Structural Dynamics of Nitric Oxide Reductases Srudied by Time-Resolved Techniques

• Project/Area Number: 19H00926
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 37:Biomolecular chemistry and related fields
• Research Institution: University of Hyogo
• Principal Investigator: Shiro Yoshitsugu 兵庫県立大学, 理学研究科, 特任教授 (70183051)
• Co-Investigator(Kenkyū-buntansha): 村本 和優 兵庫県立大学, 理学研究科, 准教授 (50305679)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥45,110,000 (Direct Cost: ¥34,700,000、Indirect Cost: ¥10,410,000); Fiscal Year 2022: ¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000); Fiscal Year 2021: ¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000); Fiscal Year 2020: ¥10,790,000 (Direct Cost: ¥8,300,000、Indirect Cost: ¥2,490,000); Fiscal Year 2019: ¥11,960,000 (Direct Cost: ¥9,200,000、Indirect Cost: ¥2,760,000)
• Keywords: 一酸化窒素還元酵素 / 一酸化窒素 / 脱窒 / 亜酸化窒素 / 抗菌ガス / 呼吸酵素の分子進化 / 抗菌剤開発 / 嫌気呼吸 / 髄膜炎菌 / 低温電子顕微鏡 / 時間分解構造解析 / 構造ダイナミクス / クライオ電子顕微鏡 / ナノディスク複合体 / 緑膿菌 / 時間分解解析 / 時間分解分光解析

Outline of Research at the Start

脱窒菌の一酸化窒素還元酵素（NO消去酵素：NOR）は、鉄を活性中心に含む膜結合酵素であり、脱窒過程で中間生成物として現れる細胞毒性の高い一酸化窒素（NO）を速やかに消去している。地球環境保全、地球上での窒素循環、呼吸酵素の分子進化、病原菌の感染増殖などに関連する鍵酵素である。緑膿菌NOR、NOR-NiR複合体、髄膜炎菌NORとそれらの変異体を研究対象として、時間分解赤外分光と時間分解X線結晶構造解析を併用した測定により、NOR酵素反応中に現れる２つの短寿命反応中間体の電子状態ならびに配位構造（Fe-N-O）を決定する。それにより、NOR反応の分子機構を構造ダイナミクスを基盤に確立する。

Outline of Final Research Achievements

Membrane-bound nitric oxide reductases (NORs), which have a binuclear complex of heme and non-heme irons at the active center, are key enzymes in anaerobic respiration, denitrification, since they catalyze reduction of toxic nitric oxide NO for its detoxification. They are also involved in life cycle of pathogenic bacteria in their host.
I determined both monomeric and dimeric structures of Neisseria meningitidis qNOR, and also analyzed its reaction with NO in the absence and presence of inhibitors. These data provided bases for design and construct of anti-bacterial drugs.
Using Pseudomonas aeruginosa cNOR as a target enzyme, I followed the enzymatic reaction with NO with time-resolved visible and IR spectroscopic, and cryo-reduction annealing ESR spectroscopic techniques. In these measurements, I proposed the molecular mechanism of NOR enzymatic reaction. The results are highly related to the molecular evolution of the respiratory enzyme.

Academic Significance and Societal Importance of the Research Achievements

髄膜炎菌のqNORは、菌が宿主内でバイオフィルム内で嫌気呼吸をおこない、なおかつ宿主が産生する抗菌ガスNOを無毒化する上で、鍵となる酵素である。本研究で分子構造と反応プロトンの移動との関係、阻害剤との相互作用の詳細が明らかになったことにより、抗菌薬設計の基礎データとなる。また、緑膿菌cNORを用いた反応解析から、酵素反応の分子機構が提案でき、好気呼吸酵素との比較が可能となり、呼吸酵素の分子進化を議論できるようになった。さらに、NOR反応の生成物が、地球環境破壊に関係していることから、環境科学の観点からも今後注目される成果となった。

Research Products

• [Int'l Joint Research] University of Liverpool/University of Bristol/University of Leicester(英国)
• [Int'l Joint Research] University of Liverpool/University of Leeds(英国)
• [Int'l Joint Research] University of Liverpool/University of Leeds(英国)
• [Journal Article] Trapping of a Mononitrosyl Nonheme Intermediate of Nitric Oxide Reductase by Cryo-Photolysis of Caged Nitric Oxide (2023)
  • Author(s): Takeda Hanae、Shimba Kanji、Horitani Masaki、Kimura Tetsunari、Nomura Takashi、Kubo Minoru、Shiro Yoshitsugu、Tosha Takehiko
  • Journal Title: The Journal of Physical Chemistry B Volume: 127 Issue: 4 Pages: 846-854
  • DOI: 10.1021/acs.jpcb.2c05852
  • Peer Reviewed / Open Access
• [Journal Article] Structural basis for heme detoxification by an ATP-binding cassette?type efflux pump in gram-positive pathogenic bacteria (2022)
  • Author(s): Nakamura Hiro、Hisano Tamao、Rahman Md. Mahfuzur、Tosha Takehiko、Shirouzu Mikako、Shiro Yoshitsugu
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 27
  • DOI: 10.1073/pnas.2123385119
  • Peer Reviewed / Open Access
• [Journal Article] Author Correction: Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases (2022)
  • Author(s): Nishida Yuya、 et al., Shiro Yoshitsugu、Honma Teruki、Shigeta Yasuteru、Kubo Minoru、Takashima Seiji、Shintani Yasunori
  • Journal Title: Nature Communications Volume: 13 Issue: 1
  • DOI: 10.1038/s41467-022-35600-y
  • Peer Reviewed / Open Access
• [Journal Article] Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion. (2022)
  • Author(s): Hieu VN, Thuy LTT, Hai H, Dat NQ, Hoang DV, Hanh NV, Phuong DM, Hoang TH, Sawai H, Shiro Y, Sato-Matsubara M, Oikawa D, Tokunaga F, Yoshizato K, Kawada N.
  • Journal Title: Redox Biol. Volume: 52 Pages: 102286-102286
  • DOI: 10.1016/j.redox.2022.102286
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography (2021)
  • Author(s): Nomura, T., Kimura, T., Kanematsu, Y., Yamada, D., Yamashita, K., Hirata, K., Ueno, G., Murakami, H., Hisano, T., Yamagiwa, R., Takeda, H., Gopalasingam, C., Kousaka, R., Yanagisawa, S., Shoji, O., Kumasaka, T., Yamamoto, M., Takano, Y., Sugimoto, H., Tosha, T., Kubo, M., Shiro, Y
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 118 Issue: 21
  • DOI: 10.1073/pnas.2101481118
  • Peer Reviewed / Open Access
• [Journal Article] XFEL Crystal Structures of Peroxidase Compound II (2021)
  • Author(s): Kwon, H., Basran, J., Pathak, C., Hussain, M., Freeman, S. L., Fielding, A. J., Bailey, A. J., Stefanou, N., Sparkes, H. A., Tosha, T., Yamashita, K., Hirata, K., Murakami, H., Ueno, G., Ago, H., Tono, K., Yamamoto, M., Sawai, H., Shiro, Y., Sugimoto, H., Raven, E., Moody, P. C. E.
  • Journal Title: Angewandte Chemie International Edition Volume: - Issue: 26 Pages: 14578-14585
  • DOI: 10.1002/anie.202103010
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Regulatory Switching by Concerted Motions on the Microsecond Time Scale of the Oxygen Sensor Protein FixL (2021)
  • Author(s): Yamawaki Takeo、Mizuno Misao、Ishikawa Haruto、Takemura Kazuhiro、Kitao Akio、Shiro Yoshitsugu、Mizutani Yasuhisa
  • Journal Title: The Journal of Physical Chemistry B Volume: 125 Issue: 25 Pages: 6847-6856
  • DOI: 10.1021/acs.jpcb.1c01885
  • Peer Reviewed
• [Journal Article] Spatially restricted substrate-binding site of cortisol-synthesizing CYP11B1 limits multiple hydroxylations and hinders aldosterone synthesis (2021)
  • Author(s): Mukai Kuniaki、Sugimoto Hiroshi、Kamiya Katsumasa、Suzuki Reiko、Matsuura Tomomi、Hishiki Takako、Shimada Hideo、Shiro Yoshitsugu、Suematsu Makoto、Kagawa Norio
  • Journal Title: Current Research in Structural Biology Volume: 3 Pages: 192-205
  • DOI: 10.1016/j.crstbi.2021.08.001
  • Peer Reviewed / Open Access
• [Journal Article] Impact of membrane protein-lipid interactions on formation of bilayer lipid membranes on SAM-modified gold electrode (2021)
  • Author(s): Kato Masaru、Masuda Yuya、Yoshida Narumi、Tosha Takehiko、Shiro Yoshitsugu、Yagi Ichizo
  • Journal Title: Electrochimica Acta Volume: 373 Pages: 137888-137888
  • DOI: 10.1016/j.electacta.2021.137888
  • Peer Reviewed / Open Access
• [Journal Article] NO Dynamics in Microbial Denitrification System (2021)
  • Author(s): Tosha Takehiko、Yamagiwa Raika、Sawai Hitomi、Shiro Yoshitsugu
  • Journal Title: Chemistry Letters Volume: 50 Issue: 2 Pages: 280-288
  • DOI: 10.1246/cl.200629
  • NAID: 130007986360
  • Peer Reviewed / Open Access
• [Journal Article] Heme controls the structural rearrangement of its sensor protein mediating the hemolytic bacterial survival (2021)
  • Author(s): Nishinaga Megumi、Sugimoto Hiroshi、Nishitani Yudai、Nagai Seina、Nagatoishi Satoru、Muraki Norifumi、Tosha Takehiko、Tsumoto Kouhei、Aono Shigetoshi、Shiro Yoshitsugu、Sawai Hitomi
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 467-467
  • DOI: 10.1038/s42003-021-01987-5
  • Peer Reviewed / Open Access
• [Journal Article] The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer (2020)
  • Author(s): Jamali M. Arif M.、Gopalasingam Chai C.、Johnson Rachel M.、Tosha Takehiko、Muramoto Kazumasa、Muench Stephen P.、Antonyuk Svetlana V.、Shiro Yoshitsugu、Hasnain Samar S.
  • Journal Title: IUCrJ Volume: 7 Issue: 3 Pages: 404-415
  • DOI: 10.1107/s2052252520003656
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Timing of NO Binding and Protonation in the Catalytic Reaction of Bacterial Nitric Oxide Reductase as Established by Time-Resolved Spectroscopy. (2020)
  • Author(s): Hanae Takeda, Tatsunari Kimura, Takashi Nomura, Masaki Horitani, Azusa Yokota, Akiko Matsubayashi, Shoko Ishii, Yoshitsugu Shiro, Minoru Kubo and Takehiko Tosha
  • Journal Title: Bulletin of the Chemical Society of Japan Volume: - Issue: 7 Pages: 825-833
  • DOI: 10.1246/bcsj.20200038
  • NAID: 130007879554
  • Peer Reviewed / Open Access
• [Journal Article] 分子構造を基盤にしたヘムタンパク質・ヘム酵素の機能解明 (2020)
  • Author(s): 城 宜嗣
  • Journal Title: BJSCC Volume: 75 Pages: 51-56
  • NAID: 130007879402
  • Peer Reviewed
• [Journal Article] UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1 (2019)
  • Author(s): Sachiko Yanagisawa , Kure’e Kayama , Masayuki Hara , Hiroshi Sugimoto , Yoshitsugu Shiro , Takashi Ogura
  • Journal Title: Biophysical Journal Volume: 117 Issue: 4 Pages: 706-716
  • DOI: 10.1016/j.bpj.2019.07.017
  • Peer Reviewed
• [Journal Article] Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo-electron microscopy (2019)
  • Author(s): Gopalasingam Chai C.、Johnson Rachel M.、Chiduza George N.、Tosha Takehiko、Yamamoto Masaki、Shiro Yoshitsugu、Antonyuk Svetlana V.、Muench Stephen P.、Hasnain S. Samar
  • Journal Title: Science Advances Volume: 5 Issue: 8
  • DOI: 10.1126/sciadv.aax1803
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Chemomechanical Coupling in the Transport Cycle of a Type II ABC Transporter (2019)
  • Author(s): K. Tamura, H. Sugimoto, Y. Shiro, Y. Sugita
  • Journal Title: J. Phys. Chem. B Volume: 123 Issue: 34 Pages: 7270-7281
  • DOI: 10.1021/acs.jpcb.9b04356
  • Peer Reviewed
• [Presentation] Elucidation of Mechanisms for Catalytic Reaction of Nitric Oxide Reductases by Time-resolved Techniques (2022)
  • Author(s): T. Tosha, H. Takeda, Y. Shiro and M. Kubo
  • Organizer: International Conference on Porphyrins and Phthalocyanines (ICPP-12)
  • Int'l Joint Research / Invited
• [Presentation] Molecular Mechanism of NO Reduction Catalyzed by Fe-containing Nitric Oxide Reductases (2022)
  • Author(s): Y. Shiro
  • Organizer: 8th Asian Conference on Coordination Chemistry (ACCC8)
  • Int'l Joint Research / Invited
• [Presentation] 鉄の生体内動態の分子科学 (2022)
  • Author(s): 城 宜嗣
  • Organizer: 第２回レドックスR&D戦略委員会 春のシンポジウム 最先端技術が切り拓くレドックスバイオロジー
  • Invited
• [Presentation] Time-resolved Techniques Provide Mechanism for Nitric Oxide Reduction by Nitric Oxide Reductase (2021)
  • Author(s): T. Tosha, H. Takeda, Y. Shiro and M. Kubo
  • Organizer: The International Chemical Congress of Pacific Basin Societies 2021 (Pacifichem 2021)
  • Int'l Joint Research / Invited
• [Presentation] 分子構造を基盤にした鉄結合タンパク質の機能解明 生命金属科学への展開をめざして (2021)
  • Author(s): 城 宜嗣
  • Organizer: 第15回バイオ関連化学シンポジウム
  • Invited
• [Presentation] 光解離性ケージド基質を利用した一酸化窒素還元酵素の反応機構の解明 (2021)
  • Author(s): 當舎武彦
  • Organizer: 第94回日本生化学大会
  • Invited
• [Presentation] Probing Functional and Structural Differences of Quinol-dependent Nitric Oxide Reductases’ (qNOR) within a Lipidic Nanodisc Environment (2021)
  • Author(s): C. Gopalasingam
  • Organizer: Protein Science Society of Japan Annual Meeting
  • Invited
• [Presentation] 分子構造を基盤にした鉄結合タンパク質の機能解明：Functional Studies of Iron-Related Proteins on Molecular Basis (2021)
  • Author(s): 城 宜嗣
  • Organizer: 日本化学会第１０１春季年会
  • Invited
• [Presentation] 『生命金属科学』のめざすところ (2020)
  • Author(s): 城 宜嗣
  • Organizer: CSJ化学フェスタ
  • Invited
• [Presentation] Dynamics of Nitric Oxide in Bacterial Cellular Systems: NO Generation and Decomposition (2019)
  • Author(s): Shiro, Y.
  • Organizer: Japan-BIOCEV Symposium: Macromolecules; Structure, Function and Beyond
  • Int'l Joint Research / Invited
• [Presentation] Dynamics of Nitric Oxide in Cellular Systems: NO Generation and Decomposition (2019)
  • Author(s): Shiro Y.
  • Organizer: KTJ Bioinorganic Chemistry Symposium 2019 (KTJ-BICS 2019)
  • Int'l Joint Research / Invited
• [Presentation] 分子構造を基盤にしたヘムタンパク質の機能解明～生命金属科学への展開をめざして～ (2019)
  • Author(s): 城 宜嗣
  • Organizer: 錯体化学会第69回討論会
  • Invited
• [Presentation] ヘム蛋白質の機能を司る構造・ダイナミクスとエネルギー流：実験と理論 (2019)
  • Author(s): 城 宜嗣
  • Organizer: 第57回日本生物物理学会年会
  • Invited
• [Book] ヘムタンパク質の科学 (2022)
  • Author(s): 城 宜嗣、青野 重利、齋藤 正男
  • Total Pages: 472
  • Publisher: エヌ・ティー・エス
  • ISBN: 9784860437787
• [Book] 生命金属ダイナミクス 生体内における金属の挙動と制御 (2021)
  • Author(s): 城 宜嗣・津本浩平
  • Total Pages: 511
  • Publisher: NTS
  • ISBN: 9784860437060
• [Remarks] 兵庫県立大学大学院理学研究科細胞制御学Ⅱ講座
  • URL: https://www.sci.u-hyogo.ac.jp/life/regulation/index-j.html
• [Remarks] 兵庫県立大学大学院理学研究科細胞膜超分子複合体機能解析学講座
  • URL: http://www2.riken.jp/biometal/index.htm
• [Remarks] 兵庫県立大学大学院理学研究科細胞制御Ⅱ講座
  • URL: https://www.sci.u-hyogo.ac.jp/life/regulation/index-j.html
• [Remarks] 兵庫県立大大学院生命理学研究科細胞制御Ⅱ講座
  • URL: https://www.sci.u-hyogo.ac.jp/life/regulation/index-j.html