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Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate
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-containin...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691058/ https://www.ncbi.nlm.nih.gov/pubmed/29147002 http://dx.doi.org/10.1038/s41467-017-01702-1 |
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author | 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 |
author_facet | 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 |
author_sort | Tosha, Takehiko |
collection | PubMed |
description | 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 Fe(3+)-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. |
format | Online Article Text |
id | pubmed-5691058 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-56910582017-11-20 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 Nat Commun Article 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 Fe(3+)-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. Nature Publishing Group UK 2017-11-17 /pmc/articles/PMC5691058/ /pubmed/29147002 http://dx.doi.org/10.1038/s41467-017-01702-1 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article 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 Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate |
title | Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate |
title_full | Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate |
title_fullStr | Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate |
title_full_unstemmed | Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate |
title_short | Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate |
title_sort | capturing an initial intermediate during the p450nor enzymatic reaction using time-resolved xfel crystallography and caged-substrate |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691058/ https://www.ncbi.nlm.nih.gov/pubmed/29147002 http://dx.doi.org/10.1038/s41467-017-01702-1 |
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