A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline
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- 作者：Miyuki Sakaguchi ; Tetsunari Kimura ; Takuma Nishida ; Takehiko Tosha ; Hiroshi Sugimoto ; Yoshihiro Yamaguchi ; Sachiko Yanagisawa ; Go Ueno ; Hironori Murakami ; Hideo Ago ; Masaki Yamamoto ; Takashi Ogura ; Yoshitsugu Shiro and Minoru Kubo
- 关键词：cytochrome P450nor ; UV&ndash ; visible absorption spectroscopy ; radiation damage ; simultaneous measurement ; X-ray crystallography
- 刊名：Journal of Synchrotron Radiation
- 出版年：2016
- 出版时间：January 2016
- 年：2016
- 卷：23
- 期：1
- 页码：334-338
- 全文大小：846K
- ISSN：1600-5775
文摘
UV–visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.