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

Author

Listed:
  • 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
    Graduate School of Life Science, University of Hyogo)

  • 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
    Japan Science and Technology Agency, PRESTO)

  • 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
    Graduate School of Life Science, University of Hyogo)

  • 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
    Graduate School of Medicine, Kyoto University)

  • Rie Tanaka

    (RIKEN SPring-8 Center)

  • Osamu Nureki

    (Graduate School of Science, The University of Tokyo)

  • Osami Shoji

    (Graduate School of Science, Nagoya University
    Japan Science and Technology Agency, CREST)

  • 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
    Graduate School of Medicine, Kyoto University)

  • Hiroshi Sugimoto

    (RIKEN SPring-8 Center
    Japan Science and Technology Agency, CREST)

  • Yoshitsugu Shiro

    (RIKEN SPring-8 Center
    Graduate School of Life Science, University of Hyogo)

  • Minoru Kubo

    (RIKEN SPring-8 Center
    Japan Science and Technology Agency, PRESTO)

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.

Suggested Citation

  • Takehiko Tosha & Takashi Nomura & Takuma Nishida & Naoya Saeki & Kouta Okubayashi & Raika Yamagiwa & Michihiro Sugahara & Takanori Nakane & Keitaro Yamashita & Kunio Hirata & Go Ueno & Tetsunari Kimur, 2017. "Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate," Nature Communications, Nature, vol. 8(1), pages 1-9, December.
  • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01702-1
    DOI: 10.1038/s41467-017-01702-1
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    Cited by:

    1. Basudev Maity & Mitsuo Shoji & Fangjia Luo & Takanori Nakane & Satoshi Abe & Shigeki Owada & Jungmin Kang & Kensuke Tono & Rie Tanaka & Thuc Toan Pham & Mariko Kojima & Yuki Hishikawa & Junko Tanaka &, 2024. "Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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