title: A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline.
abstract: 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.
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Sakaguchi
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Tetsunari
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.
authors.forename: Takuma
authors.initials: T
authors.lastname: Nishida
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Tosha
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugimoto
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yamaguchi
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Yanagisawa
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Go
authors.initials: G
authors.lastname: Ueno
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hironori
authors.initials: H
authors.lastname: Murakami
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Ago
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ogura
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kubo
mesh_terms: D023281:Genomics; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014961:X-Ray Diffraction / Q000379:methods*
keywords: UV–visible absorption spectroscopy; X-ray crystallography; cytochrome P450nor; radiation damage; simultaneous measurement