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reductase by X-ray crystallography; New insights into regulation of efficient electron transferYamada, Mitsugu*; Tamada, Taro; Takeda, Kazuki*; Matsumoto, Fumiko*; Ono, Hiraku*; Kosugi, Masayuki*; Takaba, Kiyofumi*; Shoyama, Yoshinari*; Kimura, Shigenobu*; Kuroki, Ryota; et al.
Journal of Molecular Biology, 425(22), p.4295 - 4306, 2013/11
Times Cited Count：21 Percentile：50.76(Biochemistry & Molecular Biology)NADH-Cytochrome reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68
resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD
. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78 resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.
Kimura, Tsunehisa*; Kimura, Fumiko*; Matsumoto, Kenji*; Metoki, Naoto
Neutron Diffraction, p.179 - 202, 2012/03
We report the results of the neutron scattering study on three dimensionally oriented pseudo single crystals under rotating magnetic field.
Kimura, Fumiko*; Kimura, Tsunehisa*; Matsumoto, Kenji*; Metoki, Naoto
Crystal Growth & Design, 10(1), p.48 - 51, 2009/12
Times Cited Count：13 Percentile：75.09(Chemistry, Multidisciplinary)Single-crystal neutron diffraction analysis of an L-alanine pseudo single crystal (PSC, a composite in which microcrystals are aligned three-dimensionally) prepared from its powder is presented. Microcrystals of L-alanine were suspended in an ultraviolet-curable resin precursor and subjected to three-dimensional magnetic alignment, followed by consolidation of the resin precursor to fix the achieved alignment. A cylindrical PSC of diameter ca. 8 mm and height 10 mm was obtained and then subjected to neutron diffraction measurements. Pole figures measured at (040), (120), and (002) planes showed sharp peaks, demonstrating three-dimensional alignment of microcrystals. Scans were performed for 31 diffraction peaks whose intensities showed good agreement with calculation results. The present study showed the potential use of the PSC method in single-crystalneutron diffraction analysis when a large crystal is not available.
reductaseYamada, Mitsugu; Tamada, Taro; Matsumoto, Fumiko; Takeda, Kazuki*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*
no journal, ,
no abstracts in English
Yamada, Mitsugu; Tamada, Taro; Matsumoto, Fumiko; Takeda, Kazuki*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*
no journal, ,
no abstracts in English
reductaseYamada, Mitsugu; Tamada, Taro; Matsumoto, Fumiko; Takeda, Kazuki*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*
no journal, ,
NADH-cytochrome reductase (b5R; EC 1.6.2.2) is a flavoprotein and catalyses two-electron transfer from NADH to cytochrome through FAD. Here, we present crystal structures of fully reduced and two re-oxidized forms of b5Rs determined. The crystal structure of fully reduced b5R showed that the relative location of two domains slightly shifted in comparison with that of oxidized form. This shift allowed to create a new hydrogen bonding interaction between N5 atom of isoalloxazine ring and hydroxyl oxygen atom of Thr66. The isoalloxazine ring of FAD in fully reduced form is flat and stacked with the nicotinamide ring of bound NAD. Moreover, two re-oxidized forms (form-1 and 2) were prepared using fully reduced crystals by exposure to the air. The electron densities for the nicotinamide ring of NAD was ambiguous in form-1 and was completely disappeared in form-2. These results suggested that re-oxidization follows a two-step mechanism in which nicotinamide moiety is released firstly and then whole NAD is released.
reductase by X-ray crystallographyTamada, Taro; Yamada, Mitsugu*; Takeda, Kazuki*; Matsumoto, Fumiko*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*
no journal, ,
NADH-cytochrome reductase (b5R) is a flavoprotein consisting of NADH- and FAD- domains, and catalyzes the electron transfer from two electron carriers of NADH to one electron carrier of cytochrome (Cb5). The structure of the fully reduced form of porcine liver b5R was determined using a crystal grown under anaerobic condition. In the reduced b5R structure, which was determined at 1.68 resolution, the relative location of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent accessible surface area of FAD, and created a new hydrogen bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat, which is similar to that in the oxidized form, and is stacked with the nicotinamide ring of NAD. Both of reduced and oxidized b5R structures could explain how prevents the backflow and accelerates the transfer of electrons to one-electron acceptors, such as Cb5, in the catalytic cycle. Furthermore, the crystallographic analysis by the cryo-trapping method, which controls the exposure time of the fully reduced crystals against the air, suggested that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.
