学術雑誌論文 Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate

Tosha, Takehiko  ,  Nomura, Takashi  ,  Nishida, Takuma  ,  Saeki, Naoya  ,  Okubayashi, Kouta  ,  Yamagiwa, Raika  ,  Sugahara, Michihiro  ,  Nakane, Takanori  ,  Yamashita, Keitaro  ,  Hirata, Kunio  ,  Ueno, Go  ,  Kimura, Tetsunari  ,  Hisano, Tamao  ,  Muramoto, Kazumasa  ,  Sawai, Hitomi  ,  Takeda, Hanae / Mizohata, Eiichi / Yamashita, Ayumi / Kanematsu, Yusuke / Takano, Yu / Nango, Eriko / Tanaka, Rie / Nureki, Osamu / Shoji, Osami / Ikemoto, Yuka / Murakami, Hironori / Owada, Shigeki / Tono, Kensuke / Yabashi, Makina / Yamamoto, Masaki / Ago, Hideo / Iwata, So / Sugimoto, Hiroshi / Shiro, Yoshitsugu / Kubo, Minoru

8p.1585 , 2017-11-17 , Nature Publishing Group
ISSN:2041172320411723
内容記述
Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe3+-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H- from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.
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http://www.lib.kobe-u.ac.jp/repository/90004465.pdf

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