High-resolution structural analysis of mitochondrial supercomplex and ATP synthase involved in ATP synthesis

• Project/Area Number: 17H03646
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: University of Hyogo
• Principal Investigator: Shinzawa-Itoh Kyoko 兵庫県立大学, 生命理学研究科, 特任准教授 (70206316)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥16,770,000 (Direct Cost: ¥12,900,000、Indirect Cost: ¥3,870,000); Fiscal Year 2019: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2018: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2017: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
• Keywords: 呼吸鎖超複合体 / ATP合成酵素 / ミトコンドリア / 呼吸鎖電子伝達系 / 複合体Ⅳ / supercomplex / ATP synthase / mitochondria / respiratory chain / Super complex / ミトコンドリア呼吸鎖 / ATP合成酵素（複合体V) / X線結晶構造解析 / ATP合成

Outline of Final Research Achievements

Mitochondrial respiratory complex I, III and IV organized into super complex convert energy from a series of electron transfer into a proton electrochemical gradient, harvested by complex V for synthesis of ATP. The structure and functions of the super complex and each respiratory complex, purified from bovine heart to retain their original structure and functions, were compared. In all crystals reported to date, bovine complex IV exists as a dimer. Based on the structural and functional comparison of the monomer and dimer, we propose that the monomer is the activated form. Two cardiolipins are located at the interface region in the super complex. The visible resonance Raman spectra of purified reactive super complex and mixture of its individual components enabled us to discuss the detailed active site structures, but no detectable differences were observed. This indicates that the formation of a super complex did not markedly change the structure or electronic state of heme.

Academic Significance and Societal Importance of the Research Achievements

2量体結晶構造が詳細に解明されてきた複合体Ⅳは、膜内及び超複合体内では単量体で存在し、単量体が活性型である事を示した。単量体結晶のX線結晶構造はカルジオリピンが複合体Ⅳの2量体形成ではなく複合体Ⅰ、Ⅲとの相互作用に関わることを示した。超複合体と各複合体混合溶液の振動分光学解析は会合が活性中心の大きな構造変化を伴わない事を示した。また、高い呼吸活性を示すが、比較的不飽和度が低く短鎖の脂質を含む流動性のある膜内で、密度を及び電子伝達体の割合を高くすることにより超複合体形成を伴わないで効率の良いATP合成を可能にしているショウジョウバエの適応は、ミトコンドリア内でのATP産生調節の多様性を示した。

Research Products

• [Journal Article] Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I (2020)
  • Author(s): Uno, S., Masuya, T., Shinzawa-Itoh, K., Lasham, J., Haapanen, O., Shiba, T., Inaoka, D. K., Sharma, V., Murai, M., and Miyoshi, H.
  • Journal Title: The Journal of Biological Chemistry Volume: 295 Issue: 8 Pages: 2449-2463
  • DOI: 10.1074/jbc.ra119.012347
  • Peer Reviewed / Open Access / Int'l Joint Research
• [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] Osmotic Pressure Effects Identify Dehydration upon Cytochrome c - Cytochrome c Oxidase Complex Formation Contributing to a Specific Electron Pathway Formation (2020)
  • Author(s): Sato, W., Hitaoka, S., Uchida, T., Shinzawa-Itoh, K., Yoshizawa, K., Yoshikawa, S., Ishimori, K.
  • Journal Title: Biochem. J. Volume: 印刷中 Issue: 8 Pages: 1565-1578
  • DOI: 10.1042/bcj20200023
  • NAID: 120007029079
  • Peer Reviewed
• [Journal Article] Monomeric structure of an active form of bovine cytochrome c oxidase (2019)
  • Author(s): Shinzawa-Itoh Kyoko、Sugimura Takashi、Misaki Tomonori、Tadehara Yoshiki、Yamamoto Shogo、Hanada Makoto、Yano Naomine、Nakagawa Tetsuya、Uene Shigefumi、Yamada Takara、Aoyama Hiroshi、Yamashita Eiki、Tsukihara Tomitake、Yoshikawa Shinya、Muramoto Kazumasa
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 116 Issue: 40 Pages: 19945-19951
  • DOI: 10.1073/pnas.1907183116
  • Peer Reviewed / Open Access
• [Journal Article] Purified F-ATP synthase forms a Ca2+dependent high-conductance channel matching the mitochondrial permeability transition pore. (2019)
  • Author(s): Urbani A,Giorgio V,Carrer A, Franchin C,Arrigoni G, Jiko C,Abe K,Maeda S, Sginzawa-Itoh K, Bogers JF, McMillan DG, Gerle C, Szabo I, Bernardi P
  • Journal Title: Nature communications Volume: 4341 Issue: 1 Pages: 4341-4341
  • DOI: 10.1038/s41467-019-12331-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [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] Higd1a improves respiratory function in the models of mitochondrial disorder (2019)
  • Author(s): Nagao Takemasa、Shintani Yasunori、Hayashi Takaharu、Kioka Hidetaka、Kato Hisakazu、Nishida Yuya、Yamazaki Satoru、Tsukamoto Osamu、Yashirogi Shohei、Yazawa Issei、Asano Yoshihiro、Shinzawa‐Itoh Kyoko、Imamura Hiromi、Suzuki Takeo、Suzuki Tsutomu、Goto Yu‐ichi、Takashima Seiji
  • Journal Title: The FASEB Journal Volume: 34 Issue: 1 Pages: 1859-1871
  • DOI: 10.1096/fj.201800389r
  • Peer Reviewed
• [Journal Article] Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V (2018)
  • Author(s): Shimada S, Maeda S, Hikita M, Mieda-Higa K, Uene S, Nariai Y, Shinzawa-Itoh K
  • Journal Title: Protein Expr Purif. Volume: 150 Pages: 33-43
  • DOI: 10.1016/j.pep.2018.04.015
  • Peer Reviewed / Open Access
• [Journal Article] Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH (2018)
  • Author(s): Luo Fangjia、Shinzawa-Itoh Kyoko、Hagimoto Kaede、Shimada Atsuhiro、Shimada Satoru、Yamashita Eiki、Yoshikawa Shinya、Tsukihara Tomitake
  • Journal Title: Acta Crystallographica Section F Structural Biology Communications Volume: 74 Issue: 2 Pages: 92-98
  • DOI: 10.1107/s2053230x17018532
  • Peer Reviewed / Open Access
• [Journal Article] Performance of a time-resolved IR facility for assessment of protonation T states and polarity changes in carboxyl groups in a large membrane protein, mammalian cytochrome c oxidase, under turnover conditions in a sub-millisecond time resolution (2018)
  • Author(s): Chen Li, Tatsuhito Nishiguchi, Kyoko Shinzawa-Itoh, Shinya Yoshikawa, Takashi Ogura, Satoru Nakashima
  • Journal Title: Biochimica et Biophysica Acta Volume: 1859 Issue: 10 Pages: 1045-1050
  • DOI: 10.1016/j.bbabio.2018.05.015
  • Peer Reviewed
• [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
• [Journal Article] A unique respiratory adaptation in Drosophila independent of supercomplex formation. (2018)
  • Author(s): Shimada S, Oosaki M, Takahashi R, Uene S, Yanagisawa S, Tsukihara T, Shinzawa-Itoh K.
  • Journal Title: Biochim Biophys Acta. Volume: 1859 Issue: 2 Pages: 154-163
  • DOI: 10.1016/j.bbabio.2017.11.007
  • Peer Reviewed / Open Access
• [Presentation] Monomeric structure of an active form of bovine cytochrome c oxidase. (2019)
  • Author(s): Shinzawa-Itoh K., Sugimura T., Misaki T., Tadehara Y., Yamamoto S., Hanada M., Yano N., Nakagawa T., Uene S., Yamada T., Aoyama H., Yamashita E., Tsukihara T., Yoshikawa S., and Muramoto K.
  • Organizer: 日本生化学会
• [Presentation] Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V (2018)
  • Author(s): Kyoko Shinzawa-Itoh
  • Organizer: 日本生化学会
• [Presentation] A unique respiratory adaptation in Drosophila independent of supercomplex formation. (2018)
  • Author(s): Kyoko Shinzawa-Itoh
  • Organizer: 生物物理学会
• [Presentation] Complex structure of cytochrome c-cytochrome c oxidase reveals a novel interprotein interaction mode (2017)
  • Author(s): 伊藤(新澤)恭子
  • Organizer: 第19回国際生物物理学(International Union for Pure and Applied Biophysics：IUPAB)大会
  • Int'l Joint Research
• [Presentation] Complex structure of cytochrome c-cytochrome c oxidase reveals a novel interprotein interaction mode (2017)
  • Author(s): 伊藤(新澤)恭子
  • Organizer: 第9回国際構造生物学会（9th International Conference on Structural Biology）
  • Int'l Joint Research
• [Presentation] ショウジョウバエのミトコンドリアにおける超複合体形成によらない独特の呼吸適応 (2017)
  • Author(s): 伊藤(新澤)恭子
  • Organizer: 2017年度生命科学系学会合同年次大会（ConBio2017）