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2001 Fiscal Year Final Research Report Summary

Molecular Mechanism and Structural Basis of Ubiquinone-Redox Reaction in Bacterial Respiratory Chains

Research Project

Project/Area Number 12460045
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionYamaguchi University

Principal Investigator

MATSUSHITA Kazunobu  Faculty of Agriculture, Yamaguchi University, Professor, 農学部, 教授 (50107736)

Co-Investigator(Kenkyū-buntansha) YAMADA Mamoru  Faculty of Agriculture, Yamaguchi University, Professor, 農学部, 教授 (30174741)
MOGI Tateshi  Graduate school of Science, Tokyo University, Associate Professor, 大学院・理学研究科, 助教授 (90219965)
MIYOSHI Hideto  Graduate school of Agriculture, Kyoto University, Associate Professor, 大学院・農学研究科, 助教授 (20190829)
TOYAMA Hirohide  Faculty of Agriculture, Yamaguchi University, Associate Professor, 農学部, 助教授 (60240884)
Project Period (FY) 2000 – 2001
KeywordsAzidoquinone / Photo-affinity labeling / Ubiquinone / Escherichia coli Glucose dehydrogenase / Alcohol Dehydrogenase of acetic acid bacteria / Bacterial respiratory chain
Research Abstract

In this research project, search for ubiquinone-binding site and molecular mechanism of redox reaction of ubiquinone was carried out in several ubiquinone-reacting enzymes in the respiratory chains of Escherichia coli and Gluconobacter suboxydans. The objective ubiquinone-reacting enzymes were cytochrome bo oxidase (Cyo), glucose dehydrogenase (GDH), and succinate dehydrogenases (SDH) of E. coli, and alcohol dehydrogenases (ADH) of G. suboxydans. In order to do this research, detection of the bound quinone in the enzymes and also photo-affinity labeling with azidoquinone of the enzymes followed by analysis of the labeled subunit and further peptides by MALDI-TOF-MS were mainly performed.
1) 2-azido-Q2 and also 3-azido-Q2 were successfully synthesized via the precursors, 2- and 3- metylated quinone compounds, and also the azido-moiety was found to be decomposed by UV irradiation. 2) Labeling conditions for Cyo of E. coli with both 2-azido- and 3-azido-Q2 were established. 3) Ubiquinone-reacting site of E. coli GDH was shown to be present in the C- terminal hydrophilic PQQ domain, which could be done by preparing the C-terminal domain by site-directed mutagenesis and the reactivity toward ubiquinone. 4) In E. coil SDH, ubiquinone-reacting site was shown to be present in the membrane-anchoring subunit by photo-affinity labeling with azidoquinone. 5) When purified G. suboxydans ADH with dodecyl maltoside, the enzyme retained tightly ubiquinone-10 inside the molecule, and also shown to be as the ubisemiquinone form. 6) In G. suboxydans ADH, the subunit II was shown to be labeled with azidoquinone, especially the ubiquinone reduction site, but not the ubiquinol oxidation site. And also the labeled quinone was found to work as electron mediator to pooled ubiquinone.

  • Research Products

    (34 results)

All Other

All Publications (34 results)

  • [Publications] Uchida, T.: "Resonance Raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hvdrogen peroxide"Biochemistry. 39. 6669-6678 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kobayashi, K.: "Transient tormation of ubisemiquinone radical and subsequent electron transfer processes in the Eseherichia colicytochrome bo revealed by pulse radiolysis"Biochemistry. 39. 15620-15625 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsubaki, M.: "Probing molecular structure of dioxygen reduction site of bacterial quinol oxidases through ligand binding to the redox metal centers"J.Inorg.Biochem.. 82. 19-25 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Elias, MD: "Functions of amino acid residues in the active site of Escherichia coli PQQ-containing quinoprotein glucose dehydrogenase"J.Biol.Chem.. 275. 7321-7326 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyadera, H.: "Altered quinone biosynthesis in the long-kived clk-lmutants of Caenorhabditis elegans"J.Biol.Chem.. 276. 7713-7716 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ohmura, S.: "Anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria"Proc.Natl.Acad.Sci.USA. 98. 60-62 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] O.Adachi: "Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257:A versatile enzyme for the oxidative fermentation of various ketoses"Biosci.Biotechnol.Biochem.. 65. 2755-2762 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi, H: "Probing the ubiquinone reduction site in bovine mitochondrial complex I using a series of synthetic ubiquinones and inhibitors"J.Bioenerg.Biomembr. 33. 223-231 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mitani, S: "The biochemica mode of action of the novel selective fungkide cyazofamid : specific inhibition of mitochondrial complex III in Pythium spinosum"Pestic.Biochem.Physiol.. 71. 107-115 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Matsushita: "NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH"FEMS Microbiol.Lett.. 204. 271-276 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] MD Elias: "C-terminal periplasmic domain of Escherichia coil Quinoprotein glucose dehydrogenase transfers electrons to ubiquinone"J.Biol.Chem.. 276. 48356-48361 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Matsushita: "Quinoproteins : structure, function and biotechnological applications"Appl.Micriobiol.Biotechnol.. 58. 13-22 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Sakamoto: "Synthesis and Electron-transfer Activity of Azido Ubiquinone-2"J.Pesticide Sci.. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Miyadera: "Quinones in long-lived clk-1 mutants of Caenorhabditis elegans"FEBS Lett.. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 茂木立志: "解き明かされた大腸菌の呼吸の仕組み"蛋白質核酸酵素. 47(印刷中). (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Matsushita, K.: "Biochemistry and Molecular Biology of vitamin B6 and PQQ-dependent proteins"Birkhauser Verlag AG. 377 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 茂木立志: "細胞呼吸の膜エネルギー装置:起源と分子進化"「生体膜のエネルギー装置(吉田賢右・茂木立志編)」共立出版. 21 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 松下一信: "廣川タンパク質化学 第4巻酵素 4.1オキシドレダクターゼ"廣川書店. 353 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Uchida T, T. Mogi, and T. Kitagawa: "Resonance Raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide"Biochemistry. 39. 6669-6678 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kobayashi K, S. Tagawa, and T, Mogi: "Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo revealed by pulse radiolysis"Biochemistry. 39. 15620-15625 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsubaki M, H. Hori, and T. Mogi: "Probing molecular structure of dioxygen reduction site of bacterial quinol oxidases through ligand binding to the redox metal centers"J. Inorg. Biochem. 82. 19-25 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Elias MD, M. Tanaka, H. Izu, K. Matsushita, O. Adachi, & M. Yamada: "Functions of amino acid residues in the active site of Escherichia coli PQQ-containing quinoprotein glucose dehydrogenase"J. Biol. Chem. 275. 7321-7326 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyadera H, Amino H, Hiraishi A, Taka H, Murayama K, Miyoshi H, Sakamoto K, Ishii N, Hekimi S, and Kita K: "Altered quinone biosynthesis in the long-lived clk-I mutants of Caenorhabditis. elegans"J. Biol. Chem. 276. 7713-7716 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ohmura S, Miyadera H, Ui H, Shiomi K, Yamaguchi Y, Masuma R, Nakamitsu T, Takano D, Sunazuka T, Harder A, Kolbl H, Namikoshi M, Miyoshi H, Sakamoto K, and Kita K: "Anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria"Proc. Natl. Acad. Sci. USA. 98. 60-62 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyoshi H: "Probing the ubiquinone reduction site in bovine mitochondrial complex I using a series of synthetic ubiquinones and inhibitors"J. Bioenerg. Biomembr. 33. 223-231 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mitani S, Araki S, Takii Y, Ohshima T, Matsuo N, and Miyoshi H: "The biochemical mode of action of the novel selective fungicide cyazofamid : specific inhibition of mitochondrial complex III in Pythium spinosum"Pestic. Biochem. Physiol. 71. 107-115 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Matsushita, A.Otofuji, M. Iwahashi, H. Toyama & O. Adachi: "NADH dehydrogenase of Coryneacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH"FEMS Microbiol. Lett. 204. 271-276 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Elias MD, Tanaka M, Sakai M, Toyama H, Matsushita K, Adachi O, Yamada M: "C-terminal periplasmic domain of Escherichia coli Quinoprotein glucose dehydrogenase transfers electrons to ubiquinone"J. Biol. Chem. 276. 48356-48361 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] O. Adachi, Y, Fujii, M. F. Ghaly, H. Toyama E. Shinagawa, & K. Matsushita: "Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257 : A versatile enzyme for the oxidative fermentation of various ketoses"Biosci. Biotechnol. Biochem. 65. 2755-2762 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Matsushita, H. Toyama, M. Yamada, and O. Adachi: "Quinoproteins : structure, function, and biotechnological applications"Appl. Micriobiol. Biotechnol. 58. 13-22 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyadera H, Kano K, Miyoshi H, Ishii N, Hekimi S, and Kita K: "Quinones in long-lived clk-1 mutants of Caenorhabaditis elegans"FEBS Lett. 512. 33-37 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Sakamoto, K. Nomura, H. Miyoshi: "Synthesis and Electron-transfer Activity of Azido ubiquinone-2"J. Pesticide Sci. (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Mogi: "Solved mechanism of Escherichia coli respiration"Protein, Nucleic Acid, and Enzyme. 47 (in press) (in Japanese). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Matsushita K, T. Yamashita, N. Aoki, H. Toyama, O. Adachi: "Electron transport systems for quinohemoprotein type II alcohol dehydrogenase of Pseudomonas putida HK5. Biochemistry and Molecular Biology of vitamin B6 and PQQ-deoendent proteins"Birkhauser Verlag AG. 219-224 (2000)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2003-09-17  

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