Elucidation of the chemistry of gaseous molecule at iron binuclear center using nuclear resonance spectroscopies

• Project/Area Number: 21H02064
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 37010:Bio-related chemistry
• Research Institution: University of Hyogo (2023); Institute of Physical and Chemical Research (2021-2022)
• Principal Investigator: Tosha Takehiko 兵庫県立大学, 理学研究科, 教授 (00548993)
• Co-Investigator(Kenkyū-buntansha): 堀谷 正樹 佐賀大学, 農学部, 准教授 (80532134)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2023: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2022: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2021: ¥9,230,000 (Direct Cost: ¥7,100,000、Indirect Cost: ¥2,130,000)
• Keywords: 金属酵素 / 一酸化窒素 / ヘム / 反応中間体 / 核共鳴非弾性散乱 / 低温光解離 / ケージド化合物 / 振動分光 / 一酸化窒素還元酵素 / 電子スピン共鳴 / 二核鉄中心 / 核共鳴散乱分光 / クライオフォトリシス

Outline of Research at the Start

生体内の温和な条件下で高活性を示す金属酵素の作動原理を理解するためには、触媒反応中に形成される反応中間体の構造を決定することが重要である。本課題では、光解離性のケージド基質の低温光解離と温度制御を組み合わせることで、短寿命反応中間体を捕捉し、その詳細な構造決定に挑む。ヘム鉄と非ヘム鉄からなる複核活性中心において、二分子の一酸化窒素（NO）を亜酸化窒素（N2O）に還元・無毒化するNO還元酵素（NOR）を研究対象とし、ケージドNOを利用して捕捉した反応中間体の電子・原子構造を核共鳴散乱分光法により決定することで、鉄複核中心と気体分子の化学について理解するとともに、本研究手法の有用性を実証する。

Outline of Final Research Achievements

We aimed to elucidate the molecular mechanism of nitric oxide reductase (NOR) whose active site is composed of heme and non-heme iron. Reduced NOR was mixed with caged NO, which releases NO upon UV illumination, followed by the illumination under liquid nitrogen temperature to induce the photolysis of caged NO. Subsequent thermal annealing enables NO to diffuse and react with reduced NOR, producing reaction intermediates. The characterization of first reaction intermediate by ESR spectroscopy revealed that the first reaction intermediate can be a non-heme Fe-NO species. The trapped reaction intermediate was also analyzed by synchrotron-based vibrational spectroscopy, nuclear resonance vibrational spectroscopy (NRVS), to get more insights into the chemical structure of the reaction intermediate.

Academic Significance and Societal Importance of the Research Achievements

NORによるNO還元には、鉄へのNOの結合、電子移動、プロトン輸送、化学結合の形成（N-N結合）および開裂（N-O結合）といった、化学反応の基礎が詰め込まれており、その反応機構を理解することは、化学反応の本質の理解に迫るものである。酵素そのものを使って、反応中間体を捕捉・解析した本成果は、近年、主流となっているNORの理論研究やモデル錯体を用いた研究に対して一石を投じるものであり、今後の反応機構の理解に大きく貢献する。また、NORは、病原菌の感染や地球環境とも関連が深いため、その反応機構の理解は、これらの研究分野を含む多くの他分野への影響が期待できる。

Research Products

• [Int'l Joint Research] ゴッセンベルグ大学(スウェーデン)
• [Int'l Joint Research] インペリアルカレッジロンドン(英国)
• [Int'l Joint Research] Diamond Light Source/University of Liverpool/University of Essex(英国)
• [Int'l Joint Research] University of Liverpool/University of Essex/Diamond Light Source(英国)
• [Journal Article] Catalytic Oxidation of Methane by Wild-Type Cytochrome P450BM3 with Chemically Evolved Decoy Molecules (2023)
  • Author(s): Ariyasu Shinya、Yonemura Kai、Kasai Chie、Aiba Yuichiro、Onoda Hiroki、Shisaka Yuma、Sugimoto Hiroshi、Tosha Takehiko、Kubo Minoru、Kamachi Takashi、Yoshizawa Kazunari、Shoji Osami
  • Journal Title: ACS Catalysis Volume: 13 Issue: 13 Pages: 8613-8623
  • DOI: 10.1021/acscatal.3c01158
  • Peer Reviewed
• [Journal Article] Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism (2023)
  • Author(s): Fadini Alisia et al.
  • Journal Title: Journal of the American Chemical Society Volume: 145 Issue: 29 Pages: 15796-15808
  • DOI: 10.1021/jacs.3c02313
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase (2023)
  • Author(s): Safari Cecilia et. al.
  • Journal Title: Science Advances Volume: 9 Issue: 49 Pages: 1-12
  • DOI: 10.1126/sciadv.adh4179
  • Peer Reviewed / Open Access / Int'l Joint Research
• [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] Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases (2022)
  • Author(s): Rose Samuel L.、Baba Seiki、Okumura Hideo、Antonyuk Svetlana V.、Sasaki Daisuke、Hedison Tobias M.、Shanmugam Muralidharan、Heyes Derren J.、Scrutton Nigel S.、Kumasaka Takashi、Tosha Takehiko、Eady Robert R.、Yamamoto Masaki、Hasnain S. Samar
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 30
  • DOI: 10.1073/pnas.2205664119
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature (2022)
  • Author(s): Moreno-Chicano, Carey, Axford, Beale, Doak, Duyvesteyn, Ebrahim, Henning, Monteiro, Myles, Owada, Sherrell, Straw, Srajer, Sugimoto, Tono, Tosha T., Tews, Trebbin, Strange, Weiss, Worrall, Meilleur, Owen, Ghiladi, Hough
  • Journal Title: IUCrJ Volume: 9 Issue: 5 Pages: 610-624
  • DOI: 10.1107/s2052252522006418
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Serial Femtosecond Crystallography Reveals the Role of Water in the One- or Two-Electron Redox Chemistry of Compound I in the Catalytic Cycle of the B-Type Dye-Decolorizing Peroxidase DtpB (2022)
  • Author(s): Lucic Marina、Wilson Michael T.、Tosha Takehiko、Sugimoto Hiroshi、Shilova Anastasya、Axford Danny、Owen Robin L.、Hough Michael A.、Worrall Jonathan A. R.
  • Journal Title: ACS Catalysis Volume: 12 Issue: 21 Pages: 13349-13359
  • DOI: 10.1021/acscatal.2c03754
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases. (2022)
  • Author(s): Nishida Y, Yanagisawa S, Morita R, Shigematsu H, Shinzawa-Itoh K, Yuki H, Ogasawara S, et al
  • Journal Title: Nat Commun. Volume: 13 Issue: 1 Pages: 7591-7591
  • DOI: 10.1038/s41467-022-34771-y
  • Peer Reviewed / Open Access
• [Journal Article] Mechanism of substrate inhibition in cytochrome-c dependent NO reductases from denitrifying bacteria (cNORs) (2022)
  • Author(s): Matsumura Hirotoshi、Faponle Abayomi S.、Hagedoorn Peter-Leon、Tosha Takehiko、de Visser Sam P.、Moenne-Loccoz Pierre
  • Journal Title: Journal of Inorganic Biochemistry Volume: 231 Pages: 111781-111781
  • DOI: 10.1016/j.jinorgbio.2022.111781
  • Peer Reviewed / 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] 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
• [Presentation] Mechanism of nitric oxide reduction by P450 in fungal denitrification (2023)
  • Author(s): Takehiko Tosha
  • Organizer: 日本薬物動態学会第38回年会/第23回シトクロムP450国際会議国際合同大会
  • Int'l Joint Research / Invited
• [Presentation] Characterization of the short-lived reaction intermediate of NO reductase with caged substrate (2023)
  • Author(s): Takehiko Tosha
  • Organizer: Dynamic Crystallography-XFELs and Synchrotrons to study enzyme reactions
  • Int'l Joint Research / Invited
• [Presentation] Characterization of reaction intermediates of nitric oxide reductases involved in denitrification (2023)
  • Author(s): Takehiko Tosha
  • Organizer: ThermusQ高度好熱菌研究を切り拓くネオテクノロジー
• [Presentation] ケージド基質の低温光解離を利用した金属酵素反応中間体の捕捉 (2023)
  • Author(s): 當舎武彦、武田英恵、榛葉幹治、堀谷正樹、城宜嗣
  • Organizer: 第49回生体分子科学討論会
• [Presentation] 嫌気呼吸酵素を標的とした緑膿菌の抗菌薬開発に向けて (2023)
  • Author(s): 當舎武彦、川上凌平、Chai Gopalasingam、重松秀樹、西田優也、新谷泰範
  • Organizer: 第2回生命金属科学シンポジウム
• [Presentation] NO reduction chemistry at heme/non-heme binuclear active center proved by methods using photo-sensitive caged NO (2022)
  • Author(s): Takehiko Tosha
  • Organizer: 10th Asian Biological Inorganic Chemistry Conference
  • Int'l Joint Research / Invited
• [Presentation] Mechanism of biological nitric oxide reduction proved by time-resolved structural analysis (2022)
  • Author(s): Takehiko Tosha
  • Organizer: REDOX WEEK IN SENDAI 2022, 4th International conference on persulfide and sulfur metabolism in biology and medicine
  • Int'l Joint Research / Invited
• [Presentation] Elucidation of Mechanisms for Catalytic Reaction of Nitric Oxide Reductases by Time-resolved Techniques (2022)
  • Author(s): T. Tosha, H. Takeda, Y. Shiro, M. Kubo
  • Organizer: International Conference on Porphyrins and Phthalocyanines (ICPP)-12
  • Int'l Joint Research / Invited
• [Presentation] 光解離性基質の低温光解離を利用した金属酵素短寿命反応中間体の同定 (2022)
  • Author(s): 當舎武彦
  • Organizer: 第61回電子スピンサイエンス学会年会
  • Invited
• [Presentation] 光解離性ケージド基質を用いた金属酵素反応機構の解明 (2022)
  • Author(s): 當舎武彦
  • Organizer: メタルバイオサイエンス研究会2022
  • Invited
• [Presentation] 酸素バリア性フィルムを利用した嫌気下での構造解析の試み (2022)
  • Author(s): 當舎武彦、榛葉幹治、松浦滉明、平田邦生、山本雅貴、城宜嗣
  • Organizer: 第60回日本生物物理学会年会
• [Presentation] 金属酵素活性中心における一酸化窒素還元反応の分子機構 (2022)
  • Author(s): 當舎武彦
  • Organizer: 物性研短期研究会「理論タンパク質物性科学の最前線：理論と実験との密な協働」
  • Invited
• [Presentation] 各種分光計測を駆使した鉄活性中心における一酸化窒素還元酵素の反応機構の解明 (2022)
  • Author(s): 當舎武彦
  • Organizer: 第22回日本蛋白質科学会年会
  • Invited
• [Presentation] Time-resolved techniques provide mechanism for nitric oxide reduction by nitric oxide reductase (2021)
  • Author(s): Tosha T., Takeda H., Shiro Y., Kubo M.
  • Organizer: Pacifichem 2021
  • Int'l Joint Research / 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
• [Book] 第4章、第2節 生体鉄の分子科学と細胞生物学、“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、澤井仁美、當舎武彦
  • Total Pages: 13
  • Publisher: NTS出版
• [Remarks] 反応途中の酵素を観る新手法の開発
  • URL: https://www.riken.jp/press/2023/20230111_2/index.html