Development of a micro-flow time-resolved IR spectrometer for analyzing short-lived reaction intermediates of enzymes

• Project/Area Number: 15H03841
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bio-related chemistry
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Kubo Minoru 国立研究開発法人理化学研究所, 放射光科学総合研究センター, 専任研究員 (90392878)
• Co-Investigator(Renkei-kenkyūsha): TOSHA Takehiko 国立研究開発法人理化学研究所, 放射光科学総合研究センター, 専任研究員 (00548993) ; KIMURA Tetsunari 神戸大学, 大学院理学研究科, 特命講師 (70506906)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥17,810,000 (Direct Cost: ¥13,700,000、Indirect Cost: ¥4,110,000); Fiscal Year 2017: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2016: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000); Fiscal Year 2015: ¥13,260,000 (Direct Cost: ¥10,200,000、Indirect Cost: ¥3,060,000)
• Keywords: 時間分解赤外分光 / マイクロ流路フローセル / 酵素反応 / 膜タンパク質 / 一酸化窒素還元酵素 / 超精密計測 / フェムト秒レーザー / ケージド化合物 / ポンププローブ法 / 蛋白質 / 高性能レーザー / 生物物理

Outline of Final Research Achievements

This project aims to establish a time-resolved IR spectroscopic technique for studying dynamic structures of enzymes. A novel micro flow system has been developed and incorporated into a femtosecond laser-based IR spectrometer, which allows micro flow-flash IR measurements of enzymatic reactions with the minimum sample volume of 20 nL. We applied this measurement system to NO reductase, a heme enzyme that catalyzes the reduction of NO to N2O in the nitrogen cycle, and succeeded in determining the NO binding mode in the short-lived intermediate. Furthermore, we also used the micro-flow flash method for time-resolved crystallography at SACLA, and succeeded in visualizing the 3D structure of the NO-bound intermediate during the NO reduction reaction.

Research Products

• [Journal Article] Ultraprecision cutting of single-crystal calcium fluoride for fabricating micro flow cells (2018)
  • Author(s): Ryo KOMIYA, Tetsunari KIMURA, Takashi NOMURA, Minoru KUBO, Jiwang YAN
  • Journal Title: Journal of Advanced Mechanical Design, Systems, and Manufacturing Volume: 12 Issue: 1 Pages: JAMDSM0021-JAMDSM0021
  • DOI: 10.1299/jamdsm.2018jamdsm0021
  • NAID: 130006386864
  • ISSN: 1881-3054
  • Peer Reviewed
• [Journal Article] Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate (2017)
  • Author(s): Tosha Takehiko、Kubo Minoru, et. al
  • Journal Title: Nature Communications Volume: 8 Issue: 1 Pages: 1-9
  • DOI: 10.1038/s41467-017-01702-1
  • Peer Reviewed / Open Access
• [Journal Article] Nanosecond pump probe device for time-resolved serial femtosecond crystallography developed at SACLA (2017)
  • Author(s): Kubo Minoru、Nango Eriko、Murata Michio、Neutze Richard、Yabashi Makina、Iwata So, その他１８名
  • Journal Title: Journal of Synchrotron Radiation Volume: 24 Issue: 5 Pages: 1086-1091
  • DOI: 10.1107/s160057751701030x
  • NAID: 120006363292
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A nanosecond time-resolved XFEL analysis of structural changes associated with CO release from cytochrome c oxidase. (2017)
  • Author(s): Shimada, A.†, Kubo, M.†, Baba, S.† et al. (29人中2番目)(†equal contribution)
  • Journal Title: Sci. Adv. Volume: 3 Issue: 7
  • DOI: 10.1126/sciadv.1603042
  • NAID: 120006363290
  • Peer Reviewed / Open Access
• [Journal Article] Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. (2017)
  • Author(s): Suga, M.†, Akita, F.†, Sugahara, M.†, Kubo, M.†, Nakajima, Y.† et al. (†equal contribution)(38人中1番目)
  • Journal Title: Nature Volume: 543 Issue: 7643 Pages: 131-135
  • DOI: 10.1038/nature21400
  • NAID: 120006344935
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography. (2016)
  • Author(s): Nogly, P., et al. (40人中8番目)
  • Journal Title: Nat. Commun. Volume: 7 Issue: 1 Pages: 12314-12314
  • DOI: 10.1038/ncomms12314
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A three dimensional movie of structural changes in bacteriorhodopsin. (2016)
  • Author(s): Nango, E., Royant, A.†, Kubo, M.† et al. (†equal contribution)(45名中2番目)
  • Journal Title: Science Volume: 354 Issue: 6319 Pages: 1552-1557
  • DOI: 10.1126/science.aah3497
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A Study of the Dynamics of the Heme Pocket and C-helix in CooA Upon CO Dissociation Using Time-resolved Visible and UV Resonance Raman Spectroscopy (2016)
  • Author(s): Akihiro Otomo, Haruto Ishikawa, Misao Mizuno, Tetsunari Kimura, Minoru Kubo, Yoshitsugu Shiro, Shigetoshi Aono, and Yasuhisa Mizutani
  • Journal Title: J. Phys. Chem. B Volume: 120 Issue: 32 Pages: 7836-7843
  • DOI: 10.1021/acs.jpcb.6b05634
  • Peer Reviewed
• [Journal Article] Molecular mechanism of the catalytic reaction of NO reductase revealed by novel time-resolved visible/IR absorption spectrometers with microfluidic device (2016)
  • Author(s): Kimura, T., Takeda, H., Ishii, S., Tosha, T., Shiro, Y., Kubo, M.
  • Journal Title: Biophysical Journal Volume: 110 Supp1 Issue: 3 Pages: 548a-548a
  • DOI: 10.1016/j.bpj.2015.11.2933
• [Presentation] Observation of water-channel opening of cytochrome c oxidase by time-resolved XFEL crystallography. (2017)
  • Author(s): Kubo, M., Shimada, A., Baba, S., Yamashita, K., Hirata, K., Yamamoto, M., Shinzawa-Itoh, K., Ago, H., Yoshikawa, S., Tsukihara, T.
  • Organizer: 19th International Union for Pure and Applied Biophysics Congress and 11th European Biophysics Congress
  • Int'l Joint Research
• [Presentation] Time-resolved XFEL crystallography and spectroscopy of cytochrome c oxidase. (2017)
  • Author(s): Kubo, M., Shimada, A., Baba, S., Yamashita, K., Hirata, K., Yamamoto, M., Shinzawa-Itoh, K., Ago, H., Yoshikawa, S., Tsukihara, T.
  • Organizer: 24th Congress & General Assembly of the International Union of Crystallography (IUCr 2017)
  • Int'l Joint Research
• [Presentation] 蛋白質の時間分解X線結晶構造解析における分光の役割． (2017)
  • Author(s): 久保稔
  • Organizer: 第17回日本蛋白質科学会年会，ワークショップ “SACLAにおける蛋白質構造科学研究の現状と展望”
  • Invited
• [Presentation] Combining XFEL crystallography and single-crystal spectroscopy for studying reaction dynamics of respiratory metalloenzymes． (2017)
  • Author(s): 久保稔
  • Organizer: 日本生物物理学会，第55回年会シンポジウム “構造生命科学の新しい潮流”
  • Invited
• [Presentation] XFEL結晶構造解析と赤外分光法を用いたタンパク質の動的精密構造解析． (2017)
  • Author(s): 久保稔
  • Organizer: 2017年度第1回水和ナノ構造研究会
  • Invited
• [Presentation] チトクロム酸化酵素の動的構造解析：リガンド結合と共役した水チャネルの開閉機構． (2017)
  • Author(s): 久保稔
  • Organizer: 2017年度生命科学系学会合同年次大会（ConBio2017），ワークショップ “X線自由電子レーザーが捉えるタンパク質ダイナミクス研究の最前線”
  • Invited
• [Presentation] 凍結トラップX線結晶構造解析および時間分解赤外分光法を用いた P450nor の反応中間体の構造解析． (2017)
  • Author(s): 野村高志, 當舎武彦, 杉本宏, 久野玉雄, 山際来佳, Chai Gopalasingam, 山下恵太郎, 平田邦生, 山本雅貴, 城宣嗣, 久保稔
  • Organizer: 平成29年度日本結晶学会年会
• [Presentation] 時間分解赤外分光法および凍結トラップXFEL結晶構造解析を用いたP450norの反応中間体の解析． (2017)
  • Author(s): 野村高志, 當舎武彦, 杉本宏, 久野玉雄, 山際来佳, Chai Gopalasingam, 山下恵太郎, 平田邦生, 山本雅貴, 城宣嗣, 久保稔
  • Organizer: 第44回生体分子科学討論会
• [Presentation] X線自由電子レーザーを利用した時間分解X線結晶構造解析による酵素反応の観測． (2017)
  • Author(s): 當舎武彦, 西田拓真, 野村高志, 佐伯直哉, 杉本宏, 山下恵太郎, 平田邦生, 吾郷日出夫, 山本雅貴, 中根崇智, 菅原道泰, 南後恵理子, 岩田想, 城宜嗣, 久保稔
  • Organizer: 第44回生体分子科学討論会
• [Presentation] Elucidation of the NO reduction mechanism of nitric oxide reductase using time-resolved vis/IR spectroscopy． (2017)
  • Author(s): 武田英恵，木村哲就，野村高志，松林亜希子，石井頌子，當舎武彦，城宜嗣，久保稔
  • Organizer: 日本生物物理学会，第55回年会
• [Presentation] Time-resolved crystallography and IR spectroscopy of cytochrome c oxidase. (2017)
  • Author(s): Kubo, M.
  • Organizer: The 4th Ringberg Workshop on Structural Biology with FELs
  • Place of Presentation: Ringberg Castle (83708 Kreuth, Germany)
  • Int'l Joint Research / Invited
• [Presentation] Molecular mechanism of the catalytic reaction of NO reductase revealed by novel time-resolved visible/IR absorption spectrometers with microfluidic device (2016)
  • Author(s): Kimura, T., Takeda, H., Ishii, S., Tosha, T., Shiro, Y., Kubo, M.
  • Organizer: The 60th Biophysical Society Annual Meeting
  • Place of Presentation: Los Angeles Convention Center (Los Angeles, USA)
  • Year and Date: 2016-03-02
  • Int'l Joint Research
• [Presentation] ダイナミクス研究における未解決問題と振動分光からのアプローチ (2016)
  • Author(s): 久保稔
  • Organizer: 蛋白研セミナー “構造を基盤とする蛋白質科学における未解決問題”
  • Place of Presentation: 東京大学先端科学技術研究センター（東京都目黒区）
  • Year and Date: 2016-03-01
  • Invited
• [Presentation] フェムト秒赤外レーザーを用いた生体分子ダイナミクスの観測 (2016)
  • Author(s): 久保稔，木村哲就，武田英恵，石井頌子，當舎武彦，城宜嗣
  • Organizer: 分子研研究会“高輝度・高強度赤外光源の現状と展望”
  • Place of Presentation: 岡崎コンファレンスセンター（愛知県岡崎市）
  • Year and Date: 2016-02-11
• [Presentation] SACLAが拓く応用研究：生体分子の動的構造観察． (2016)
  • Author(s): 久保稔
  • Organizer: 日本分光学会関西支部，平成28年度 最近の分光学の進歩に関する講演会 “自由電子レーザーの進歩と実用”
  • Place of Presentation: 大阪電気通信大学 駅前キャンパス（寝屋川市早子町）
  • Invited
• [Presentation] Observation of water channel opening upon CO photolysis in cytochrome c oxidase by time-resolved XFEL crystallography and IR spectroscopy. (2016)
  • Author(s): Kubo, M., Shimada, A., Baba, S., Yamashita, K., Hirata, K., Yamamoto, M., Shinzawa-Itoh, K., Tsukihara, T., Ago, H., Yoshikawa, S.
  • Organizer: The 42nd Naito Conference "In the Vanguard of Structural Biology: Revolutionizing Life Sciences"(ポスター賞受賞)
  • Place of Presentation: シャトレーゼガトーキングダム（札幌市北区）
  • Int'l Joint Research
• [Presentation] 膜タンパク質の反応解析に向けた赤外分光技術の開発と膜内在性金属酵素への応用． (2016)
  • Author(s): 久保稔
  • Organizer: 蛋白研セミナー “膜タンパク質の構造ダイナミクス”
  • Place of Presentation: 大阪大学吹田キャンパス 蛋白質研究所（吹田市山田丘）
  • Invited
• [Presentation] Kinetic analysis of the NO reduction reaction of nitric oxide reductase using nanoliter-flow time-resolved visible/IR spectroscopy. (2016)
  • Author(s): Takeda, H., Kimura, T., Ishii, S., Tosha, T., Shiro, Y., Kubo, M.
  • Organizer: The 4th International Symposium for Young Chemists on Stimuli-Responsive Chemical Species for the Creation of Functional Molecules.
  • Place of Presentation: 大阪大学吹田キャンパス サントリーメモリアルホール（吹田市山田丘）
  • Int'l Joint Research
• [Presentation] マイクロ流路フロー・フラッシュ時間分解赤外分光法による一酸化窒素還元酵素の反応機構解析． (2016)
  • Author(s): 木村哲就, 武田英恵, 石井頌子, 當舎武彦, 城宜嗣, 久保稔
  • Organizer: 第43回生体分子科学討論会
  • Place of Presentation: 名古屋大学野依記念学術交流館（名古屋市千種区）
• [Presentation] Development of a highly-sensitive time-resolved IR spectrometer and its application to high-background aqueous biological samples (2015)
  • Author(s): Kubo, M., Kimura, T., Yamaguchi, Y., Yanagisawa, S., Komiya, R., Yan, J., Nakashima, S., Ogura, T., Shiro, Y.
  • Organizer: Pacifichem 2015
  • Place of Presentation: Marriott Waikiki Beach (Hawaii, USA)
  • Year and Date: 2015-12-18
  • Int'l Joint Research
• [Presentation] Development of a time-resolved visible/IR absorption spectrometer with a novel microfluidic device and its application to catalytic reaction of NO reductase (2015)
  • Author(s): Kimura, T., Ishii, S., Tosha, T., Shiro, Y., Kubo, M.
  • Organizer: Pacifichem 2015
  • Place of Presentation: Hawaii Convention Center (Hawaii, USA)
  • Year and Date: 2015-12-16
  • Int'l Joint Research
• [Presentation] マイクロ流体フロー・フラッシュ赤外分光測定系による一酸化窒素還元酵素の触媒反応の実時間観察 (2015)
  • Author(s): 木村哲就, 石井頌子, 當舎武彦, 城宜嗣, 久保稔
  • Organizer: 第9回分子科学討論会
  • Place of Presentation: 東京工業大学大岡山キャンパス（東京都目黒区）
  • Year and Date: 2015-09-19
• [Presentation] Development of micro-channel flow-flash infrared absorption spectroscopy and its application to early intermediate of nitric oxide reductase (2015)
  • Author(s): 木村哲就，石井頌子，當舎武彦，城宜嗣，久保稔
  • Organizer: 日本生物物理学会，第53回年会
  • Place of Presentation: 金沢大学角間キャンパス（石川県金沢市）
  • Year and Date: 2015-09-14
• [Presentation] マイクロ流体フローを用いた一酸化窒素還元酵素の触媒反応の動的分光観察 (2015)
  • Author(s): 木村哲就, 石井頌子, 當舎武彦, 城宜嗣, 久保稔
  • Organizer: 第15回日本蛋白質科学会年会 若手奨励賞シンポジウム
  • Place of Presentation: あわぎんホール（徳島県徳島市）
  • Year and Date: 2015-06-25
  • Invited