Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate

Takehiko Tosha, Takashi Nomura, Takuma Nishida, Naoya Saeki, Kouta Okubayashi, Raika Yamagiwa, Michihiro Sugahara, Takanori Nakane, Keitaro Yamashita, Kunio Hirata, Go Ueno, Tetsunari Kimura, Tamao Hisano, Kazumasa Muramoto, Hitomi Sawai, Hanae Takeda, Eiichi Mizohata, Ayumi Yamashita, Yusuke Kanematsu, Yu Takano, Eriko Nango, Rie Tanaka, Osamu Nureki, Osami Shoji, Yuka Ikemoto, Hironori Murakami, Shigeki Owada, Kensuke Tono, Makina Yabashi, Masaki Yamamoto, Hideo Ago, So Iwata, Hiroshi Sugimoto (), Yoshitsugu Shiro () and Minoru Kubo ()
Additional contact information
Takehiko Tosha: RIKEN SPring-8 Center
Takashi Nomura: RIKEN SPring-8 Center
Takuma Nishida: Graduate School of Life Science, University of Hyogo
Naoya Saeki: Graduate School of Life Science, University of Hyogo
Kouta Okubayashi: Graduate School of Life Science, University of Hyogo
Raika Yamagiwa: RIKEN SPring-8 Center
Michihiro Sugahara: RIKEN SPring-8 Center
Takanori Nakane: Graduate School of Science, The University of Tokyo
Keitaro Yamashita: RIKEN SPring-8 Center
Kunio Hirata: RIKEN SPring-8 Center
Go Ueno: RIKEN SPring-8 Center
Tetsunari Kimura: Graduate School of Science, Kobe University
Tamao Hisano: RIKEN SPring-8 Center
Kazumasa Muramoto: Graduate School of Life Science, University of Hyogo
Hitomi Sawai: Graduate School of Life Science, University of Hyogo
Hanae Takeda: RIKEN SPring-8 Center
Eiichi Mizohata: Graduate School of Engineering, Osaka University
Ayumi Yamashita: RIKEN SPring-8 Center
Yusuke Kanematsu: Hiroshima City University
Yu Takano: Hiroshima City University
Eriko Nango: RIKEN SPring-8 Center
Rie Tanaka: RIKEN SPring-8 Center
Osamu Nureki: Graduate School of Science, The University of Tokyo
Osami Shoji: Graduate School of Science, Nagoya University
Yuka Ikemoto: Japan Synchrotron Radiation Research Institute
Hironori Murakami: Japan Synchrotron Radiation Research Institute
Shigeki Owada: RIKEN SPring-8 Center
Kensuke Tono: Japan Synchrotron Radiation Research Institute
Makina Yabashi: RIKEN SPring-8 Center
Masaki Yamamoto: RIKEN SPring-8 Center
Hideo Ago: RIKEN SPring-8 Center
So Iwata: RIKEN SPring-8 Center
Hiroshi Sugimoto: RIKEN SPring-8 Center
Yoshitsugu Shiro: RIKEN SPring-8 Center
Minoru Kubo: RIKEN SPring-8 Center

Nature Communications, 2017, vol. 8, issue 1, 1-9

Abstract: Abstract 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.

Date: 2017
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DOI: 10.1038/s41467-017-01702-1

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