Elucidations of the catalytic cycle of NADH-cytochrome reductase by X-ray crystallography; New insights into regulation of efficient electron transfer

Yamada, 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

https://doi.org/10.1016/j.jmb.2013.06.010

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.

Model experiment of cosmic ray acceleration due to an incoherent wakefield induced by an intense laser pulse

Kuramitsu, Yasuhiro*; Nakanii, Nobuhiko*; Kondo, Kiminori; Sakawa, Yoichi*; Mori, Yoshitaka*; Miura, Eisuke*; Tsuji, Kazuki*; Kimura, Kazuya*; Fukumochi, Shuji*; Kashihara, Mamoru*; et al.

Physics of Plasmas, 18(1), p.010701_1 - 010701_4, 2011/01

https://doi.org/10.1063/1.3528434

Substituting an intensive laser pulse for the large amplitude light waves, we performed a model experiment of the shock environments in a laboratory plasma. An intensive laser pulse was propagated in a plasma tube created by imploding a hollow polystyrene cylinder, as the large amplitude light waves propagated in the upstream plasma at an astrophysical shock. Nonthermal electrons were generated, and the energy distribution functions of the electrons have a power-law component with an index of 2.

Long-term endurance test of PNC Type in-sodium hydrogen meter; Studies of leak detector development on LMFBR's SG (2)

Kuroha, Mitsuo; Takeda, Kunio; Iitsuka, Shoji; Sasaki, Shuichi; Okada, Toshio; Isozaki, Mikio; Daigo, Yoshimichi; Sato, Minoru

PNC TN941 81-49, 204 Pages, 1981/05

PNC-TN941-81-49.pdf:22.56MB

PNC type in-sodium hydrogen meters have been developed as leak detectors for LMFBR MONJU steam generators. In order to confirm the long-term reliability and the durability of the meters, the four meters were installed in three sodium loops at the O-arai Engineering Center, and they had been tested over a long time in flowing sodium. A period of the test was from oct. 1977 to Feb. 1980. They are called type II. The dynamic chamber of the vacuum system can separate from the static one, and be also connected with it using one flexible tube. Important findings from the test are; (1) The operating time of two meters exceeded 10,000 hours, and the total of all meters was about 35,000 hours. No trouble had been experienced in the sodium systems and the nickel membranes of them during the period, which had the good durability. Air leaks, however, occured three times in the two vacuum systems. (2) Any secular changes had hardly happened in the permeability of hydrogen through the nickel membrane and the relationship between ion pump current and hydrogen pressure. (3) The pumping speeds had decreased with increasing the amount of absorbed hydrogen. The decreasing rates differed among four ion pumps, and those of two pumps were considerably large at the beginning of absorbing hydrogen. (4) The calibration curves, which describe the relationship between hydrogen concentration in sodium and ion pump current, had changed with time. The largest cause was the decrement of the pumping speeds. (5) The UHV gauges were superior to the ion pumps from the point of the signal-to-noise ratio as the hydrogen sensor.

Sodium impurities measurement and control experiments (No.5); Determination of the solubility of oxygen in liquid sodium by vacuum distillation

Takeda, Kunio*; Ito, Kazumoto*; Mizutani, Tomonori*; Wada, Juichi*; Takani, Satoru*; Atsumo, Hideo*

PNC TN951 76-04, 64 Pages, 1976/03

PNC-TN951-76-04.pdf:1.62MB

The solubility of oxygen in liquid sodium was determined by vacuum distillation. Oxygen concentration was controlled by a cold trap in a dynamic sodium loop, and sodium was sampled from an expansion tank by dipping cup method and analysed by on line vacuum distillation method. A least-squares fit of the 37 experimental data over the range from 150 to 250C of cold trap temperature yielded a correlating equation of log S = 6.118-(2383/T) where S= wt ppm of oxygen in sodium assumed to be NaO. T = absolute temperature (K) The heat of solution H of NaO in liquid sodium was calculated from this equation to be 10902 cal/mole. The reproducibility of results was obtained at equilibrium temperature of 150, 200, and 250C with a standard deviation of 1.2, 1.8, and 4.1 ppm, respectively. The oxygen analysis results with cold trapping sodium from various sodium test loops in Oarai Engineering Center almost correspond with the solubility curve of this work.

Crystallization of NADH-cytochrome reductase aimed to neutron crystallography

Shoyama, Yoshinari; Tamada, Taro; Kuroki, Ryota; Kimura, Shigenobu*; Takeda, Kazuki*; Hayashi, Takuro*; Miki, Kunio*

no journal, , 

NADH-cytochrome reductase (b5R) is a pyridine nucleotide-dependent flavin reductase which contains a FAD in a molecule. The b5R catalyzes the electron transfer from NADH to cytochrome b5. The b5R is related to fatty acid metabolism and reduction of cytochrome P450. In order to clarify detailed structure of b5R, we carried out purification of b5R from E coli cell according to the method previously reported. Since a large-size crystal is necessary for the neutron structure analysis. We addressed enlargement of crystal by macroseeding method and periodical addition of protein sample to the crystallization solution. We succeeded in a preparation of large-scale crystal with volume of 4 mm, and we did preliminary neutron diffraction study using this crystal. Then we got 2 resolution neutron diffraction data at BIX4.

Structural analysis of reaction intermediates of cytochrome b5 reductase

Yamada, Mitsugu; Tamada, Taro; Matsumoto, Fumiko; Takeda, Kazuki*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*

no journal, , 

no abstracts in English

Elucidations of the catalytic cycle of NADH-cytochrome reductase by X-ray crystallography

Tamada, 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.

Ultra-high resolution structure of high-potential iron-sulfur protein

Hirano, Yu; Takeda, Kazuki*; Kurihara, Kazuo; Tamada, Taro; Miki, Kunio*

no journal, , 

It is important for understanding the electron transfer reaction to include the information about valence shell electrons and hydrogen atoms into crystal structure refinement. Recently, we have successfully collected 0.48 resolution data of high-potential iron-sulfur protein (HiPIP) in BL41XU beamline of SPring-8. We performed multipolar refinement with the program to consider valence shell electrons in the structure refinement of HiPIP. After multipolar refinement, the deformation map clearly displays the distribution of valence shell electrons such as lone-pair electrons of carbonyl oxygen atoms, bonding electrons in aromatic rings, and -orbital electrons of Fe atoms in the FeS cluster. In addition, we performed preliminary neutron diffraction experiment at iBIX beamline of Japan Proton Accelerator Research Complex (J-PARC) and observed diffraction spots up to 1.17 resolution using HiPIP crystal with the size of 2.3 mm.

Structure of hydrogen atoms in high-resolution neutron structure of protein

Hirano, Yu; Tamada, Taro; Kurihara, Kazuo; Kusaka, Katsuhiro*; Ono, Hiraku*; Takeda, Kazuki*; Miki, Kunio*

no journal, , 

Hydrogen atoms are involved in protein folding and enzymatic reaction. Structures of hydrogen atoms in proteins have been discussed based on the ideal bond distances and angles obtained by small molecular crystallography. However, structural information about hydrogen atoms without geometric restraints is important for understanding structures and functions of proteins. High-potential iron-sulfur protein (HiPIP) is an electron carrier protein which functions in photosynthetic electron transfer chain of purple bacteria. In this work, we determined high-resolution neutron structure of HiPIP. The neutron diffraction experiment was performed at the BL03 beamline (iBIX) of J-PARC/MLF. We have collected the highest resolution data at 1.1 angstrom in the protein neutron structures. After structure refinement, we have observed many deviations in positions and bond lengths of hydrogen atoms from the ideal geometries.