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; Minoru Kubo

doi:10.1038/s41467-017-01702-1

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 TakanoEriko 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, Minoru Kubo

Research output: Contribution to journal › Article › peer-review

58 Citations (Scopus)

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.

Original language	English
Article number	1585
Journal	Nature communications
Volume	8
Issue number	1
DOIs	https://doi.org/10.1038/s41467-017-01702-1
Publication status	Published - 2017 Dec 1
Externally published	Yes

ASJC Scopus subject areas

Chemistry(all)
Biochemistry, Genetics and Molecular Biology(all)
General
Physics and Astronomy(all)

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10.1038/s41467-017-01702-1

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Tosha, T., Nomura, T., Nishida, T., Saeki, N., Okubayashi, K., Yamagiwa, R., Sugahara, M., Nakane, T., Yamashita, K., Hirata, K., Ueno, G., Kimura, T., Hisano, T., Muramoto, K., Sawai, H., Takeda, H., Mizohata, E., Yamashita, A., Kanematsu, Y., ... Kubo, M. (2017). Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate. Nature communications, 8(1), [1585]. https://doi.org/10.1038/s41467-017-01702-1

Tosha, T, Nomura, T, Nishida, T, Saeki, N, Okubayashi, K, Yamagiwa, R, Sugahara, M, Nakane, T, Yamashita, K, Hirata, K, Ueno, G, Kimura, T, Hisano, T, Muramoto, K, Sawai, H, Takeda, H, Mizohata, E, Yamashita, A, Kanematsu, Y, Takano, Y, Nango, E, Tanaka, R, Nureki, O, Shoji, O, Ikemoto, Y, Murakami, H, Owada, S, Tono, K, Yabashi, M, Yamamoto, M, Ago, H, Iwata, S, Sugimoto, H, Shiro, Y & Kubo, M 2017, 'Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate', Nature communications, vol. 8, no. 1, 1585. https://doi.org/10.1038/s41467-017-01702-1

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

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 {\AA}. 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.",

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

note = "Funding Information: We thank the staff at SACLA and the members of Shiro laboratory at University of Hyogo for their technical support. We also acknowledge the computational support from SACLA HPC system and Mini-K supercomputer system. X-ray diffraction experiments were conducted at BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (2015B8042, 2016A8052, 2016A8043, 2016B8068, 2017A8047), at BL26B2 of SPring-8 with the approval of RIKEN (20150042, 20160040), and at BL32XU and BL41XU of SPring-8 with the approval of JASRI (2014B1528, 2015A1122, 2015B2122, 2016A2555). IR spectroscopic experiments were conducted at BL43IR of SPring-8 with the approval of JASRI (2015B1247, 2016A1346, 2016B1146, 2017A1170). The QM/MM computations were performed at the Research Center for Computational Science, Okazaki, Japan. This work was supported by MEXT XFEL Priority Strategy Program (to H. Sugimoto, H.A., S.I.), MEXT KAKENHI GRANT 26220807 (to Y.S.), 17H05896 (to H. Sugimoto), JSPS KAKENHI GRANT 15H03841, 15H01055 (to M.K.), 15H00965 (to T.T.), JP26105012 (to Y.T.), JST-CREST JPMJCR15P3 (to O.S., H. Sugimoto), JST-PRESTO JPMJPR12L1 (to M.K.) JPMJPR14L9 (to K.H.), JST-Research Acceleration Program (to S.I.), and Pioneering Project “Dynamic Structural Biology” of RIKEN (to M.K. and M.Y.). R.Y. and H.T. were supported by the RIKEN Junior Research Associate Program. Publisher Copyright: {\textcopyright} 2017 The Author(s).",

year = "2017",

month = dec,

day = "1",

doi = "10.1038/s41467-017-01702-1",

language = "English",

volume = "8",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Nature Publishing Group",

number = "1",

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TY - JOUR

T1 - Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate

AU - Tosha, Takehiko

AU - Nomura, Takashi

AU - Nishida, Takuma

AU - Saeki, Naoya

AU - Okubayashi, Kouta

AU - Yamagiwa, Raika

AU - Sugahara, Michihiro

AU - Nakane, Takanori

AU - Yamashita, Keitaro

AU - Hirata, Kunio

AU - Ueno, Go

AU - Kimura, Tetsunari

AU - Hisano, Tamao

AU - Muramoto, Kazumasa

AU - Sawai, Hitomi

AU - Takeda, Hanae

AU - Mizohata, Eiichi

AU - Yamashita, Ayumi

AU - Kanematsu, Yusuke

AU - Takano, Yu

AU - Nango, Eriko

AU - Tanaka, Rie

AU - Nureki, Osamu

AU - Shoji, Osami

AU - Ikemoto, Yuka

AU - Murakami, Hironori

AU - Owada, Shigeki

AU - Tono, Kensuke

AU - Yabashi, Makina

AU - Yamamoto, Masaki

AU - Ago, Hideo

AU - Iwata, So

AU - Sugimoto, Hiroshi

AU - Shiro, Yoshitsugu

AU - Kubo, Minoru

N1 - Funding Information: We thank the staff at SACLA and the members of Shiro laboratory at University of Hyogo for their technical support. We also acknowledge the computational support from SACLA HPC system and Mini-K supercomputer system. X-ray diffraction experiments were conducted at BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (2015B8042, 2016A8052, 2016A8043, 2016B8068, 2017A8047), at BL26B2 of SPring-8 with the approval of RIKEN (20150042, 20160040), and at BL32XU and BL41XU of SPring-8 with the approval of JASRI (2014B1528, 2015A1122, 2015B2122, 2016A2555). IR spectroscopic experiments were conducted at BL43IR of SPring-8 with the approval of JASRI (2015B1247, 2016A1346, 2016B1146, 2017A1170). The QM/MM computations were performed at the Research Center for Computational Science, Okazaki, Japan. This work was supported by MEXT XFEL Priority Strategy Program (to H. Sugimoto, H.A., S.I.), MEXT KAKENHI GRANT 26220807 (to Y.S.), 17H05896 (to H. Sugimoto), JSPS KAKENHI GRANT 15H03841, 15H01055 (to M.K.), 15H00965 (to T.T.), JP26105012 (to Y.T.), JST-CREST JPMJCR15P3 (to O.S., H. Sugimoto), JST-PRESTO JPMJPR12L1 (to M.K.) JPMJPR14L9 (to K.H.), JST-Research Acceleration Program (to S.I.), and Pioneering Project “Dynamic Structural Biology” of RIKEN (to M.K. and M.Y.). R.Y. and H.T. were supported by the RIKEN Junior Research Associate Program. Publisher Copyright: © 2017 The Author(s).

PY - 2017/12/1

Y1 - 2017/12/1

N2 - 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.

AB - 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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JF - Nature Communications

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ER -

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

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