-variant which was prepared by the biosynthetic metal substitution., Springer
Advanced Topics in Science and Technology in China, 2013年

Structure Analysis and Comparative Characterization of the Cytochrome c ' and Flavocytochrome c from Thermophilic Purple Photosynthetiic Bacterium Thermochromatium tepidum
Yu Hirano; Yukihiro Kimura; Hideaki Suzuki; Kunio Miki; Zheng-Yu Wang, The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 degrees C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins., AMER CHEMICAL SOC
BIOCHEMISTRY, 2012年08月, ［査読有り］

Structure analysis and comparative characterization of the cytochrome c’ and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum
Yu Hirano; Yukihiro Kimura; Hideaki Suzuki; Kunio Miki; Zheng-Yu Wang, The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 degrees C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins., AMER CHEMICAL SOC
Biochemistry, 2012年08月, ［査読有り］

Metal cations modulate the bacteriochlorophyll-protein interaction in the light-harvesting 1 core complex from Thermochromatium tepidum
Yukihiro Kimura; Yuta Inada; Tomoko Numata; Teruhisa Arikawa; Yong Li; Jian-Ping Zhang; Zheng-Yu Wang; Takashi Ohno, The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium (Tch.) tepidum exhibits unusual Q(y) absorption by LH1 bacteriochlorophyll-a (BChl-a) molecules at 915 nm, and the transition energy is finely modulated by the binding of metal cations to the LH1 polypeptides. Here, we demonstrate the metal-dependent interactions between BChl-a and the polypeptides within the intact LH1-RC complexes by near-infrared Raman spectroscopy. The wild-type LH1-RC (B915) exhibited Raman bands for the C3-acetyl and C13-keto C=0 stretching modes at 1637 and 1675 cm(-1), respectively. The corresponding bands appeared at 1643 and 1673 cm(-1) when Ca2+ was biosynthetically replaced with Sr2+ (B888) or at 1647 and 1669 cm(-1) the mesophilic counterpart, Allochromatium vinosum. These results indicate the significant difference in the BChl-polypeptide interactions between B915 and B888 and between 8915 and the mesophilic counterpart. The removal of the original metal cations from B915 and B888 resulted in marked band shifts of the C3-acetyl/C13-carbonyl nu C=0 modes to similar to 1645/similar to 1670 cm(-1), supporting a model in which the metal cations are involved in the fine-tuning of the hydrogen bonding between the BChl-a and LH1-polypeptides. Interestingly, the interaction modes were almost identical between the Ca2+-depleted B915 and Sr2+-depleted B888 and between B915 and Ca2+-substituted B888, despite the significant differences in their LH1 Q(y) peak positions and the denaturing temperatures, as revealed by differential scanning calorimetry. These results suggest that not only the BChl-polypeptide interactions but some structural origin may be involved in the unusual Q(y) red-shift and the enhanced thermal stability of the LH1-RC complexes from Tch. tepidum. (C) 2012 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2012年07月, ［査読有り］

Gene sequencing and characterization of the light-harvesting complex 2 from thermophilic purple sulfur bacterium Thermochromatium tepidum
Fumie Sekine; Kentaro Horiguchi; Yasuhiro Kashino; Yuuki Shimizu; Long-Jiang Yu; Masayuki Kobayashi; Zheng-Yu Wang, In this study, gene sequences coding for the light-harvesting (LH) 2 polypeptides from a thermophilic purple sulfur bacterium Thermochromatium tepidum are reported and characterization of the LH2 complex is described. Three sets of pucBA genes have been identified, and the gene products have been analyzed by electrophoresis and reversed-phase chromatography. The result shows that all of the genes are expressed but the distribution of the expression is not uniform. The gene products undergo post-translational modification, where two of the beta-polypeptides appear to be N-terminally methylated. Absorption spectrum of the purified LH2 complex exhibits Q(y) transitions at 800 and 854 nm in dodecyl beta-maltopyranoside solution, and the circular dichroism spectrum shows a "molischianum''-like characteristic. No spectral change was observed for the LH2 when the bacterium was cultured under different conditions of light intensity. In lauryl dimethylamine N-oxide (LDAO) solution, significant changes in the absorption spectrum were observed. The B850 peak decreased and blue-shifted with increasing the LDAO concentration, whereas the B800 intensity increased without change in the peak position. The spectral changes can be partially or almost completely reversed by addition of metal ions, and the divalent cations seem to be more effective. The results indicate that ionic interactions may exist between LH2, detergent molecules and metal ions. Possible mechanisms involved in the detergent-and cation-induced spectral changes are discussed., SPRINGER
PHOTOSYNTHESIS RESEARCH, 2012年03月, ［査読有り］

3PT234 好熱性紅色硫黄細菌Thermochromatium tepidum由来光捕集1複合体におけるBChl-aとTrp残基間の水素結合相互作用(日本生物物理学会第50回年会(2012年度))
Li Yong; Kimura Yukihiro; Numata Tomoko; Inada Yuta; Arikawa Teruhisa; Otomo Seiu; Ohno Takashi, 一般社団法人 日本生物物理学会
生物物理, 2012年

3PT233 好熱性紅色硫黄細菌Thermochromatium tepidum由来cytochrome c'における耐熱化メカニズムの検討(日本生物物理学会第50回年会(2012年度))
Kasuga Sachiko; Kimura Yukihiro; Furusawa Kei; Ohno Takashi; Otomo Seiu, 一般社団法人 日本生物物理学会
生物物理, 2012年

Effects of Aggregation on the Excitation Dynamics of LH2 from Thermochromatium tepidum in Aqueous Phase and in Chromatophores
Fan Yang; Long-Jiang Yu; Peng Wang; Xi-Cheng Ai; Zheng-Yu Wang; Jian-Ping Zhang, We carried out femtosecond magic-angle and polarized pump probe spectroscopies for the light-harvesting complex 2 (LH2) from Thermochromatium (Tch.) tepidum in aqueous phase and in chromatophores. To examine the effects of LH2 aggregation on the dynamics of excitation energy transfer, dominant monodispersed and aggregated LH2s were prepared by controlling the surfactant concentrations. The aqueous preparations solubilized with different concentrations of n-dodecyl-beta-D-maltoside (DDM) show similar visible-to-near-infrared absorption spectra, but distinctively different aggregation states, as revealed by using dynamic light scattering. The B800 -> B850 intra-LH2 energy transfer time was determined to be 1.3 Ps for isolated LH2, which, upon aggregation in aqueous phase or clustering in chromatophores, shortened to 1.1 or 0.9 Ps, respectively. The light-harvesting complex 1 (LH1) of this thermophilic purple sulfur bacterium contains bacteriochlorophyll a absorbing at 915 nm (B915), and the LH2(B850)-> LH1(B915) intercomplex transfer time in chromatophores was found to be 6.6 ps. For chromatophores, a depolarization time of 21 ps was derived from the anisotropy kinetics of B850*, which is attributed to the migration of B850* excitation before being trapped by LH1. In addition, the B850* annihilation is accelerated upon LH2 aggregation in aqueous phase, but it is much less severe upon LH2 clustering in the intracytoplasmic membrane. These results are helpful in understanding the light-harvesting function of a bacterial photosynthetic membrane incorporating different types of antenna complexes., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2011年06月, ［査読有り］

A Spectroscopic Variant of the Light-Harvesting 1 Core Complex from the Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum
Yukihiro Kimura; Yuta Inada; Long-Jiang Yu; Zheng-Yu Wang; Takashi Ohno, Thermochromatium tepidum is a purple sulfur photosynthetic bacterium, and its light-harvesting 1 reaction center (LH1RC) complexes exhibit an unusual LH1 Q(y) absorption at 915 nm (B915) and possess enhanced thermal stability. These unique properties are closely related to an inorganic cofactor, Ca(2+). Here, we report a spectroscopic variant of LH1RC complexes from Tch. tepidum cells in which Ca(2+) was biosynthetically replaced with Sr(2+). The photosynthetic growth of wild-type cells cannot be maintained without Ca(2+) and is heavily inhibited when the Ca(2+) is replaced with other metal cations. Interestingly, only Sr(2+) supported photosynthetic growth instead of Ca(2+) with slightly reduced rates. The resulting Sr-tepidum cells exhibited characteristic absorption spectra in the LH1 Q(y) region with different LH1RC:LH2 ratios depending on the growth conditions. LH1RC complexes purified from the Sr-tepidum cells exhibited a Q(y) maximum at 888 nm (B888) that was blue-shifted after removal of Sr(2+) to similar to 870 nin (B870). Reconstitution of Sr(2+) and Ca(2+) into B870 resulted in red shifts of the Q(y) peak to 888 and 908 nm, respectively. The thermal stability of B888 was slightly lower than that of B915 as revealed by differential scanning calorimetry analysis. Effects of other divalent metal cations on the Q(y) peak position and thermal stability of B888 were similar but not identical to those of B915. This study provides the first evidence of a purple bacterium in which LH1RC complexes alter spectroscopic and thermodynamic properties in vivo by utilizing exogenous metal cations and improve the ability to adapt to the environmental changes., AMER CHEMICAL SOC
BIOCHEMISTRY, 2011年05月, ［査読有り］

Examination of the putative Ca2+-binding site in the light-harvesting complex 1 of thermophilic purple sulfur bacterium Thermochromatium tepidum
Long-Jiang Yu; Shogo Kato; Zheng-Yu Wang, The core light-harvesting complex (LH1) of purple sulfur photosynthetic bacterium Thermochromatium tepidum exhibits an unusual absorption maximum at 915 nm for the Q (y) transition, and is highly stable when copurified with reaction center (RC) in a LH1-RC complex form. In previous studies, we demonstrated that the calcium ions are involved in both the large red shift and the enhanced thermal stability, and possible Ca2+-binding sites were proposed. In this study, we further examine the putative binding sites in the LH1 polypeptides using purified chromatophores. Incubation of the chromatophores in the presence of EDTA revealed no substantial change in the absorption maximum of LH1 Q (y) transition, whereas further addition of detergents to the chromatophores-EDTA solution resulted in a blue-shift for the LH1 Q (y) peak with the final position at 892 nm. The change of the LH1 Q (y) peak to shorter wavelengths was relatively slow compared to that of the purified LH1-RC complex. The blue-shifted LH1 Q (y) transition in chromatophores can be restored to its original position by addition of Ca2+ ions. The results suggest that the Ca2+-binding site is exposed on the inner surface of chromatophores, corresponding to the C-terminal region of LH1. An Asp-rich fragment in the LH1 alpha-polypeptide is considered to form a crucial part of the binding network. The slow response of LH1 Q (y) transition upon exposure to EDTA is discussed in terms of the membrane environment in the chromatophores., SPRINGER
PHOTOSYNTHESIS RESEARCH, 2010年12月, ［査読有り］

Excitation Dynamics of the Light-Harvesting Complex 2 from Thermochromatium Tepidum
Yang Fan; Yu Long-Jiang; Wang Peng; Ai Xi-Cheng; Wang Zheng-Yu; Zhang Jian-Ping, Purple photosynthetic bacterium Thermochromatium (Tch.) tepidum is a moderate thermophile growing in an optimal temperature range of 48-50 degrees C. Its light-harvesting complex 2 (LH2) possesses heterogeneous compositions of apoprotein and carotenoid (Car), but the high. resolution crystallographic structure remains unknown. We have attempted an ultrafast time. resolved spectroscopic study of the isolated LH2 complex from Tch. tepidum. The spectral dynamics and population kinetics of n-dodecyl-beta-D-moltoside (DDM) and lauryldimethylamine oxide (LDAO) preparations of LH2 reveal efficient S-2-state mediated Car-to-Car and Car-to-bacteriochlorophyll (BChl) singlet excitation energy transfer (EET) occurring in a time scale of similar to 100 fs, as well as the Q(y)-state mediated B800-to-B850 singlet EET for the DDM preparation proceeding with a time constant of similar to 1.2 ps. These ultrafast EET processes suggest that the Cars with 11 and 12 conjugated C=C double bonds (N-C=C) coexist in the LH2 complex, and that the B800-B850 mutual orientation in LH2 differs considerably from those in the LH2s from some of the previously investigated bacterial species. In addition, anhydrorhodovibrin (N-C=C=12) as a minor Car composition is found to act as an efficient trap of excitation energy, which is considered to be an important photoprotection mechanism. Furthermore, based on the results of ultrafast Car band shift in response to BChl excitation, we propose that, compared to other Car compositions, (OH-) spirilloxanthin (N-C=C=13) locates in closer proximity to BChl. Our results may facilitate to understand the light. harvesting and the photoprotection mechanisms of Tch. tepidum living under harsh natural conditions., PEKING UNIV PRESS
ACTA PHYSICO-CHIMICA SINICA, 2010年07月, ［査読有り］

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome c(z) Subunit from Green Photosynthetic Bacterium Chlorobium tepidum
Yu Hirano; Makoto Higuchi; Chihiro Azai; Hirozo Oh-oka; Kunio Miki; Zheng-Yu Wang, In green sulfur photosynthetic bacteria, the cytochrome c(z) (cyt c(z)) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c(z) subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c(z) subunit (C-cyt c(z)) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-angstrom resolution. The overall fold of C-cyt c(z) consists of four a-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c(z) supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c(z). (C) 2010 Elsevier Ltd. All rights reserved., ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
JOURNAL OF MOLECULAR BIOLOGY, 2010年04月, ［査読有り］

2P288 生合成的にSr置換された好熱性紅色硫黄細菌Thermochromatium tepidumにおける色素蛋白質複合体の特性評価(光生物-光合成,第48回日本生物物理学会年会)
Kimura Yukihiro; Inada Yuta; Kasuga Sachiko; Sasaki Yuko; Yu Long-Jiang; Wang Zheng-Yu; Ohno Takashi, 一般社団法人 日本生物物理学会
生物物理, 2010年

Overexpression, characterization, and crystallization of the functional domain of cytochrome c(z) from Chlorobium tepidum
Makoto Higuchi; Yu Hirano; Yukihiro Kimura; Hirozo Oh-oka; Kunio Miki; Zheng-Yu Wang, Cytochrome c(z) is found in green sulfur photosynthetic bacteria, and is considered to be the only electron donor to the special pair P840 of the reaction center. It consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. Large scale expression of the C-terminal functional domain of the cytochrome c(z) (C-cyt c(z)) from the thermophilic bacterium Chlorobium tepidum has been achieved using the Escherichia coli expression system. The C-cyt c(z) expressed has been highly purified, and is stable at room temperature over 10 days of incubation for both reduced and oxidized forms. Spectroscopic measurements indicate that the heme iron in C-cyt c(z) is in a low-spin state and this does not change with the redox state. H-1-NMR spectra of the oxidized C-cyt c(z) exhibited unusually large paramagnetic chemical shifts for the heme methyl protons in comparison with those of other Class I ferric cytochromes c. Differences in the H-1-NMR linewidth were observed for some resonances, indicating different dynamic environments for these protons. Crystals of the oxidized C-cyt c(z) were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to a maximum resolution of 1.2 angstrom, and the diffraction data were collected to 1.3 angstrom resolution., SPRINGER
PHOTOSYNTHESIS RESEARCH, 2009年10月, ［査読有り］

Specific Ca2+-binding motif in the LH1 complex from photosynthetic bacterium Thermochromatium tepidum as revealed by optical spectroscopy and structural modeling
Fei Ma; Yukihiro Kimura; Long-Jiang Yu; Peng Wang; Xi-Cheng Ai; Zheng-Yu Wang; Jian-Ping Zhang, Native and Ca2+-depleted light-harvesting-reaction center core complexes (LH1-RC) from the photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibit maximal LH1-Q(y) absorption at 915 and 889 nm, respectively. To understand the structural origins of the spectral variation, we performed spectroscopic and structure modeling investigations. For the 889 nm form of LH1-RC, bacteriochlorophyll a (BChl a) in the native form was found by means of near-infrared Fourier-transform Raman spectroscopy, a higher degree of macrocycle distortion and a stronger hydrogen bond with the beta-Trp(-8) residue. SWISS-MODEL structure modeling suggests the presence of a specific coordination motif of Ca2+ at the C-terminus of the alpha-subunit of LH1, while MODELLER reveals the tilt of alpha- and beta-polypeptides with reference to the structural template, as well as a change in the concentric orientation of BChl a molecules, both of which may be connected to the long-wavelength LH1-Q(y) absorption of the 915 nm form. The carotenoid spirilloxanthin shows a twisted all-trans configuration in both forms of LH1 as evidenced by the resonance Raman spectroscopic results. With regard to the thermal stability, the 915 nm form was shown by the use of temperature-dependent fluorescence spectroscopy to be approximately 20 K more stable than the 889 nm form, which may be ascribed to the specific Ca2+-binding motif of LH1., WILEY-BLACKWELL
FEBS JOURNAL, 2009年03月, ［査読有り］

On the excitation-trap dynamics, red absorption and thermal stability of the lh1-rc complex from photosynthetic bacterium thermochromatium tepidum　　　　　　　　　　　　　　　
Fei Ma; Yukihiro Kimura; Long-Jiang Yu; Peng Wang; Xi-Cheng Ai; Zheng-Yu Wang; Jian-Ping Zhang
Optics InfoBase Conference Papers, 2009年

Calcium Ions Are Required for the Enhanced Thermal Stability of the Light-harvesting-Reaction Center Core Complex from Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum
Yukihiro Kimura; Long-Jiang Yu; Yu Hirano; Hiroaki Suzuki; Zheng-Yu Wang, sThermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057 - 1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca(2+) as a cofactor. Removal of the Ca(2+) from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca(2+) to the Ca(2+)-depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Q(y) transition between 915 nm for the native and 880 nm for the Ca(2+)-depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15 degrees C higher and the enthalpy change is about 28% larger than the Ca(2+)-depleted LH1-RC. Substitution of the Ca(2+) with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca(2+) ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
JOURNAL OF BIOLOGICAL CHEMISTRY, 2009年01月, ［査読有り］

Excitation dynamics of two spectral forms of the core complexes from photosynthetic bacterium Thermochromatium tepidum
Fei Ma; Yukihiro Kimura; Xiao-Hui Zhao; Yi-Shi Wu; Peng Wang; Li-Min Fu; Zheng-Yu Wang; Jian-Ping Zhang, The intact core antenna-reaction center (LH1-RC) core complex of thermophilic photosynthetic bacterium Thermochromatium(Tch.) tepidum is peculiar in its long-wavelength LH1-Q(y) absorption (915 nm). We have attempted comparative studies on the excitation dynamics of bacteriochlorophyll (BChl) and carotenoid (Car) between the intact core complex and the EDTA-treated one with the Q(y) absorption at 889 nm. For both spectral forms, the overall Car-to-BChl excitation energy transfer efficiency is determined to be similar to 20%, which is considerably lower than the reported values, e. g.,similar to 35%, for other photosynthetic purple bacteria containing the samekind of Car (spirilloxanthin). The RC trapping time constants are found to be 50 similar to 60 ps (170 similar to 200 ps) forRCin open (closed) state irrespective to the spectral forms and the wavelengths of Q(y) excitation. Despitethe low-energy LH1-Q(y) absorption, the RC trapping time are comparable to those reported for other photosynthetic bacteria with normal LH1-Q(y) absorption at 880 nm. Selective excitation to Car results in distinct differences in the Q(y)- bleaching dynamics between the two different spectral forms. This, together with the Car band-shift signals in response to Q(y) excitation, reveals the presence of two major groups of BChls in the LH1 of Tch. tepidum with a spectral heterogeneity of similar to 240 cm(-1), as well as an alteration in BChl-Car geometry in the 889-nm preparation with respect to the native one., BIOPHYSICAL SOC
BIOPHYSICAL JOURNAL, 2008年10月, ［査読有り］

Calcium ions are involved in the unusual red shift of the light-harvesting 1 Q(y) transition of the core complex in thermophilic purple sulfur bacterium Thermochromatium tepidum
Yukihiro Kimura; Yu Hirano; Long-Jiang Yu; Hiroaki Suzuki; Masayuki Kobayashi; Zheng-Yu Wang, Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 C and exhibits an unusual Q(y) absorption at 915 nm for the core light-harvesting complex (LH1), an similar to 35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Qy transition is strongly dependent on metal cations and Ca2+ is involved in the unusual red shift. Removal of the Ca2+ resulted in formation of a species with the LH1 Qy absorption at 880 nm, and addition of the Ca2+ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca2+ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca2+/alpha beta-subunit = 1: 1 and the binding constant was in 10(5) M-1 order of magnitude, which is comparable with those for EF-hand Ca2+-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca2+ binding were small and occurred slowly, with a typical time constant of similar to 6 min. Replacement of the Ca2+ with other metal cations caused blue shifts of the Qy bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca2+-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the alpha-polypeptide and the N-terminal region of the beta-polypeptide., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
JOURNAL OF BIOLOGICAL CHEMISTRY, 2008年05月, ［査読有り］

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum.
Hiroaki Suzuki; Yu Hirano; Yukihiro Kimura; Shinichi Takaichi; Masayuki Kobayashi; Kunio Miki; Zheng-Yu Wang, A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A.
Biochimica et biophysica acta, 2007年08月, ［査読有り］

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum
Hiroaki Suzuki; Yu Hirano; Yukihiro Kimura; Shinichi Takaichi; Masayuki Kobayashi; Kunio Miki; Zheng-Yu Wang, A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A. (c) 2007 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2007年08月, ［査読有り］

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum
Hiroaki Suzuki; Yu Hirano; Yukihiro Kimura; Shinichi Takaichi; Masayuki Kobayashi; Kunio Miki; Zheng-Yu Wang, A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A. (c) 2007 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2007年08月

Solution structure of the Rhodobacter sphaeroides PufX membrane protein: Implications for the quinone exchange and protein-protein interactions
Zheng-Yu Wang; Hiroaki Suzuki; Masayuki Kobayashi; Tsunenori Nozawa, PufX membrane protein is found in Rhodobacter species of purple photosynthetic bacteria and has been known to play an essential role in ubiquinone/ubiquinol exchange between the reaction center and cytochrome bc(1) complex and also contribute to the dimerization of the reaction center-light-harvesting core complex. We have determined the solution structure of the Rhodobacter sphaeroides PufX using multidimensional NMR spectroscopy. The PufX, functionally expressed in Escherichia coli, forms a stable alpha helix consisting of 21 residues over the central transmembrane domain. The overall structure of the PufX is very similar to those of the LH1 alpha- and beta-polypeptides from Rhodospirillum rubrum and LH2 polypeptides. A short segment (Lys28-Gly35) rich in Gly and Ala residues revealed a relatively fast exchange between the backbone amide protons and deuteriums in the hydroxyl groups of the solvent, indicating that the backbone of this segment is more easily accessible to the surrounding solvent molecules compared to those of its neighboring portions. The Gly- and Ala-rich segment is located in the middle of the central helix and forms an extensive groove-like conformation on the surface with the neighboring residues, where the residues with large side chains are aligned on one side of the helix, and small residues are aligned on the other face. Such a structural motif may serve as a functional site responsible for ubiquinol transport from the core complex to the membrane phase and for sequence-specific helix-helix interactions with the neighboring polypeptides., AMER CHEMICAL SOC
BIOCHEMISTRY, 2007年03月, ［査読有り］

Overexpression and characterization of the Rhodobacter sphaeroides PufX membrane protein in Escherichia coli
Shiho Onodera; Hiroaki Suzuki; Yuichiro Shimada; Masayuki Kobayashi; Tsunenori Nozawa; Zheng-Yu Wang, Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodohacter sphaeroidcs was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had alpha-helix contents of 29% in organic solvents and 22-26% in 0.8-2.0% (W/V) n-octyl beta-D-glucopyranoside solutions, suggesting that the PufX contains a substantial alpha-helical region composed of 18-22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 alpha- and beta-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional Puff membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system., WILEY
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 2007年01月, ［査読有り］

2P344 Roles of calcium ions on the structural stability of the LH1-RC core complex from thermophilic purple bacterium Thermochromatium tepidum(43. Photosynthesis,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)
Kimura yukihiro; Suzuki Hiroaki; Dao rina; Nakamura Masao; Otomo Seiu, 一般社団法人 日本生物物理学会
生物物理, 2006年

Isotopic labeling of proteins by utilizing photosynthetic bacteria
H Suzuki; Y Shimada; M Kobayashi; M Kudo; T Nozawa; ZY Wang, ACADEMIC PRESS INC ELSEVIER SCIENCE
ANALYTICAL BIOCHEMISTRY, 2005年12月, ［査読有り］

Electronic properties and thermal stability of soluble redox proteins from a thermophilic purple sulfur photosynthetic bacterium, Thermochromatium tepidum
M Kobayashi; T Saito; K Takahashi; ZY Wang; T Nozawa, The electronic properties of soluble redox proteins from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium (T) tepidum have been studied in terms of electronic absorption, magnetic CD, midpoint redox potential, and electron-transfer behavior. T tepidum possesses one high potential iron-sulfur protein (HiPIP), one cytochrome c', and two low potential cytochrome c-552's. The two low potential cytochrome c-552's can be separated further into a higher and a lower potential species. The HiPIP has a midpoint redox potential of +340 mV at pH7. From electron-transfer ability shown by flash experiments, the HiPIP was confirmed to be the possible electron-transfer protein to the reaction center under physiological conditions. The HiPIP is thermally stable up to 60 degrees C, which is much higher than that of the mesophilic photosynthetic bacterium, Allochromatium (A.) vinosum. Cytochrome c' has a high-spin protoheme with a redox midpoint potential of +100 mV at pH 7, similar to those of many other photosynthetic bacteria. The molecular weight was determined to be 14147 Da by MALDI-TOF/MS experiments. The higher potential cytochrome c552's have hemes c's with midpoint redox potentials of -15 and +85 mV, and the lower one of -15 mV. The molecular weights for the higher potential cytochrome c-552 and the lower potential c-552 were found to be 20702 and 47757 Da, respectively. The higher potential cytochrome c-552 has an attached flavoprotein with a molecular mass of 42000 Da. The lower potential cytochrome c-552 has been found for the first time in T. tepidum., CHEMICAL SOC JAPAN
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 2005年12月, ［査読有り］

Selective detection of the solid-state NMR signals from the bacteriochlorophyll a dimers in a reconstituted light-harvesting 1 complex
M Umetsu; T Kadota; ZY Wang; Y Tanaka; T Adschiri; T Nozawa, High resolution solid-state C-13 NMR spectra have been obtained for bacteriochlorophyll (BChl) a in a light-harvesting (LH) 1 complex utilizing a reconstitution method. The LH I complexes were reconstituted with C-13-enriched BChl a and the apo-LHI polypeptide from Rhodospirillum rubrum. Measurement of the 2-dimentional C-13-C-13 dipole correlation NMR spectra enabled the selective assignment of the 13 C resonance from the BChl a molecules in the reconstituted LH1., CHEMICAL SOC JAPAN
CHEMISTRY LETTERS, 2005年07月, ［査読有り］

Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability
M Kobayashi; Y Fujioka; T Mori; M Terashima; H Suzuki; Y Shimada; T Saito; ZY Wang; T Nozawa, Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LM-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or-by the core light-harvesting complexes. The. results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile., TAYLOR & FRANCIS LTD
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 2005年06月, ［査読有り］

Solution structures of the core light-harvesting α and β polypeptides from Rhodospirillum rubrum: Implications for the pigment-protein and protein-protein interactions
Wang; Z.-Y; Gokan; Kobayashi, M; Nozawa, T, We have determined the solution structures of the core light-harvesting (LH1) alpha and beta-polypeptides from wild-type purple photosynthetic bacterium Rhodospirillum rubrum using multidimensional NMR spectroscopy. The two polypeptides form stable a helices in organic solution. The structure of a-polypeptide consists of a long helix of 32 amino acid residues over the central transmembrane domain and a short helical segment at the N terminus that is followed by a three-residue loop. Pigment-coordinating histidine residue (His29) in the alpha-polypeptide is located near the middle of the central helix. The structure of beta-polypeptide shows a single helix of 32 amino acid residues in the membrane-spanning region with the pigment-coordinating histidine residue (His38) at a position close to the C-terminal end of the helix. Strong hydrogen bonds have been identified for the backbone amide protons over the central helical regions, indicating a rigid property of the two polypeptides. The overall structures of the R. rubrum LH1 alpha and beta-polypeptides are different from those previously reported for the LH1 beta-polypeptide of Rhodobacter sphaeroides, but are very similar to the structures of the corresponding LH2 alpha and beta-polypeptides determined by X-ray crystallography. A model constructed for the structural subunit (B820) of LH1 complex using the solution structures reveals several important features on the interactions between the LH1 alpha and beta-polypeptides. The significance of the N-terminal regions of the two polypeptides for stabilizing both B820 and LH1 complexes, as clarified by many experiments, may be attributed to the interactions between the short N-terminal helix (Trp2-Gln6) of alpha-polypeptide and a GxxxG motif in the beta-polypeptide. (c) 2005 Elsevier Ltd. All rights reserved., ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
J. Mol. Biol., 2005年03月

3P122 大腸菌発現の光捕集膜タンパク質の立体構造は天然のものと同じか?(膜蛋白質))
大友 征宇; 望月 有志; 嶋田 友一郎; 鈴木 宏昭, 一般社団法人 日本生物物理学会
生物物理, 2005年

Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems
Y Shimada; ZY Wang; Y Mochizuki; M Kobayashi; T Nozawa, Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E. coli extracts. The alpha-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His(6)) tag added to the carboxyl terminus. Both of the expression systems produced alpha-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native beta-apoprotein and the pigment molecule bacteriochlorophyll a. The expression product in E. coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100. Circular dichroism measurement indicated that the expressed alpha-apoproteins from both systems had alpha-helical contents essentially identical with that of the native one. About two thirds of the alpha-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the alpha-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue. Functional expression of the alpha-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis., TAYLOR & FRANCIS LTD
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 2004年09月, ［査読有り］

NMR relaxation study of the bacteriochlorophyll c in solutions
ZY Wang; T Kadota; M Kobayashi; A Kasuya; T Nozawa, Bacteriochlorophyll (BChl) c is a major light-harvesting pigment family in green photosynthetic bacteria. In organic solvents, the pigment molecules are capable of forming stable dimers and self-assembling into high aggregates which have been used as a model for the native chlorosome antenna. NMR relaxation times were measured for the intact farnesyl (3(1)R)-[E, E]BChl c(F) in methanol, acetone, and carbon tetrachloride. The spin-lattice relaxation times (T-1(H)) were determined to be 0.3-1.2 s for the macrocyclic protons and 0.73-3.3 s for the farnesyl protons in methanol and acetone in which the BChl c exists as a monomer. Strong hydrogen bonding between the BChl c and solvent molecules resulted in a significant reduction in the spin-spin relaxation times (T-2(H)) for the protons close to the hydrogen-bonding sites. This result can be interpreted in terms of a combined effect of scalar coupling with the hydroxyl proton and dipolar interaction with the solvent molecules. Formation of BChl c dimer in carbon tetrachloride led to an increase in T-1(H) and a large decrease in T-2(H) with respect to the values of monomer, indicating that the correlation time became longer as a result of the much reduced molecular motion. With the use of a highly randomly C-13-labeled sample, we were able to measure the C-13 relaxation times. The T-1(C) were determined in a range of 0.26-3.3 s for the macrocyclic carbons in methanol, and these values decreased as BChl c formed a dimer but remained in the same order of magnitude. The (3(1)R)-[E, E]BChl c(F) is demonstrated as an ideal molecule for studying the hydrogen-bonding property and the dynamic exchange behavior between the individual molecules within a dimer., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2004年09月

Magic-angle spinning nuclear magnetic resonance under ultrahigh field reveals two forms of intermolecular interaction within CH2Cl2-treated (3(1)R)-type bacteriochlorophyll c solid aggregate
M Umetsu; JG Hollander; J Matysik; ZY Wang; T Adschiri; T Nozawa; HJM de Groot, (3(1)R)-bacteriochlorophyll (BChl) c solid aggregates with an absorbance around 740 nm were formed from BChl c dimers, and 2-D homonuclear C-13-C-13 radio frequency-driven dipolar recoupling as well as proton-driven spin diffusion dipolar correlation NMR spectra have been collected in ultrahigh magnetic field. Doubling of signals is observed for most carbons in the BChl c macrocycle, leading to two correlation networks. In this way, two major fractions denoted types A and B are identified. Some of the ring carbons show multiple resonances, revealing additional slight differences in microstructural environment. 2-D heteronuclear H-1-C-13 correlation data have been recorded using the frequency- and phase-switched Lee-Goldburg technique to assign the H-1 response. N-15 chemical shifts are assigned from 2-D heteronuclear N-15-C-13 correlation experiments using spectrally induced filtering in combination with cross polarization. Also the nitrogen atoms in the pyrrole rings I, II, and IV (N-I, N-II, and N-IV, respectively) show two sets of resonances, each of which is connected to a single C-13 correlation network A or B. The C-13 chemical shifts are compared with the signals from antiparallel dimers in solution and with the response from chlorosomes previously reported. The data clearly show that the stacking in CH2Cl2-treated aggregates is different from the stacking in the chlorosomes and hexane-treated aggregates. Some degree of similarity with the antiparallel dimer form in solution transpires, in particular for the type A species. It is proposed that the CH2Cl2 precipitate represents a structural intermediate between the antiparallel dimer and the parallel stack as found in the chlorosome., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2004年02月, ［査読有り］

1P119 光合成光捕集膜タンパク質の立体構造解析(膜蛋白質)
大友 征宇; 後閑 和孝; 小林 正幸; 野澤 庸則, 一般社団法人 日本生物物理学会
生物物理, 2004年

Dynamic exchange properties of the antiparallel bacteriochlorophyll c dimers
M Umetsu; R Seki; T Kadota; ZY Wang; T Adschiri; T Nozawa, The dynamic exchange behavior of the (3(1)R)-type bacteriochlorophyll (BChl) c dimer with an antiparallel piggy-back conformation has been investigated by two-dimensional nuclear magnetic resonance exchange spectroscopy (EXSY). The exchange rate between two BChl c molecules in the antiparallel dimer was evaluated from the integrated intensities of cross-peaks due to chemical exchange in the EXSY spectra, and its values were found to dramatically increase with the rise of solvent polarity. The temperature dependence of the exchange rates can be well expressed by the Arrhenius equation, the parameters of which show the correlation of the fast exchange rate with the stability of a transition state in the exchange process. Thermodynamic analysis was also applied to investigate the substituent effect on the exchange rate of the antiparallel dimer. The increase of the exchange rate upon addition of substituents at the peripheral 8-position was mainly attributed to the rise in frequency factor rather than activation energy. A high exchange of the (3(1)R)-type BChl c dimer by a small amount of (3(1)S)-type BChl c also resulted from the significant increase of the frequency factor. The substituent effects demonstrate that the antiparallel dimer formed by the most abundant BChl c homologue in the native light-harvesting entity, called chlorosome, can be highly exchanged by minor BChl c components. The results suggest that minor BChl c homologues in the chlorosome could bring about a phase transition of the most abundant BChl c from the stable dimer to a large aggregate., AMER CHEMICAL SOC
JOURNAL OF PHYSICAL CHEMISTRY B, 2003年09月, ［査読有り］

Determination of the B820 subunit size of a bacterial core light-harvesting complex by small-angle neutron scattering
Wang, Z. Y.; Muraoka, Y.; Nagao, M.; Shibayama, M.; Kobayashi, M.; Nozawa, T.
Biochemistry, 2003年, ［査読有り］

Purification and characterization of the polypeptides of core light-harvesting complexes from purple sulfur bacteria
WANG Z.-Y.
Photosynth. Res., 2003年

Circular and magnetic circular dichroism studies of bacteriochlorophyll c aggregates: T-shaped and antiparallel dimers
Mitsuo Umetsu; Ryoichi Seki; Zheng-Yu Wang; Izumi Kumagai; Tsunenori Nozawa, Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra have been measured on (31R)-bacteriochlorophyll (BChl) c aggregates in organic solvents. BChl c exists as a monomer in acetone with a Qy(0-0) transition at 661 nm. In a mixed solvent system of methanol and dichloromethane (CH3OH/CH2Cl2 = 1/4000), another Qy(0-0) transition appeared at 680 nm with a nondispersion-type CD signal. The ratio of MCD intensity to its dipole strength (B/D) for the Qy(0-0) transition is half the value of its monomer species, indicating a dimerization of BChl c. The apparent inconsistency between the CD and MCD results has been reconciled by assuming the formation of BChl c dimer with a T-shaped conformation. In neat CCl4, a dispersion-type CD signal was observed for the Qy(0-0) exciton components at 680 and 710 nm. MCD spectra of the exciton-type Qy(0-0) components suggest that two BChl c molecules are parallel with their macrocycle planes as judged by the mixing effect between the excited states of Qx(0-0) and Qy(0-0). The CD and MCD results are consistent with the fact that a BChl c dimer with an antiparallel conformation is dominant in the neat CCl4 as reported previously. Corresponding Qx(0-0) transitions were determined around 650 nm by the simultaneous deconvolution of absorption, CD, and MCD spectra. It is demonstrated that the antiparallel BChl c dimer shows two exciton components for both Qx(0-0) and Qy(0-0) transitions. Finally, we evaluated solvent dependence of the stability of the BChl c dimer by two-dimensional exchange spectroscopy (EXSY) experiments.
Journal of Physical Chemistry B, 2002年04月18日, ［査読有り］

Interaction of photosynthetic pigments with various organic solvents 2. Application of magnetic circular dichroism to bacteriochlorophyll a and light-harvesting complex 1
M Umetsu; ZY Wang; K Yoza; M Kobayashi; T Nozawa, Magnetic circular dichroism (MCD) and absorption spectra of metal bacteliochlorin complexes have been measured on bacteriochlorophyll (BChl) a in various solvents and different forms of light-harvesting complexes 1 (LH1 complexes). In hydrophilic organic solvents, the MCD intensity of the Q(y)(0-0) transition of BChl a was sensitive to the wavelength of absorption maximum of Q(x)(0-0), and the ratio of MCD Q(y)(0-0) intensity to the dipole strength (B/D) was inversely proportional to the difference in energy between the Q(x)(0-0) and Q(y)(0-0). The similar correlation has been observed in metal chlorin derivatives as previously reported. The correlation depends on the coordination number of the Mg atom in BChl a and the molecules ligating to it. In a hydrophobic solvent such as carbon tetrachloride (CCl4), however, the correlation did trot hold because of the existence of aggregates. Hence, the col relation between the values of B/D and the energy difference can be used to estimate the type and number of the molecules ligated to the Mg atom and to disclose the existence of aggregated pigments. We further apply the correlation to the LH 1 complex treated with n-octyl beta-D-glucopyranoside. (C) 2000 Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2000年04月, ［査読有り］

2E1415 耐熱性光合成反応中心の熱安定化構造の検討
森 貴幸; 小林 正幸; 禾 晃和; 王 征宇; 三木 邦夫; 野澤 庸則, 一般社団法人 日本生物物理学会
生物物理, 2000年

13C- and 15N-NMR studies on the intact bacteriochlorophyll c dimers in solutions
Zheng-Yu Wang; Mitsuo Umetsu; Masayuki Kobayashi; Tsunenori Nozawa, 13C and 15N chemical shifts of the intact farnesyl (31R)- bacteriochlorophyll (BChl) c have been measured in methanol and carbon tetrachloride solutions. Two sets of resonances have been observed in carbon tetrachloride for all carbon and nitrogen atoms, indicating a formation of highly stable dimeric species with asymmetric configurations. Complete assignments have been made based on a combination of homonuclear and heteronuclear correlation experiments using the 13C- and 15N-labeled BChl c samples. Changes of the 13C chemical shift in the two solvents can be interpreted in terms of mixed effects arising from (a) ring current due to the overlap of the macrocycles, (b) coordination state of the central magnesium, (c) excitation state of the π-electron system, (d) polarity of the solvents used, and (e) hydrogen bonding. Substantial ring current effect is observed on the 13C chemical shifts for the carbon atoms around pyrrolic ring I upon the dimer formation. Remarkable differences in the line widths observed for all propionic carbons and some carbons of the farnesyl group suggest that the propionic-famesyl side chains may adopt a 'return' structure over the region from 171 to f2 carbons with much different conformation and mobility in the dimer. No clear evidence is obtained for a hydrogen bond formed with the C131 carbonyl group in CCl4 solution, nor for ring overlap over the ring V. Comparison between the 15N chemical shifts in both solvents indicates thai the paramagnetic shielding effect is predominant and N(IV) nitrogen is most sensitive to the dimer formation, followed by N(II), N(I), and N(III), respectively. The result reveals a high sensitivity of 15N chemical shift to the electronic state and N-Mg bond length for each nitrogen atom in the dimer.
Journal of the American Chemical Society, 1999年10月13日, ［査読有り］

Complete Assignment of 1H NMR Spectra and Structural Analysis of Intact Bacteriochlorophyll C Dimer in Solution
Zheng-Yu Wang; Mitsuo Umetsu; Masayuki Kobayashi; Tsunenori Nozawa
The Journal of Physical Chemistry B, 1999年04月, ［査読有り］

Interaction of photosynthetic pigments with various organic solvents. Magnetic circular dichroism approach and application to chlorosomes
Mitsuo Umetsu; Zheng-Yu Wang; Masayuki Kobayashi; Tsunenori Nozawa, Magnetic circular dichroism (MCD) and absorption spectra have been measured on three intact photosynthetic pigments with the chlorin ring as macrocycle: chlorophyll a, bacteriochlorophyll c and d, in various hydrophilic organic solvents. The MCD intensity of a Q(y)(0-0) transition for the Mg chlorin derivative was sensitive to the coordination state of the central Mg atom by the solvent molecules. The coordination number has been characterized in terms of the relationship between the ratio of Q(y)(0-0) MCD intensity to its dipole strength (B/D) and the difference in energies of Q(x)(0-0) and Q(y)(0-0) transitions. This relationship depends not only on the coordination number of the magnesium (Mg) atom but also on the coordination interaction of the solvent molecules to the Mg atom, and can clarify the spectroscopic change of chlorosomes by alcohol treatment. We propose that the correlation between the MCD intensity of Q(y)(0-0) transition and the energy difference can be used as a new measure for determining the coordination number of the Mg atom and for estimating the interaction strength of the Mg atom with solvent molecules. Copyright (C) 1999 Elsevier Science B.V.
Biochimica et Biophysica Acta - Bioenergetics, 1999年01月27日, ［査読有り］

1PB046 アンテナ器官クロロゾーム中の主成分色素が形成する高次会合体のCP/MAS NMRによる測定
梅津 光央; 王 征宇; 山内 一夫; 小林 正幸; 野澤 庸則, 一般社団法人 日本生物物理学会
生物物理, 1999年

Interaction of photosynthetic pigments with various organic solvents as revealed from magnetic circular dichroism measurements
M Umetsu; ZY Wang; M Kobayashi; T Nozawa, SPRINGER
PHOTOSYNTHESIS: MECHANISMS AND EFFECTS, VOLS I-V, 1998年, ［査読有り］

A small-angle neutron scattering study on the small aggregates of bacteriochlorophylls in solutions
Zheng-Yu Wang; Mitsuo Umetsu; Kenji Yoza; Masayuki Kobayashi; Masayuki Imai; Yushu Matsushita; Nobuo Niimura; Tsunenori Nozawa, Small-angle neutron scattering measurements have been made to determine the size of small aggregates of bacteriochlorophyll chlorophyll (BChl) a and c formed in non-polar solution. Both BChl a and c form dimers and oligomers in equilibrium with each other at high concentrations in benzene-d6 solutions. The size and molecular weight for each dimer have been quantitatively evaluated based on Guinier approximation. The radii of gyration of the dimers are determined to be 17.0 ± 0.5 A and 16.5 ± 0.5 W for the BChl a and c, respectively. Limited observations in the very small angle region suggest that the oligomers are of considerable dimension, approximately as large as a hundred angstroms for the radius of gyration. The SANS results can be well correlated with the electronic absorption and NMR spectra and are in good agreement with other spectroscopic behavior of the small aggregates reported previously.
Biochimica et Biophysica Acta - Bioenergetics, 1997年05月16日, ［査読有り］

New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1',2'-dihydro-γ-carotene, 1',2'-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains
Shinichi Takaichi; Zheng-Yu Wang; Mitsuo Umetsu; Tsunenori Nozawa; Keizo Shimada; Michael T. Madigan, The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1',2'-dihydro derivatives (1',2'-dihydro-γ-carotene and 1',2'-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those inthe thermophilic green nonsulfur bacterium Chloroflexus aurantiacus.
Archives of Microbiology, 1997年, ［査読有り］

Molecular structures and optical properties of aggregated forms of chlorophylls analyzed by means of magnetic circular dichroism
Masayuki Kobayashi; Zheng-Yu Wang; Kenji Yoza; Mitsuo Umetsu; Hideo Konami; Mamuro Mimuro; Tsunenori Nozawa, Molecular structures and optical properties of aggregated forms of chlorophylls analyzed by means of magnetic circular dichroism. Magnetic circular dichroism (MCD) spectra were measured on chlorophylls in different aggregation states formed in organic solvents and in pigment-protein complexes. An index which reflects the magnetic dipole contribution of a specific transition is introduced, that is, a ratio of MCD intensity of Faraday B-term (B) to that of dipole strength (D) (B/D). The absolute value of the index is higher in monomeric chlorophyll a and that in monomeric bacteriochlorophyll a. This index changed depending on the aggregation state of bacteriochlorophyll
in the case of the dimer of bacteriochlorophyll a and bacteriochlorophyll d, the value was nearly the half of that of respective monomer, and it decreases further for a higher aggregate of bacteriochlorophyll d. Based on this index, a molecular structure of the primary electron donor in reaction center complexes is discussed., Elsevier
Spectrochimica Acta - Part A Molecular Spectroscopy, 1996年, ［査読有り］

Morphology and spectroscopy of chlorosomes from Chlorobium tepidum by alcohol treatments
Zheng-Yu Wang; Giselher Marx; Mitsuo Umetsu; Masayuski Kobayashi; Mamoru Mimuro; Tsunenori Nozawa, Chlorosomes from Chlorobium tepidum have been treated with alcohol-saturated buffers, followed by dilution to the buffers with half the saturated concentrations. Morphologic changes during this process have been statistically investigated by dynamic light scattering technique combined with electron microscopy to obtain the complete information on shape, size and distribution, while spectral properties have been studied by absorption, CD and magnetic circular dichroism. Three alcohols (1-hexanol, 1-butanol and phenol) have been found to produce nearly reversible conversion of absorption spectra despite the more than 15-times difference in the alcohol concentration. It is shown that the degree of saturation, not the alcohol concentration, is the key factor for the complete conversion of bacteriochlorophyll c in chlorosomes from the aggregated state to the monomeric form. We have observed substantial changes in the shape, size and distribution at each step of the treatment with 1-hexanol, indicating that the whole process is morphologically irreversible. Comparison of the morphologic changes with the corresponding spectroscopic behaviour suggests that the relative overall size rather than the shape and distribution may be a more important factor affecting the spectral properties. © 1995.
BBA - Bioenergetics, 1995年12月12日, ［査読有り］

MAGNETIC CIRCULAR-DICHROISM INVESTIGATION ON CHROMOPHORES IN REACTION CENTERS OF PHOTOSYSTEM-I AND PHOTOSYSTEM-II OF GREEN PLANT PHOTOSYNTHESIS
T NOZAWA; M KOBAYASHI; ZY WANG; S ITOH; M IWAKI; M MIMURO, Magnetic circular dichorism (MCD) of chlorophylls (Chl) in P700-enriched (12 Chl/P700) photo-system (PS) I particles and D1-D2-cyt b559 (PSII reaction center) particles isolated from spinach are measured in the wavelength region between 450 and 750 nm. The relative magnitude of MCD to absorption intensity ([MCD]/[Abs]) is found to be a good measure to estimate the interaction of chromophores (dimer or monomer). The difference spectrum between reduced and oxidized state for the 12 Chl/P700 particles can be interpreted to be composed of a positive MCD from P700 and a negative MCD for P700+. MCD signals for the primary acceptor Chl a monomer (A0) and the reduced cytochrome f are also observed. The MCD signal from pheophytin a in the PSII reaction center is remarkably in the Q(x) region as strong as the signal from the reaction center chlorophylls (P680 and accessory) and other attached chlorophylls. Based on the data, the MCD bands, as well as the absorption bands in the Q(y) region, are deconvoluted into the contribution from reaction center chlorophylls and the other chlorophylls. The [MCD]/[Abs] ratio of P680 or P700 is small and similar to that of special pair bacteriochlorophylls in the reaction center of purple bacteria, indicating the specific feature of MCD signals that originate from a dimer-type interaction., PERGAMON-ELSEVIER SCIENCE LTD
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, 1995年01月, ［査読有り］

Molecular networks and funneling process of energy transfer in green photosynthetic bacteria
M Mimuro; K Matsuura; K Shimada; Y Nishimura; Yamazaki, I; M Kobayashi; ZY Wang; T Nozawa, KLUWER ACADEMIC PUBL
PHOTOSYNTHESIS: FROM LIGHT TO BIOSPHERE, VOL I, 1995年, ［査読有り］

好熱性紅色光合成細菌Halorhodospira halochlorisの特性解析
能島紳吾; 小林正幸; 竹中慎治; M.T.Madaigan; 大友征宇; 木村行宏
日本農芸化学会 関西・中部支部2019合同神戸大会（神戸）, 2019年09月

紅色光合成細菌電子伝達系におけるキノン輸送の追跡
岸利華子; 今西三千絵; 小林正幸; 永島賢治; 竹中慎治; M.T.Madaigan; 大友征宇; 木村行宏
日本農芸化学会 関西・中部支部2019合同神戸大会（神戸）, 2019年09月

紅色光合成細菌におけるキノン動態の解析
岸利華子; 今西三千絵; 小林正幸; 永島賢治; 竹中慎治; M.T.Madaigam; 大友征宇; 木村行宏
第27回「光合成セミナー2019：反応中心と色素系の多様性」（豊中）, 2019年07月

好熱性および好塩性を示す Halorhodospira halochloris由来光捕集反応中心複合体の特性解析
能島紳吾; 小林正幸; 竹中慎治; Michael; T. Madigan; 大友征宇; 木村行宏
第26回「光合成セミナー2018：反応中心と色素系の多様性」（神戸）, 2018年07月

光誘起FTIR分光法を用いた紅色細菌由来LH1-RC複合体におけるキノン分子の追跡
岸利華子; 今西三千絵; 小林正幸; 永島賢治; Michael T. Madigan; 大友征宇; 木村 行宏
第26回「光合成セミナー2018：反応中心と色素系の多様性」（神戸）, 2018年07月

紅色光合成細菌 Thiorhodovibrio Strain 970が示す異常なLH1Qyレッドシフトの起源
今西三千絵; 竹之内瑠貴; 小林正幸; 竹中慎治; ࣭Michael T. Madigan; Jorg Overmann; 大友征宇; 木村 行宏
第26回「光合成セミナー2018：反応中心と色素系の多様性」（神戸）, 2018年07月

Monitoring of quinone reduction in the thermophilic purple bacterium Thermochromatium tedium by means of isotope-edited FTIR spectroscopy
今西 三千絵; 岸 利華子; 小林 正幸; 大友 征宇; 木村 行宏
第55回日本生物物理学会年会（熊本）, 2017年09月

同位体標識化された好熱性紅色細菌Thermochromatium tediumにおけるキノール分子の振動分光学的検出
岸利華子; 今西三千絵; 小林正幸; 大野 隆; 大友征宇; 木村 行宏
第25回「光合成セミナー2017：反応中心と色素系の多様性」（神戸）, 2017年07月

FTIR 分光法を用いた好熱性紅色細菌 Thermochromatium tepidumにおける光合成電子伝達反応のモニタリング
今西三千絵; 橋本佳奈子; 永島賢治; 小林正幸; 由良優季; 大野 隆; 大友征宇; 木村 行宏
第58回日本植物生理学会年会, 2017年03月

ハイブリッド型光捕集反応中心複合体の機能解析
後藤健太; 佐々木舞; 阿部悠人; 小沼里沙; 永島賢治; 永島咲子; 大友征宇
生体分子科学討論会講演要旨集, 2017年

好熱性紅色光合成細菌Thermochromatium tepidum 由来反応中心複合体におけるカルシウムイオンの機能的、構造的役割
Michie Imanishi; Masayuki Kobayashi; Manami Kobayashi; Mari Matsuzaki; Yuki Yura; Takashi Ohno; Seiu Otomo; Yukihiro Kimura
第54回日本生物物理学会年会（東京）, 2016年11月

光合成細菌，光合成膜を用いた光電変換特性について
小林正幸; 高木洋介; 豊福航平; 大友征宇; 野澤庸則
第21回「光合成セミナー2013：光合成の色素系と反応中心に関するセミナー」（名古屋）, 2013年06月

光合成膜顆粒懸濁液を用いた光電変換特性
小林正幸; 高木洋介; 野沢庸則; 大友征宇
第19回光合成の色素系と反応中心に関するセミナー（大阪）, 2011年07月

紅色光合成細菌の光捕集複合体LH2の特性評価
関根文恵; 堀口健太郎; 菓子野康浩; 清水佑記; 于龍江; 小林正幸; 大友征宇
第19回光合成の色素系と反応中心に関するセミナー（大阪）, 2011年07月

好熱性紅色光合成細菌Thermochromatium tepidumシトクロム複合体の単離精製
小林正幸; Y.Xin; A.M.Collin; J.Wen; P.Bell; 齊藤貴之; 大友征宇; R. E. Blankenship
第18回光合成の色素系と反応中心に関するセミナー（京都）, 2010年07月

Optical properties of light-harvesting complex 2 from thermophilic purple sulfur bacterium, Thermochromatium tepidum
小林正幸; 鈴木宏昭; 大友征宇; 野澤庸則
13th International Symposium on Phototrophic Prokaryotes, 2009年08月

タンパク質複合体の単離 光合成細菌の反応中心・アンテナ複合体 (光合成研究法) -- (単離・精製・活性測定)
大友 征宇; 小林 正幸; 大岡 宏造
低温科学, 2008年

Reconstitution and replacement of bacteriochlorophyll a molecules in photosynthetic reaction centers.
Masayuki Kobayashi; Atsuo Takaya; Nobuyuki Kanai; Yosuke Ota; Takayuki Saito; Zheng-Yu Wang; Tsunenori Nozawa
Journal of biochemistry, 2004年09月, ［査読有り］

Probing the Transmembrane Potential of Bacterial Cells by Voltage-Sensitive Dyes
Suzuki Hiroaki; Wang Zheng-Yu; Yamakoshi Mie; KOBAYASHI Masayuki; NOZAWA Tsunenori
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 2003年

A dimeric structure of bacteriochlorophyllide c molecules studied by scanning tunneling microscopy
Qing-Min Xu; Li-Jun Wan; Shu-Xia Yin; Chen Wang; Chun-Li Bai; Takasada Ishii; Kaku Uehara; Zheng-Yu Wang; Tsunenori Nozawa
Journal of Physical Chemistry B, 2002年03月28日

N-terminal methylation of the core light-harvesting complex in purple photosynthetic bacteria
WANG Z.-Y.
FEBS Lett., 2002年

Selective detection and assignment of the solution NMR signals of bacteriochlorophyll a in a reconstituted subunit of a light-harvesting complex
WANG Z.-Y.
J. Am. Chem. Soc., 2002年

Combination of conditioned medium and elicitation enhances taxoid production in bioreactor cultures of Taxus chinensis cells
WANG Z. Y.
Biochem. Eng. J., 2002年

The dimerization of folded monomers of ribulose 1,5-bisphosphate carboxylase/oxygenase
S Luo; ZY Wang; M Kobayashi; T Nozawa
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001年03月

Re-identification of the N-terminal amino acid residue and its modification of β -polypeptide of light-harvesting complex I from #I Rhodospirillum #N rubrum #IR
ZY Wang; M Shimonaga; Y Muraoka; M Kobayashi; T Nozawa
Photosynthesis Research, 2001年

Methionine oxidation and its effect on the stability of reconstituted subunit of light-harvesting complex from Rhodospirillum rubrum
WANG Z.-Y.
Eur. J. Biochem., 2001年

Molecular assembly of covalently-linked mesoporphyrin dimers with light-harvesting polypeptides
A Kashiwada; Y Takeuchi; H Watanabe; T Mizuno; H Yasue; K Kitagawa; K Iida; ZY Wang; T Nozawa; H Kawai; T Nagamura; Y Kurono; M Nango
TETRAHEDRON LETTERS, 2000年03月

How the formation process influences the structure of BChl c aggregates
M Umetsu; ZY Wang; J Zhang; T Ishii; K Uehara; Y Inoko; M Kobayashi; T Nozawa
PHOTOSYNTHESIS RESEARCH, 1999年05月

Biochemical and spectral characterization of the core light harvesting complex 1 (LH1) from the thermophilic purple sulfur bacterium Chromatium tepidum
Fathir, I; M Ashikaga; K Tanaka; T Katano; T Nirasawa; M Kobayashi; ZY Wang; T Nozawa
PHOTOSYNTHESIS RESEARCH, 1998年11月

In situ measurements of ribulose-1,5-bisphosphate carboxylase activity by nuclear magnetic resonance
ZY Wang; S Luo; K Sato; M Kobayasi; T Nozawa
ANALYTICAL BIOCHEMISTRY, 1998年03月

Measurements of the CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase by 31P- and 1H-NMR
Zheng-Yu Wang; Shen Luo; Kazuhiro Sato; Masayuki Kobayashi; Tsunenori Nozawa
Photosynthesis Research, 1998年

The genes coding for the L, M and cytochrome subunits of the photosynthetic reaction center from the thermophilic purple sulfur bacterium Chromatium tepidum
Fathir, I; K Tanaka; K Yoza; A Kojima; M Kobayashi; ZY Wang; F Lottspeich; T Nozawa
PHOTOSYNTHESIS RESEARCH, 1997年01月

Size distribution and photophosphorylation of chromatophores from Rhodospirillum rubrum
ZY Wang; M Kudoh; Y Ohama; H Nakatani; M Kobayashi; T Nozawa
PHOTOSYNTHESIS RESEARCH, 1997年01月

High-resolution solid-state 13C NMR of the LH1 from Rhodospirillum rubrum
Kenji Yoza; Zheng-Yu Wang; Masayuki Kobayashi; Tsunenori Nozawa
Photosynthesis Research, 1997年

Excitation energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus: A specific effect of 1-hexanol on the optical properties of baseplate and energy transfer processes
M Mimuro; Y Nishimura; Yamazaki, I; M Kobayashi; ZY Wang; T Nozawa; K Shimada; K Matsuura
PHOTOSYNTHESIS RESEARCH, 1996年05月

A SMALL-ANGLE X-RAY-SCATTERING STUDY OF ALGINATE SOLUTION AND ITS SOL-GEL TRANSITION BY ADDITION OF DIVALENT-CATIONS
ZY WANG; JW WHITE; M KONNO; S SAITO; T NOZAWA
BIOPOLYMERS, 1995年02月

Magnetic circular dichroism investigation on chromophores in reaction centers of photosystem I and II of green plant photosynthesis
Tsunenori Nozawa; Masayuki Kobayashi; Zheng-Yu Wang; Shigeru Itoh; Masayo Iwaki; Mamoru Mimuro; Kimiyuki Satoh
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 1995年

STRUCTURES OF CHLOROSOMES AND AGGREGATED BCHL-C IN CHLOROBIUM-TEPIDUM FROM SOLID-STATE HIGH-RESOLUTION CP/MAS C-13 NMR
T NOZAWA; K OHTOMO; M SUZUKI; H NAKAGAWA; Y SHIKAMA; H KONAMI; ZY WANG
PHOTOSYNTHESIS RESEARCH, 1994年07月