1994 年度実績報告書

呼吸鎖電子伝達酵素の分子構築

研究課題

研究課題/領域番号	04266105
研究機関	大阪大学
研究代表者	松原央大阪大学, 理学部, 名誉教授 (00028242)
研究分担者	福山恵一大阪大学, 理学部, 教授 (80032283) 北潔東京大学, 医科学研究所, 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, 分子科学研究所, 助手 (70183770)
キーワード	bc_1の結晶化 / bc_1低分解能結晶構造 / 回虫の複合体II / オキシダーゼ反応機構 / チトクロム酸化酵素の反応速度論 / ヒドロゲナーゼの活性中心 / 好アルカリ性細菌のチトクロムc酸化酵素
研究概要	ウシ心筋チトクロムbc_1複合体の精製標品を再結晶することにより、結晶が析出する条件範囲がこれまでより広くなり、より高分解能の回折点を与える結晶がえられた。(松原)チトクロムbc_1複合体の六方晶について、2種類の誘導体を調製し、同型置換法で10Å分解能電子密度を計算した。(福山)酸化型チトクロム酸化酵素とH_2O_2を反応させると607nmに吸収極大を与える反応中間体が生成する。これがFe=O型ヘムを持つことを共鳴ラマン分光法で証明した。(小倉)回虫複合体IIの、補欠分子族としてFADを含むFpサブユニットについてstage-specificに発現される2種のcDNAクローンを見つけた。また大腸菌複合体IIの機能的アッセンブリーにcytochrome b_<556>サブユニットのN末端45残基が必須であった。(北)ヒドロゲナーゼのX線解析から、活性部位のクラスターはFe _4S_4,Fe_3S_4,Fe_4S_4,Niの順に、分子の表面近くに直線状の約13Å間隔で配列していた。(安岡)臨界ミセル濃度は等しいが親水性部分の大きさの異なる種々の界面活性剤の、チトクロム酸化酵素の結晶化に及ぼす効果を検討した結果、界面活性剤の最適の大きさがあった。さらに分解能3.5Å近いX線回折を示す結晶を得た。(吉川)ヘム生合成系の幾つかの新しい遺伝子を見つけ、大腸菌のhemAやhemHを相捕する植物のcDNAクローン、hemGを相捕できるヒトのcDNAクローンを分離できた。これらの酵素の性質や遺伝子発現制御と細胞エネルギー生産の関わりを明らかにした。(井口)チトクロム酸化酵素の三次元構造をチューブ状結晶から15Å分解能で求めた。脂質二重膜の位置をも同定した。(豊島)チトクロム酸化酵素の電子受容部位から酸素還元部位への分子内電子移動が、約100倍の速度差で2段階的に進むことを明らかにした。また、大腸菌ユビキノール酸化酵素の極めて速い分子内電子移動に、塩素イオンが必須であることを見つけた。(折井)大腸菌、酢酸菌の呼吸鎖末端酵素であるユビキノール酸化酵素の複核中心の構造を EPR,FT-IR法により解析し、ヘム-銅末端酸化酵素としての共通の性質を持つことを明らかにした。(鍔木)好アルカリ性細菌Bacillus YN-2000のaco型チトクロムc酸化酵素の3種類のサブユニットのアミノ酸配列を決定した。サブユニットIのアミノ酸配列は他のBacillus属のcaa_3型チトクロムc酸化酵素と類似しており、サブユニットIIのチトクロムc部分にはアルカリ環境に適応するための特異的アミノ酸置換が見られた。(福森)

研究成果

(29件)

すべてその他

すべて文献書誌 (29件)

[文献書誌] M.Kawamoto: "New Crystal Forms and Preliminary X-ray Diffraction Studies of Mitochondrial Cytochrome bc_1 Complex from Bovine Heart" J.Mol.Biol.244. 238-241 (1994)
[文献書誌] H.Hirawake: "Human Complex II(succinate-ubiquinone oxidoreductase):cDNA cloning of the flavoprotein (Fp)subunit of liver mitochondria" J.Biochem. 116. 221-227 (1994)
[文献書誌] F.Saruta: "Stage-specific isoforms of Complex II(succinate-ubiquinone oxidoreductase)in mitochondria from the parasitic nematode,Ascaris suum" J.Biol.Chem.270. 928-932 (1995)
[文献書誌] T.Kuramochi: "Sequence comparison between the flavoprotein subunit of the fumarate reductase(Complex II)of the anaerobic parasitic nematode,Ascaris suum and the succinate dehydrogenase of the aerobic,free-living nematode,Caenorhabditis elegans" Mol.Biochem.Parasitol.68. 177-187 (1994)
[文献書誌] 豊島近: "アセチルコリン受容体の構造と機能" ブレインサイエンス. 5. 149-155 (1994)
[文献書誌] 豊島近: "電子顕微鏡で膜蛋白質を見る" 化学と生物. 32. 791-796 (1994)
[文献書誌] N.Sone: "Proton Pumping and Visible Absorption and Resonance Raman Spectra of a cao-Type Cytochrome c Oxidase from the Thermophilic Bacterium Bacillus PS3" Biochemistry. 33. 849-855 (1994)
[文献書誌] S.Hirota: "Observation of the Fe-O_2 and Fe^<IV>=O stretching Raman Bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli" FEBS Letts.352. 67-70 (1994)
[文献書誌] D.A.Proshlyakov: "Selective Resonance Raman Observation of s Ferryloxo Intermediate in the Reaction of Cytochrome Oxidase with Hydrogenperoxide" J.Biol.Chem.269. 29385-29388 (1994)
[文献書誌] I.Fujii: "An Integrated X-ray Measurement and Computation System in Protein Crystallography" Bioimages. 2. 143-148 (1994)
[文献書誌] Y.Higuchi: "A detailed comparison of the refined structures of cytochrome c3 moleculesfrom two strainsin Desulfovibrio vulgaris:The relationship between the heme structures and their redox propeties" Biochimie. 76. 537-545 (1994)
[文献書誌] Y.Higuchi: "Location of Active Sites of NiFe Hydrogenase determined by the Combination of Multiple Isomorphous Replacement and Multiwavelength Anomalous Diffraction Methods" Acta Crystallographica. D50. 781-785 (1994)
[文献書誌] Y.Kumazawa: "Cytochrome bc Purified from the Methanogen Methanosarcina barkeri" Curr.Microbiology. 29. 53-56 (1994)
[文献書誌] K.Denda: "Structure-function studies on the heme-copper terminal oxidases using chimeric subunit I of Halobacterium halobium cytochrome aa_3 and Escherichia coli cytochrome bo" EBEC Short Reports. 8. 44- (1994)
[文献書誌] H.Tamegai: "Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans:Purification and properties" FEMS Microbiol.Lett.119. 147-154
[文献書誌] K.Miyamoto: "Overproduction,Purification,and Characterization of Ferrochelatase from Escherichia coli" J.Biochem.115. 545-551 (1994)
[文献書誌] K.Miyamoto: "Nucleotide Sequences of cDNA Clones Encoding Ferrochelatase from Barley and Cucumber" Plant Physiol.105. 769-770 (1994)
[文献書誌] Robert K.Poole: "Reactions of the Escherichia coli flavohaemoglobin(Hmp)with oxygen and reduced nicotinamide adenine dinucleotide:evidence for oxygen switching of flavin oxidoreduction and a mechanism for oxygen sensing" Proc.R.Soc.Lond.B255. 251-258 (1994)
[文献書誌] T.Miki: "Membrane Potential-linked Reversed Electron Transfer in the Beef Heart Cytochrome bc_1 Complex Reconstituted into Potassium-loaded Phospholipid Vesicles" J.Biol.Chem.269. 1827-1833 (1994)
[文献書誌] Y.Orii: "Facilitated intramolecular electron transfer in cytochrome bo-type ubiquinol oxidase initiated upon reaction of the fully reduced enzyme with dioxygen" FEBS Letters. 352. 151-154 (1994)
[文献書誌] Y.Orii: "Kinetic characterization of the redox components in solubilized membranes from porcine neutrophils:Reduction with dithionite and photoexcited NAD(P)H" Photochem.and Photobiol.61. in press (1995)
[文献書誌] Y.Orii: "Facilitated Intramolecular Electron Transfer in the Escherichia coli bo-type Ubiquinol Oxidase Requires Chloride" Biochemistry. in press (1995)
[文献書誌] S.Hirota: "Vibrational assignments of the FeCO unit of CO-bound heme proteins revisited:observation of a new CO-isotope-sensitive reman band assignable to the FeCO bending fundamental" J.Phys.Chem.98. 6652-6660 (1994)
[文献書誌] S.Hirota: "Observation of a new oxygen-isotope-sensitive Raman band for oxyhemoproteins and its implications in heme pocket structure" J.Am.Chem.Soc.116. 10564-10570 (1994)
[文献書誌] K.Shinzawa-Itoh: "Effects of ethyleneglycol chain length of dodecyl polyethy leneglycol monoether on the crystallization of bovine heart cytochrome c oxidase" J.Mol.Biol.246. 572-575 (1995)
[文献書誌] S.Yoshikawa: "Effects of overall oxidatio state on infrared spectra of Heme a_3 cyanide bovine heart cytochrome c oxidase" J.Biol.Chem.270. 4270-4279 (1995)
[文献書誌] M.Tsubaki: "Molecular Structure of Redox Metal Centers of the Cytochrome bo Complex from Escherichia coli" J.Biol.Chem.269. 30861-30868 (1994)
[文献書誌] M.Tsubaki: "Structure of the Heme-Copper Binuclear Center of the Cytochrome bo Complex of Escherichia coli:EPR and Fourier Transform Infrared Spectroscopic Studies" Biochemistry. 32. 6065-6072 (1993)
[文献書誌] 折井豊: "蛋白質の時代-構造物性機能研究の新局面(蛋白質核酸酵素)39" 共立出版, 1152-1160 (1994)

1994 年度 実績報告書

呼吸鎖電子伝達酵素の分子構築

研究代表者

松原 央 大阪大学, 理学部, 名誉教授 (00028242)

研究成果

[文献書誌] M.Kawamoto: "New Crystal Forms and Preliminary X-ray Diffraction Studies of Mitochondrial Cytochrome bc_1 Complex from Bovine Heart" J.Mol.Biol.244. 238-241 (1994)

[文献書誌] H.Hirawake: "Human Complex II(succinate-ubiquinone oxidoreductase):cDNA cloning of the flavoprotein (Fp)subunit of liver mitochondria" J.Biochem. 116. 221-227 (1994)

[文献書誌] F.Saruta: "Stage-specific isoforms of Complex II(succinate-ubiquinone oxidoreductase)in mitochondria from the parasitic nematode,Ascaris suum" J.Biol.Chem.270. 928-932 (1995)

[文献書誌] 豊島 近: "アセチルコリン受容体の構造と機能" ブレインサイエンス. 5. 149-155 (1994)

[文献書誌] 豊島 近: "電子顕微鏡で膜蛋白質を見る" 化学と生物. 32. 791-796 (1994)

[文献書誌] N.Sone: "Proton Pumping and Visible Absorption and Resonance Raman Spectra of a cao-Type Cytochrome c Oxidase from the Thermophilic Bacterium Bacillus PS3" Biochemistry. 33. 849-855 (1994)

[文献書誌] S.Hirota: "Observation of the Fe-O_2 and Fe^<IV>=O stretching Raman Bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli" FEBS Letts.352. 67-70 (1994)

[文献書誌] D.A.Proshlyakov: "Selective Resonance Raman Observation of s Ferryloxo Intermediate in the Reaction of Cytochrome Oxidase with Hydrogenperoxide" J.Biol.Chem.269. 29385-29388 (1994)

[文献書誌] I.Fujii: "An Integrated X-ray Measurement and Computation System in Protein Crystallography" Bioimages. 2. 143-148 (1994)

[文献書誌] Y.Higuchi: "A detailed comparison of the refined structures of cytochrome c3 moleculesfrom two strainsin Desulfovibrio vulgaris:The relationship between the heme structures and their redox propeties" Biochimie. 76. 537-545 (1994)

[文献書誌] Y.Higuchi: "Location of Active Sites of NiFe Hydrogenase determined by the Combination of Multiple Isomorphous Replacement and Multiwavelength Anomalous Diffraction Methods" Acta Crystallographica. D50. 781-785 (1994)

[文献書誌] Y.Kumazawa: "Cytochrome bc Purified from the Methanogen Methanosarcina barkeri" Curr.Microbiology. 29. 53-56 (1994)

[文献書誌] K.Denda: "Structure-function studies on the heme-copper terminal oxidases using chimeric subunit I of Halobacterium halobium cytochrome aa_3 and Escherichia coli cytochrome bo" EBEC Short Reports. 8. 44- (1994)

[文献書誌] H.Tamegai: "Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans:Purification and properties" FEMS Microbiol.Lett.119. 147-154

[文献書誌] K.Miyamoto: "Overproduction,Purification,and Characterization of Ferrochelatase from Escherichia coli" J.Biochem.115. 545-551 (1994)

[文献書誌] K.Miyamoto: "Nucleotide Sequences of cDNA Clones Encoding Ferrochelatase from Barley and Cucumber" Plant Physiol.105. 769-770 (1994)

[文献書誌] Robert K.Poole: "Reactions of the Escherichia coli flavohaemoglobin(Hmp)with oxygen and reduced nicotinamide adenine dinucleotide:evidence for oxygen switching of flavin oxidoreduction and a mechanism for oxygen sensing" Proc.R.Soc.Lond.B255. 251-258 (1994)

[文献書誌] T.Miki: "Membrane Potential-linked Reversed Electron Transfer in the Beef Heart Cytochrome bc_1 Complex Reconstituted into Potassium-loaded Phospholipid Vesicles" J.Biol.Chem.269. 1827-1833 (1994)

[文献書誌] Y.Orii: "Facilitated intramolecular electron transfer in cytochrome bo-type ubiquinol oxidase initiated upon reaction of the fully reduced enzyme with dioxygen" FEBS Letters. 352. 151-154 (1994)

[文献書誌] Y.Orii: "Kinetic characterization of the redox components in solubilized membranes from porcine neutrophils:Reduction with dithionite and photoexcited NAD(P)H" Photochem.and Photobiol.61. in press (1995)

[文献書誌] Y.Orii: "Facilitated Intramolecular Electron Transfer in the Escherichia coli bo-type Ubiquinol Oxidase Requires Chloride" Biochemistry. in press (1995)

[文献書誌] S.Hirota: "Vibrational assignments of the FeCO unit of CO-bound heme proteins revisited:observation of a new CO-isotope-sensitive reman band assignable to the FeCO bending fundamental" J.Phys.Chem.98. 6652-6660 (1994)

[文献書誌] S.Hirota: "Observation of a new oxygen-isotope-sensitive Raman band for oxyhemoproteins and its implications in heme pocket structure" J.Am.Chem.Soc.116. 10564-10570 (1994)

[文献書誌] K.Shinzawa-Itoh: "Effects of ethyleneglycol chain length of dodecyl polyethy leneglycol monoether on the crystallization of bovine heart cytochrome c oxidase" J.Mol.Biol.246. 572-575 (1995)

[文献書誌] S.Yoshikawa: "Effects of overall oxidatio state on infrared spectra of Heme a_3 cyanide bovine heart cytochrome c oxidase" J.Biol.Chem.270. 4270-4279 (1995)

[文献書誌] M.Tsubaki: "Molecular Structure of Redox Metal Centers of the Cytochrome bo Complex from Escherichia coli" J.Biol.Chem.269. 30861-30868 (1994)

[文献書誌] M.Tsubaki: "Structure of the Heme-Copper Binuclear Center of the Cytochrome bo Complex of Escherichia coli:EPR and Fourier Transform Infrared Spectroscopic Studies" Biochemistry. 32. 6065-6072 (1993)

[文献書誌] 折井 豊: "蛋白質の時代-構造 物性 機能研究の新局面(蛋白質 核酸 酵素)39" 共立出版, 1152-1160 (1994)

1994 年度実績報告書

松原央大阪大学, 理学部, 名誉教授 (00028242)

[文献書誌] 豊島近: "アセチルコリン受容体の構造と機能" ブレインサイエンス. 5. 149-155 (1994)

[文献書誌] 豊島近: "電子顕微鏡で膜蛋白質を見る" 化学と生物. 32. 791-796 (1994)

[文献書誌] 折井豊: "蛋白質の時代-構造物性機能研究の新局面(蛋白質核酸酵素)39" 共立出版, 1152-1160 (1994)