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

2017 Fiscal Year Final Research Report

• 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
• 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

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.

Free Research Field

生物物理学