Interheme Electron Transfer in cytochrome C_3 and The Role of Aromatic Amino Acids.

细胞色素 C_3 中的组间电子转移和芳香族氨基酸的作用。

• 批准号: 01430024
• 负责人: AKUTSU Hideo
• 金额: $ 16.77万
• 依托单位: Yokohama National University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01430024/
• 关键词: Electrontransport Protein; Cytochrome C3; Nuclear Magnetic Resonance; Redox Potential; Tetraheme Protein; Interheme Electron Transfer; ELectron Transfer Rate; 電子伝達; 電子移動反応; ^1HーNMR; 電極界面反応; 遺伝子のクロ-ニング; ^1H-NMR

Cytochrome c_3 is a tetraheme protein that participates in the electron-transfer system involved in the metabolism of sulfur compounds and hydrogen in the sulfate reducing bacteria. This protein is of great interest not only from a biological point of view but also because of its physicochemical properties. In this project, the mechanism of interheme electron transfer of cytochrome c_3 from D. vulgaris Miyazaki F was investigated with a special enphasis on the roles of aromatic amino acid residues. Since the crystal structure of cytochrome c_3 is established, nuclear magnetic resonance was used for this investigation with close refference to the crystal structure. In the first place, the proton signals due to the aromatic residues were identified by amino acid-selective deuterataion of the protein in vivo. To carry out the deuteration, a chemical medium for the growth of the sulfate reducing bacteria was developed. Most of the aromatic signals were successfully identified by this method. Furthermore, many of them were site-specifically assigned by the nuclear Overhauser effect experiments and two-dimensional NMR. On the basis of this assignment, the role of some aromatic residues in the electron transfer was partially elucidated. One of the fundamental parameters in the electron transfer is the redox potential of a redox site. A method to estimate the 32 microscopic redox potentials of cytochrome c_3 on the basis of NMR and electrochemical experiments were developed, leading to the successful estimation of the 32 redox potentials. Furthermore, the microscopic potentials were ascribed to specifichemes in the crystal structure on the basis of the assignment of the heme methyl signals. This would provide the firm basis for the further development in the elucidation of the mechanism of electron transfer in terms of physicochemical and structural factors.

细胞色素C3是硫酸盐还原菌中参与硫化物和氢代谢的电子传递系统中的一种四氢血红素蛋白。这种蛋白质不仅从生物学角度来看很有价值，而且还因为它的物理化学性质。本课题研究了宫崎氏假丝酵母细胞色素C3的电子转移机理，特别是芳香族氨基酸残基的作用。由于确定了细胞色素C_3的晶体结构，因此用核磁共振对其晶体结构进行了研究。首先，通过蛋白质在体内的氨基酸选择性氢代反应识别芳香族残基产生的质子信号。为了进行氢化反应，研制了硫酸盐还原菌生长的化学培养基。该方法成功地识别了大部分芳香族信号。此外，它们中的许多是通过原子核Overhauser效应实验和二维核磁共振进行定点指定的。在此基础上，部分阐明了某些芳香族残基在电子转移中的作用。电子转移的基本参数之一是氧化还原位的氧化还原电势。建立了一种基于核磁共振和电化学实验的细胞色素C3的32个微观氧化还原电位的估算方法，成功地估算了32个氧化还原电位。此外，根据血红素甲基信号的归属，将微观电位归因于晶体结构中的特定血红素。这将为进一步从物理化学和结构因素方面阐明电子转移的机理提供坚实的基础。

项目成果

T. Sagara, S. Nakajima, H. Akutsu, K. Niki & G. Wilson: "Heterogeneous electron transfer rate measurements of cytochrome c_<C3> at mercury electrodes." J. Electroanal. Chem.297. 271-282 (1991)

T.相良、S.中岛、H.阿久津、K. Niki

T.Sagara: "Heterogeneous electron transfer rate measurements of Cytochrome C_3 at mercury electrodes" J.Electroanal.Chem.297. 271-282 (1991)

T.Sagara：“汞电极上细胞色素 C_3 的异质电子转移率测量”J.Electroanal.Chem.297。

T.Sagara,K.Niwa,A.Sone,C.Hinnen&K.Niki: "Reclox Reaction Mechanism of Cytochrome at Moclified Gold Electrodes" Langmuir. 6. 254-262 (1990)

T.相良,K.丹羽,A.曾根,C.Hinnen

J.-S.Park,K.Kano.K.Niki,H.Akutsu: "Full assignment of heme redox potentials of cytochrome C_3 of D.vulgaris Miyazaki F by ^1H NMR" FEBS Lett.285. 149-151 (1991)

J.-S.Park、K.Kano.K.Niki、H.Akutsu：“通过 ^1H NMR 完全分配 D.vulgaris Miyazaki F 细胞色素 C_3 的血红素氧化还原电位”FEBS Lett.285。

T.ーS.ParK,,K.Kano,,Y.Morimoto,Y.Higuchi,,N.Yasuoka,,M.Oguta,K.Niki,,H.Akutsu: " ^1 NMR studies on ferricytochrome C_3 from D^1 vulgaris Miyazaki F and its interaction with ferredoxin I." J.Biomol.NMR. 1. 271-282 (1991)

T.-S.ParK,,K.Kano,,Y.Morimoto,Y.Higuchi,,N.Yasuoka,,M.Oguta,K.Niki,,H.Akutsu：“^1 对来自 D 的铁细胞色素 C_3 进行 NMR 研究^1 vulgaris Miyazaki F 及其与铁氧还蛋白 I 的相互作用。” J.Biomol.NMR. 1. 271-282 (1991)