1992 Fiscal Year Annual Research Report

呼吸鎖電子伝達酵素の分子構築

Research Project

Project/Area Number	04266105
Research Institution	Osaka University
Principal Investigator	松原央大阪大学, 理学部, 教授 (00028242)
Co-Investigator(Kenkyū-buntansha)	福山恵一大阪大学, 理学部, 助教授 (80032283) 北潔東京大学, 医科研, 助教授 (90134444) 折井豊京都大学, 医学部, 助教授 (60028149) 吉川信也姫路工業大学, 理学部, 教授 (40068119) 小倉尚志岡崎国立共同研究機構, 分子科学研, 助手 (70183770)
Keywords	チトクロム酸化酵素 / 結晶構造解析 / チトクロムbc_1 / O_2還元反応中間体 / コハク酸脱水素酵素 / グラフィックス / 電子伝達複合体
Research Abstract	チトクロム酸化酵素の結晶のX線回折の分解能を原子レベルにまで高めるため結晶化条件を検討し、7種の界面活性剤が有効と伴った。その中でBL8SYで安定化した標品の結晶は分解能6Åで相関係数0.5以上を示した。重原子誘導体も水銀やタングステンクラスターを用いた浸漬法により作成しえた。低分解能での三次元構造の決定に必要なデータ収集が可能となった。この酵素の反応を時間分解共鳴ラマン分光法で追跡した。ラーマン散乱とコンピューターを媒介する検出器インターフェイスを設置し、スペクトルの高速加算が可能となり、世界で初めてO_2分子の還元反応の中間状態を捕捉することに成功した。一方高速ラピッドスキャン法を用いてこの酵素の2分子のヘムaの還元過程を反応動力学的かつスペクトル的に分離し、絶体吸収スペクトルに新しいものを見出し、今後の解析に期待がもてる。チトクロムbc_1複合体の結晶化条件を検討し、シュクロースモノラルレート、PEGとK一燐酸緩衝液pH8の最適条件を見出し、約6.5Å分解能までの回折を与える六方晶系の結晶をえた。この結晶のX線回折パターンをシンクロトロン放射光と巨大分子用ワイセンベルグカメラを用いてイメージングプレート上に記録し、WEISプログラムシステムで、これまでに4セットの回折強度をえた。また重原子同型置換結晶の調製も試み、強度を測定し、その中の1つはネイティブ結晶に対し大きい強度変化を示したが、重原子結合部位の決定には至っていない。線虫2種のコハク酸-ユビキノン還元酵素のIpサブユニットの全一次構造を決定し、寄生性と自由生活性両者に高い相同性を認め生物進化のエネルギー代謝系での表現に興味ある事実を提供した。さらにグラフィックスワークステーションを用いた蛋白質の立体構造図示システムを構築し、ヒドロゲナーゼやルブレドキシンに応用し、電子伝達反応の解析に応用可能な形にした。

Research Products

(25 results)

All Other

All Publications (25 results)

[Publications] M.Kawamoto: "X-Ray Crystallographic Studies of Bovine Heart Mitochondrial Cytochrome bc_1 Complex" The IVth International Conference on Biophysics and Synchrotron Radiation. A205. 203 (1992)
[Publications] 松原央: "UQチトクロム還元酵素の構造と機能" 日本応用酵素協会誌. 18. 2 (1992)
[Publications] I.Fujii: "XELE-a polypeptide model-building program for a graphics workstation" J.Molecular Graphics. 10. 185-189 (1992)
[Publications] 中川敦史: "タンパク質結晶構造解析計算のベンチマークテスト" 日本結晶学会誌. 34. 201-206 (1992)
[Publications] A.Yoneda: "Structures of Diethyl 2-((8-Hydroxy-κO-2-quinolyl-κN)methyl malonato(2-)-κC)(pyridine)palladium (II) and the Analogous Propyl Complex" Acta Cryst.C49. (1993)
[Publications] S.Yoshikawa: "Strategies for Crystallization of Large Membrane Protein Complexes" J.Cryxtal Growth. 122. 298-302 (1992)
[Publications] K.Kita: "cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinateubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer" Electrophoresis. 13. 506-511 (1992)
[Publications] Hua Wang: "Comparative Study on the Primary Structure of the Iron-sulfur subunit of Complex II in Ascaris suum Mitochondria" Jpn.J.Parasitol.41. 122-131 (1992)
[Publications] K.Kita: "Electron-transfer Complexes of Mitochondria in Ascaris suum" Parasitology Today. 8. 155-159 (1992)
[Publications] Y.Orii: "The Oxygenated flavohaemoglobin from E.coli:Evidence from photodissociation and rapid-scan studies for two kinetic and spectral forms" Biochem.Biophys.Res.Commun.187. 94-100 (1992)
[Publications] Y.Orii: "Acceleration of the oxygen reaction in CuA-deficient Nitrosomonas europaea cytochrome c oxidase as revealed by the flow-flash measurement" Biochem.Biophys.Res.Commun.182. 1264-1269 (1992)
[Publications] Y.Isogai: "Superoxide-producing b-cytochromes Enzymatic and electron paramagnetic resonance properties of cytochrome b_<558> purified from Neutrophils" J.Biol.Chem.(1993)
[Publications] 折井豊: "チトクロム酸化酵素ヘムの成分の絶対吸収スペクトル" 生化学. 64. 727 (1992)
[Publications] 折井豊: "ヘムたんぱく高速反応のラピッドスキャン法による解析" 生物物理. 32. S181 (1992)
[Publications] T.Kubota: "Hydrazine and hydroxylamine as probes for O_2 reduction site of mitochondrial cytochrome c oxidase" Biochem.J.(1993)
[Publications] W.S.Caughey: "Probing heart cytochrome c oxidase structure and function by infrared spectroscopy" J.Bioenergetics. (1993)
[Publications] M.Tsubaki: "Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl" Biophys.J.(1993)
[Publications] K.Shinzawa-Itoh: "Single crystals of bovine heart cytochrome c oxidase at fully oxidized resting,fully reduced and CO bound fully reduced states are isomorphous with each other" J.Mol.Biol.228. 987-991 (1992)
[Publications] M.Ataka: "Phase diagrams of a membrane protein,bovine heart cytochrome c oxidase" J.Cryst.Growth. 122. 60-65 (1992)
[Publications] S.Tuji: "A high resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme:dynamic behavior of protein and detergent in the complex" Eur.J.Biochem.208. 713-720 (1992)
[Publications] S.Takahashi: "Observation of the copper A ligand stretching resonance Raman band for cytochrome c oxidase" J.Am.Chm.Soc.113. 9400-9401 (1992)
[Publications] S.Yoshikawa: "Infrared evidence of azide binding to iron,copper and non-metal sites in heart cytochrome c oxidase" J.Biol.Chem.267. 9757-9766 (1992)
[Publications] T.Egawa: "Observation of the Fe(IV)=O Stretching Raman Band for a Thiolate-liganded Heme Protein;Compound I of Chloroperoxidase" FEBS Letts.305. 206-208 (1992)
[Publications] Y.Sakan: "Detection of the Fe-CO Stretching Raman Band for Recombination Transient Species of Photodissociated Carbonmonoxy Myoglobin by Using...." Chem.Phys.Letts.196. 150-154 (1992)
[Publications] 小倉尚志: "新生化学実験講座5生体酸化・薬物代謝" (株)東京化学同人, 9 (1992)

1992 Fiscal Year Annual Research Report

呼吸鎖電子伝達酵素の分子構築

Principal Investigator

松原 央 大阪大学, 理学部, 教授 (00028242)

Research Products

[Publications] M.Kawamoto: "X-Ray Crystallographic Studies of Bovine Heart Mitochondrial Cytochrome bc_1 Complex" The IVth International Conference on Biophysics and Synchrotron Radiation. A205. 203 (1992)

[Publications] 松原 央: "UQチトクロム還元酵素の構造と機能" 日本応用酵素協会誌. 18. 2 (1992)

[Publications] I.Fujii: "XELE-a polypeptide model-building program for a graphics workstation" J.Molecular Graphics. 10. 185-189 (1992)

[Publications] 中川 敦史: "タンパク質結晶構造解析計算のベンチマークテスト" 日本結晶学会誌. 34. 201-206 (1992)

[Publications] A.Yoneda: "Structures of Diethyl 2-((8-Hydroxy-κO-2-quinolyl-κN)methyl malonato(2-)-κC)(pyridine)palladium (II) and the Analogous Propyl Complex" Acta Cryst.C49. (1993)

[Publications] S.Yoshikawa: "Strategies for Crystallization of Large Membrane Protein Complexes" J.Cryxtal Growth. 122. 298-302 (1992)

[Publications] K.Kita: "cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinateubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer" Electrophoresis. 13. 506-511 (1992)

[Publications] Hua Wang: "Comparative Study on the Primary Structure of the Iron-sulfur subunit of Complex II in Ascaris suum Mitochondria" Jpn.J.Parasitol.41. 122-131 (1992)

[Publications] K.Kita: "Electron-transfer Complexes of Mitochondria in Ascaris suum" Parasitology Today. 8. 155-159 (1992)

[Publications] Y.Orii: "The Oxygenated flavohaemoglobin from E.coli:Evidence from photodissociation and rapid-scan studies for two kinetic and spectral forms" Biochem.Biophys.Res.Commun.187. 94-100 (1992)

[Publications] Y.Orii: "Acceleration of the oxygen reaction in CuA-deficient Nitrosomonas europaea cytochrome c oxidase as revealed by the flow-flash measurement" Biochem.Biophys.Res.Commun.182. 1264-1269 (1992)

[Publications] Y.Isogai: "Superoxide-producing b-cytochromes Enzymatic and electron paramagnetic resonance properties of cytochrome b_<558> purified from Neutrophils" J.Biol.Chem.(1993)

[Publications] 折井 豊: "チトクロム酸化酵素ヘムの成分の絶対吸収スペクトル" 生化学. 64. 727 (1992)

[Publications] 折井 豊: "ヘムたんぱく高速反応のラピッドスキャン法による解析" 生物物理. 32. S181 (1992)

[Publications] T.Kubota: "Hydrazine and hydroxylamine as probes for O_2 reduction site of mitochondrial cytochrome c oxidase" Biochem.J.(1993)

[Publications] W.S.Caughey: "Probing heart cytochrome c oxidase structure and function by infrared spectroscopy" J.Bioenergetics. (1993)

[Publications] M.Tsubaki: "Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl" Biophys.J.(1993)

[Publications] K.Shinzawa-Itoh: "Single crystals of bovine heart cytochrome c oxidase at fully oxidized resting,fully reduced and CO bound fully reduced states are isomorphous with each other" J.Mol.Biol.228. 987-991 (1992)

[Publications] M.Ataka: "Phase diagrams of a membrane protein,bovine heart cytochrome c oxidase" J.Cryst.Growth. 122. 60-65 (1992)

[Publications] S.Tuji: "A high resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme:dynamic behavior of protein and detergent in the complex" Eur.J.Biochem.208. 713-720 (1992)

[Publications] S.Takahashi: "Observation of the copper A ligand stretching resonance Raman band for cytochrome c oxidase" J.Am.Chm.Soc.113. 9400-9401 (1992)

[Publications] S.Yoshikawa: "Infrared evidence of azide binding to iron,copper and non-metal sites in heart cytochrome c oxidase" J.Biol.Chem.267. 9757-9766 (1992)

[Publications] T.Egawa: "Observation of the Fe(IV)=O Stretching Raman Band for a Thiolate-liganded Heme Protein;Compound I of Chloroperoxidase" FEBS Letts.305. 206-208 (1992)

[Publications] Y.Sakan: "Detection of the Fe-CO Stretching Raman Band for Recombination Transient Species of Photodissociated Carbonmonoxy Myoglobin by Using...." Chem.Phys.Letts.196. 150-154 (1992)

[Publications] 小倉 尚志: "新生化学実験講座5生体酸化・薬物代謝" (株)東京化学同人, 9 (1992)

松原央大阪大学, 理学部, 教授 (00028242)

[Publications] 松原央: "UQチトクロム還元酵素の構造と機能" 日本応用酵素協会誌. 18. 2 (1992)

[Publications] 中川敦史: "タンパク質結晶構造解析計算のベンチマークテスト" 日本結晶学会誌. 34. 201-206 (1992)

[Publications] 折井豊: "チトクロム酸化酵素ヘムの成分の絶対吸収スペクトル" 生化学. 64. 727 (1992)

[Publications] 折井豊: "ヘムたんぱく高速反応のラピッドスキャン法による解析" 生物物理. 32. S181 (1992)

[Publications] 小倉尚志: "新生化学実験講座5生体酸化・薬物代謝" (株)東京化学同人, 9 (1992)