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酸素結合ヘムタンパク質の鉄 - リガンド振動及び末端酸化酵素の構造 - 機能相関の共鳴ラマン分光法による研究
https://ir.soken.ac.jp/records/278
https://ir.soken.ac.jp/records/278cbc2f734-3933-40ad-a9b6-bd5feb3cf193
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (405.1 kB)
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本文 / Thesis (5.8 MB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | 酸素結合ヘムタンパク質の鉄 - リガンド振動及び末端酸化酵素の構造 - 機能相関の共鳴ラマン分光法による研究 | |||||
| タイトル | ||||||
| タイトル | Resonance Raman Studies of Fe-Ligand Vibrationsof Oxygen Binding Heme Proteins and Structure-Function Relationship of Terminal Oxidases | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者名 |
廣田, 俊
× 廣田, 俊 |
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| フリガナ |
ヒロタ, シュン
× ヒロタ, シュン |
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| 著者 |
HIROTA, Shun
× HIROTA, Shun |
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| 学位授与機関 | ||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||
| 学位名 | ||||||
| 学位名 | 博士（理学） | |||||
| 学位記番号 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | 総研大甲第127号 | |||||
| 研究科 | ||||||
| 値 | 数物科学研究科 | |||||
| 専攻 | ||||||
| 値 | 08 機能分子科学専攻 | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 1995-03-23 | |||||
| 学位授与年度 | ||||||
| 値 | 1994 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | In biological systems, many proteins possess prosthetic groups which are indispensable for their functions. Myoglobin (Mb) and hemoglobin (Hb) are oxygen-storage and -carrier proteins, respectively, whereas terminal oxidases are oxygen reducing proteins. A11 of these proteins contain a heme as a prosthetic group where oxygen molecules bind, even though they play different roles in biological systems. There are some similarities and differences in the physicochemical properties of these heme proteins which are very important for understanding the functions of these proteins. Spectroscopic techniques are very useful to obtain kinetic as well as steady state information of biomolecules, including heme proteins. Especially, resonance Raman (RR) spectroscopy provides information on bond characters of the heme group if assignments have been established. In this thesis, RR technique is adopted to examine these oxygen binding heme proteins, namely, Mb, Hb, and terminal oxidases. One of the most useful information for ligand-bound heme proteins obtained by RR spectroscopy is the ligand bond characters. The force constant of the ligand bending mode reflects the energy required to bend the Fe-ligand unit in the protein. Therefore, the assignment of the ligand bending mode is very important to know the ligand character in ligand-bound heme proteins. This thesis consists of two parts, PartsI and II . Parts I treats reassignment of the Fe-ligand stretching and bending vibrational modes of various ligand-bound heme proteins. In chapter I-1, the assignments of the ligand related vibrational modes, especially those obtained by RR spectroscopy, are reviewed. Chapter I-2 describes the first detection of δFeOO at 425 and 435 cm -1 for HbO2 and CcO･O2 respectively. The δ FeOO frequencies for HbO2 and CcO･O2 were very similar, suggesting that HbO2 and CcO･O2 have similar Fe-O-O geometries for their FeO2 units even though they differ in functions. The v Fe-O2 bandwidth of CcO･O2 was narrower than those of HbO2 and MbO2. This indicates that the Fe-O-O geometry is more fixed in CcO･O2 which could have relation with its oxygen reactivity, although these three O2-bound heme proteins seem to have similar Fe-O-O geometries. O2-and NO-bound heme proteins have very similar ligand-binding geometries, and thus vFe-O2 and δFeOO frequencies of O2-bound heme proteins have frequencies similar to frequencies of NO-bound heme proteins. Chapters I-3, I-4,and I-5 discuss the reassignment of the δFeco RR band. The CO-isotope-sensitive band around 575 cm -1 has been assigned heretofore to δFeco for CO-bound heme proteins, but the frequency is higher than the vFe-co frequency. Chapter I-3 describes the detection of a new CO-isotope-sensitive band around 365 cm-1 for various CO-bound heme proteins. This CO-isotope-sensitive band at 365 cm-1 was undetectable for MbCO, while it was detected for all other CO-bound heme proteins examined, including HbCO, its isolated chains, CcO･CO, and P-450･CO. In Chapter I-4 the 54Fe and 15N isotope shifts of this new CO-isotope-sensitive band, the vFe-co band, and the 575 cm-1 band for CO-bound cytochrome bo from Escherichia coli (E. coli) are discussed. The 54Fe-isotope shifts of the 575 cm-1 and vFe-co bands were 1.5 and 3.5 cm -1, respectively. These isotope shifts were unable to be reproduced by normal coordinate calculation of the isolated FeCO unit if the 575 cm-1 band was assigned to δFeco , but were well reproduced when the new CO-isotope-sensitive band around 365 cm-1 was assigned to δFeco. The force constants for vFe-O2 and vFe-co were very similar,while that of δFeOO was larger than that of δFeco. The non-equilibrium geometry of the Fe-C-O unit in CO-bound heme proteins would have a reduced bond-strength and a flatter potential curve for the bending mode. This would lower the δFeco force constant than that in the equilibrium geometry. The detection of nonfundamental Fe-O2 and Fe-CO vibrations are discussed in chapter I-5. The overtone mode of the 365 cm-1 band and a comblnatlon mode of this band with vFe-co were detected for HbCO and MbCO, but the overtone mode of the 575 cm-1 band was undetectable. These results support the assignment of the new CO-isotope sensitive band around 365 cm-1 to δFeco. The δFeoo band was also undetectable for MbO2, although they were detected for HbO2 and CcO･O2. There must be some structural origins that make the ligand bending mode undetectable in the heme pocket of Mb, although they are not known at the present stage. Chapter I-6 discusses the vFe-cN- and δFecN- frequencies of several CN--bound heme proteins systematically. The CN<sup>~</sup>-isotope-sensitive band around 452 cm-1 is assigned to vFe-cN- , and the difference peaks present in a range from 340 to 440 cm-1 of the CN--isotope difference spectra are attributed to δFeCN- coupled with porphyrln modes for the CN-- bound heme protelns examined. As the ligand-binding geometries of CN- and CO-bound heme proteins are very similar and their electronic characters are also alike, their ligand vibrational frequencies should have similarities. As δFecN- appeared around 340-440 cm-1, it is more reasonable to assign the CO-isotope-sensitive band around 365 cm-1 to δFeOO rather than to assign the 575 cm-1 band to it. Chapter I-7 discusses the observation of the vFE-OH- bands of the low-spin species for hydroxy-Mb and -Hb at 549 and 552 cm-1, respectively. The vFe-oH- frequencies of the low-spin species are reported to be higher by 60 cm-1 than those of the high-spin species. This character was similar to that obtained for a hydroxy model compound. Fe(TMPPyP) (OH) 2 (aq)3+ (Fe(TMPPyP); [tetrakls 5, 10, 15, 20 (2-N-methyl-pyrldyl)porpyriato]iron(III), to have the vFe-OH- frequency of the low-spin species higher by 50 cm-1 than that of the high-spin species. PartII treats some similarities and differences in the physicochemical properties of terminal oxidases. One of the major differences of terminal oxidases from Hb and Mb is that they have a heme-copper binuclear center at the oxygen binding site. Another special character is that the oxidases involve the intramolecular heme to heme electron transfer during the oxygen reduction. Chapter II-1 gives a review of the terminal oxidases. Chapters II-2, II-3, and II-4 each treat RR spectra of a different kind of terminal oxidases. Chapter II-2 discusses the observation of vco for CO-bound bovine aa3-type cytochrome c oxidase (CcO･CO) by RR spectroscopy, and the measurement of the CO-recombination of CO-photodissociated CcO･CO by time-resolved RR spectroscopy. The bandwidths of the vFe-CO and vCO RR bands of CcO･CO were narrower than those of CO-bound myoglobin (MbCO). This character was the same as the vFe-CO and vCO bands of CO-bound E. coli cytochrome bo-type ubiquinol oxidase, having very narrow bandwidths. This suggests that CO takes a more fixed CO conformation in the heme pocket for CO-bound terminal oxidases than for MbCO. The CO-recombination rate was well fitted with a single exponential curve, and the lifetime of the photodissociated species was 30 ms. This lifetime was very long compared with those of MbCO and HbCO which were in the order of ms. No new vFe-CO RR band was observed during the CO-recombination. This suggests that CO relaxes to its equilibrium form as soon as CO binds to the heme, although the CO binding rate is slower than those of MbCO and HbCO by an order. Chapter II-3 describes the observation of the reaction intermediates in dioxygen reduction by the E. coli cytochrome bo-type ubiquinol oxidase detected by time-resolved RR spectroscopy using the artificial cardiovascular system, and compares the results with those obtained with bovine aa3-type cytochrome coxidase. At O～20 μs following photolysis of the enzyme-CO adduct in the presence of O2, the Fe-O2 stretching Raman band was observed at 568 cm-1 which was shifted to 535 cm-1 with 18O2. These frequencies were remarkably close to those of other oxyhemoproteins, including O2-bound hemoglobin and aa3-type cytochrome c oxidase. In the later time range (20～40μs), other O2-isotope-sensitive Raman bands were observed at 788 and 361 cm-1. The 781 cm-1 band was asslgned to the FeIV=0 stretchlng mode since it exhibited a downshift by 37 cm-l upon 18O2 substitution, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (>100 μs). The 361 cm-1 band showed the 16O/18O isotopic frequency shift of 14 cm-1 similar to the case of bovine aa3-type cytochrome c oxidase reaction. The detection of the intermediates for E. coli cytochrome bo-type ubiquinol oxidase has significance since it enables us to apply the time-resolved investigation of the reaction to enzymes obtained by site-directed mutagenesis. This will be a future subject. Chapter II-4 describes the RR spectra of the 54Fe- and 56Fe-labeled E.coli cytochrome bd-type ubiquinol oxidase at the reduced and oxidized states. For the reduced enzyme, the 227 and 250 cm-1 bands detected in the 441.6 nm excitation and the 397 cm-1 bands detected in the 427.0 nm excitation were Fe-isotope-sensitive. For the 406.7 nm excitation of the oxidized enzyme, bands at 391 and 349 cm-1 were Fe-isotope-sensitive. The band at 227 and 349 cm-1 are assignable to the Fe2+-His and Fe3+-S-(Cys) stretching vibrations, respectively. Accordingly, these results suggest that a histidine is the axial ligand of heme d similar to that of heme a3 of CcO, and a cystein is the axial ligand of one of the heme b, and the heme iron of each cytochrome adopts a five coordinated structure. |
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| 値 | 有 | |||||
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| 内容記述タイプ | Other | |||||
| 内容記述 | application/pdf | |||||
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| 出版タイプ | AM | |||||
| 出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||
