Time-resolved study on the respiratory energy-conversion mechanism

• Project/Area Number: 19H03171
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: University of Hyogo
• Principal Investigator: Minoru Kubo 兵庫県立大学, 理学研究科, 教授 (90392878)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,680,000 (Direct Cost: ¥13,600,000、Indirect Cost: ¥4,080,000); Fiscal Year 2021: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000); Fiscal Year 2020: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000); Fiscal Year 2019: ¥11,440,000 (Direct Cost: ¥8,800,000、Indirect Cost: ¥2,640,000)
• Keywords: チトクロム酸化酵素 / 動的構造生物学 / 時間分解測定 / ケージド化合物 / 時間分解X線結晶構造解析 / 時間分解振動分光 / 蛋白質 / 動的構造解析 / 赤外分光 / X線自由電子レーザー

Outline of Research at the Start

呼吸と生体エネルギー発生の機構は古くから人々の興味の中心の一つである。呼吸によって取り込まれた酸素のほとんどは、細胞内のミトコンドリアという場所で、水素イオンの汲み出し反応に利用されている。本研究では、その反応を最先端の計測技術を用いて詳細に追跡し、呼吸のしくみを原子レベルで解き明かす。

Outline of Final Research Achievements

Cytochrome c oxidase catalyzes the final step of food oxidation, coupled with proton pump to store the energy. This is one of the most important steps for the respiratory energy conversion; however, its mechanism is still mystery. In this study, to elucidate the proton-pump machinery of cytochrome c oxidase at the atomic level, we have developed the method that can control the enzymatic reaction by light and the instrument that can track the reaction in real time.

Academic Significance and Societal Importance of the Research Achievements

呼吸と生体エネルギー発生の機構は古くから人々の興味を惹きつけてきた生命科学の中心課題の一つであるが、その本質はミトコンドリアで行われているプロトンポンプにある。しかしその機構はまだ謎のままであり、教科書にも載っていない。本研究ではこの謎に挑む研究基盤を確立した。なお、近年、チトクロム酸化酵素の活性化因子が発見されており、ミトコンドリア病の治療薬開発の観点で注目されている。チトクロム酸化酵素の機構を原子レベルで解明しさらにそれを制御することは、将来社会の健康保持・増進に寄与するであろう。

Research Products

• [Journal Article] A synthetic model for the possible FeIV2(μ-O)2 core of methane monooxygenase intermediate Q derived from a structurally characterized FeIIIFeIV(μ-O)2 complex. (2022)
  • Author(s): Mikata, Y., Aono, Y., Yamamoto, C., Nakayama, H., Matsumoto, A., Kotegawa, F., Harada, M., Katano, H., Kobayashi, Y., Yanagisawa, S., Kubo, M., Kajiwara, A., Kodera, M
  • Journal Title: Inorganic Chemistry Volume: 61 Issue: 2 Pages: 786-790
  • DOI: 10.1021/acs.inorgchem.1c02699
  • Peer Reviewed
• [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] Revisiting Alkane Hydroxylation with m‐CPBA (mChloroperbenzoic Acid) Catalyzed by Nickel(II) Complexes (2021)
  • Author(s): Shinke, T., Itoh, M., Wada, T., Morimoto, Y., Yanagisawa, S., Sugimoto, H., Kubo, M., Itoh S.
  • Journal Title: Chemistry A European Journal Volume: 27 Issue: 59 Pages: 14730-14737
  • DOI: 10.1002/chem.202102532
  • Peer Reviewed
• [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] SACLA時分割結晶構造解析による動的構造生物学研究～酵素反応の可視化に向けた分子動画～. (2020)
  • Author(s): 久保 稔
  • Journal Title: 放射光 Volume: 33 Pages: 266-270
  • Peer Reviewed
• [Journal Article] Syntheses, characterizations, and crystal Structures of dinitrogen-divanadium complexes bearing triamidoamine ligands. (2020)
  • Author(s): Kokubo, Y., Wasada-Tsutsui, Y., Yomura, S., Yanagisawa, S., Kubo, M., Kugimiya, S., Kajita, Y., Ozawa, T., Masuda, H.
  • Journal Title: Eur. J. Inorg. Chem. Volume: 2020 Issue: 15-16 Pages: 1456-1464
  • DOI: 10.1002/ejic.201901123
  • Peer Reviewed / Open Access
• [Journal Article] Oxidative DNA cleavage, formation of μ-1,1-hydroperoxo species, and cytotoxicity of dicopper(II) complex supported by a p-cresol-derived amide-tether ligand (2019)
  • Author(s): Kadoya, Y., Fukui, K., Hata, M., Miyano, R., Hitomi, Y., Yanagisawa, S., Kubo, M., Kodera, M.
  • Journal Title: Inorg. Chem. Volume: 58 Issue: 21 Pages: 14294-14298
  • DOI: 10.1021/acs.inorgchem.9b02093
  • Peer Reviewed
• [Journal Article] Fundamental electron-transfer and proton-coupled electron-transfer properties of Ru(iv)-oxo complexes (2019)
  • Author(s): Hiroaki Kotani, Hinatsu Shimomura, Momoka Horimoto, Tomoya Ishizuka, Yoshihito Shiota, Kazunari Yoshizawa, Sachiko Yanagisawa, Yuka Kawahara-Nakagawa, Minoru Kubo, Takahiko Kojima
  • Journal Title: Dalton Transactions Volume: 48 Issue: 35 Pages: 13154-13161
  • DOI: 10.1039/c9dt02734c
  • NAID: 120007133077
  • Peer Reviewed
• [Presentation] Time-resolved infrared and XFEL analyses of P450nor reaction intermediates using caged-substrate. (2021)
  • Author(s): Kubo, M.
  • Organizer: Pacifichem2021
  • Int'l Joint Research / Invited
• [Presentation] Combining time-resolved XFEL crystallography with IR spectroscopy for capturing P450nor reaction intermediates. (2021)
  • Author(s): Kubo, M.
  • Organizer: Pacifichem2021
  • Int'l Joint Research / Invited
• [Presentation] Spectroscopic study on the action mechanism of Higd1a for activating cytochrome c oxidase (2021)
  • Author(s): 松村和香, 柳澤幸子, 西田優也, 長尾壮将, 新谷泰範, 久保稔
  • Organizer: 第59回日本生物物理学会年会
• [Presentation] Investigation of reaction intermediates of indoleamine 2,3-dioxygenase by using a stopped-flow Raman/absorption spectrometer (2021)
  • Author(s): 河村 味奈, 名定加峰, 柳澤 幸子, 久保 稔
  • Organizer: 第59回日本生物物理学会年会
• [Presentation] Structure of a heme-oxy intermediate of cytochrome P450BM3 catalyzing a non-natural substrate (2021)
  • Author(s): 桑野わ子, 長尾聡, 當舎武彦, Joshua Kyle Stanfield, 笠井千枝, 有安真也, 荘司長三, 杉本宏, 久保稔
  • Organizer: 第59回日本生物物理学会年会（学生発表賞）
• [Presentation] SACLAを用いたタンパク質の動的構造生物学 (2020)
  • Author(s): 久保稔
  • Organizer: 光・量子デバイス研究会 "医療・バイオ応用を目指したナノ構造・ナノ界面"，姫路・西はりま地場産業センター（兵庫県・姫路市），2020年1月8日
  • Invited
• [Presentation] XFELと赤外レーザーを用いた酵素反応中間体の時間分解計測 (2019)
  • Author(s): 久保稔
  • Organizer: 第13回バイオ関連化学シンポジウム "フォーカスドセッション：先端分光分析・計算科学を活用したバイオ関連化学最前線"，東北大学青葉山東キャンパス（宮崎県・仙台市），2019年9月5日
  • Invited