The elucidation of reaction mechanism of cytochrome c oxidase using XFEL time-resolved structure analysis

• Project/Area Number: 18K14635
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: Gifu University
• Principal Investigator: Shimada Atsuhiro 岐阜大学, 応用生物科学部, 准教授 (80723874)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2020: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 膜タンパク質 / プロトンポンプ / 金属タンパク質 / ヘム / 酸素還元 / 生体エネルギー / 酸素代謝 / 電子伝達 / 中間体構造 / X線自由電子レーザー / 時分割構造解析

Outline of Final Research Achievements

Cytochrome c oxidase (CcO), which generates bioenergy by catalyzing O2 for maintain biological activity, is one of the most important protein in bioenergetics field. Recently, it was reported that the regulation of CcO activity prevents the production of excess energy and reactive oxygen species, which induce type-II diabetes and obesity. In this study, I succeeded in determining the catalytic intermediate structures of CcO using huge CcO crystal. Furthermore, 3.0 A resolution diffraction images and X-ray emission spectrum (XES) data are successfully obtained by X-ray free electron laser. Since XES shows the electron states of metal centers in CcO, I can evaluate the accurate structure of CcO based on the coordinates of atoms and electron states.

Academic Significance and Societal Importance of the Research Achievements

シトクロム酸化酵素（CcO）はミトコンドリア内膜に存在しており、酸素を水へと還元することで生命活動に必須のATPを合成する駆動力を生み出す。そのため、生体エネルギー論分野において、CcOの反応機構解明は最重要課題であり続けている。また、CcOによる酸素還元反応過程でほとんど活性酸素種といった有害な物質を産生しない。そのため、CcOの酸素還元中心の構造をもとに高効率で安全な人工触媒の設計も期待される。CcOの反応機構を理解するためには、その反応中間体構造の解明が不可欠である。本研究では、初めてCcOの生理学的な反応中間体の構造を高分解能で決定した。

Research Products

• [Journal Article] X-ray structures of catalytic intermediates of cytochrome c oxidase provide insights into its O2 activation and unidirectional proton-pump mechanisms (2020)
  • Author(s): A. Shimada, Y. Etoh, R. Kitoh-Fujisawa, A. Sasaki, K. Shinzawa-Itoh, T. Hiromoto, E. Yamashita, K. Muramoto, T. Tsukihara, S. Yoshikawa
  • Journal Title: J. Biol. Chem. Volume: 295 Issue: 17 Pages: 5818-5833
  • DOI: 10.1074/jbc.ra119.009596
  • Peer Reviewed
• [Journal Article] Low-dose X-ray structure analysis of cytochrome c oxidase utilizing high-energy X-rays. (2019)
  • Author(s): G. Ueno, A. Shimada, E. Yamashita, K. Hasegawa, T. Kumasaka, K. Shinzawa-Itoh, S. Yoshikawa, T. Tsukihara, M. Yamamoto.
  • Journal Title: J Synchrotron Radiat. Volume: 26 Issue: 4 Pages: 912-921
  • DOI: 10.1107/s1600577519006805
  • Peer Reviewed / Open Access
• [Journal Article] A temperature-controlled cold-gas humidifier and its application to protein crystals with the humid-air and glue-coating method (2019)
  • Author(s): Baba Seiki、Shimada Atsuhiro、Mizuno Nobuhiro、Baba Junpei、Ago Hideo、Yamamoto Masaki、Kumasaka Takashi
  • Journal Title: Journal of Applied Crystallography Volume: 52 Issue: 4 Pages: 699-705
  • DOI: 10.1107/s1600576719006435
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structure Changes Induced by O₂-binding Tightly Regulate the Proton-pumping of Cytochrome <i>c</i> Oxidase (2019)
  • Author(s): SHIMADA Atsuhiro
  • Journal Title: Seibutsu Butsuri Volume: 59 Issue: 2 Pages: 106-107
  • DOI: 10.2142/biophys.59.106
  • NAID: 130007620410
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed / Open Access
• [Journal Article] X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity. (2018)
  • Author(s): 1.Shimada A, Hatano K, Tadehara H, Yano N, Shinzawa-Itoh K, Yamashita E, Muramoto K, Tsukihara T, Yoshikawa S.
  • Journal Title: J Biol Chem. Volume: 293 Issue: 38 Pages: 14868-14879
  • DOI: 10.1074/jbc.ra118.003123
  • Peer Reviewed / Open Access
• [Presentation] O2-activation and unidirectional proton transfer mechanism by cytochrome c oxidase elucidated by X-ray structures of its catalytic intermediate (2020)
  • Author(s): Shimada, A
  • Organizer: 第20回日本蛋白質科学会年会
• [Presentation] The structural analysis of cytochrome c oxidase complexed with cytochrome c using cryo-electron microscopy (2020)
  • Author(s): Shimada, A., Kozai, D., Nishikawa, K., Fujiyoshi, Y., Sho, G., Mizutani, T. and Tani, K
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] O2-activation and unidirectional proton-pump mechanisms of cytochrome c oxidase elucidated by X-ray structures of its catalytic intermediates (2019)
  • Author(s): Shimada Atsuhiro、Etoh Yuki、Shinzawa-Itoh Kyoko、Yamashita Eiki、Muramoto Kazumasa、Tsukihara Tomitake、Yoshikawa Shinya
  • Organizer: International Symposium on Diffraction Structural Biology
  • Int'l Joint Research
• [Presentation] チトクロム酸化酵素反応中間体の構造解析による、酸素還元と共役したプロトンポンプ機構の解明 (2019)
  • Author(s): 島田敦広、村本和優、伊藤-新澤恭子、月原冨武、吉川信也
  • Organizer: 第19回日本蛋白質科学会年会
• [Presentation] X線照射量の違いがチトクロム酸化酵素の結晶構造へ与える影響 (2019)
  • Author(s): Gyokucho Sho、Akari Miyamoto、Kazuma ashida、Kyoko Ito-Shinzawa、Astuhiro Shimada、Tomitake Tsukihara、Shinnya yoshikawa
  • Organizer: 第19回日本蛋白質科学会年会
• [Presentation] チトクロム酸化酵素によるプロトンポンプは、酸素結合によって誘起されるタンパク質内構造変化によって厳密に制御されている (2018)
  • Author(s): 島田敦広、久保稔、馬場清喜、吾郷日出夫、吉川信也、月原冨武
  • Organizer: 第56回日本生物物理学会年会
  • Invited
• [Presentation] A nanosecond time-resolved XFEL structure analysis reveals the structural changes associated with CO release from Cytochroem c Oxidase (2018)
  • Author(s): Atsuhiro Shimada, Minoru Kubo, Seiki Baba, Hideo Ago, Tomitake Tsukihara, Shinya Yoshikawa
  • Organizer: 第18回日本蛋白質科学会年会