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c_3是一种四血红素蛋白，参与电子转移系统，参与硫酸盐还原菌中硫化合物和氢的代谢。这种蛋白质不仅从生物学角度而且由于其物理化学特性而引起人们极大的兴趣。在本项目中，研究了 D. vulgaris Miyazaki F 细胞色素 c_3 的血素间电子转移机制，特别强调了芳香族氨基酸残基的作用。由于细胞色素c_3的晶体结构已确定，因此使用核磁共振来密切参考晶体结构。首先，通过体内蛋白质的氨基酸选择性氘化来鉴定芳香族残基产生的质子信号。为了进行氘化，开发了一种用于硫酸盐还原菌生长的化学培养基。通过该方法成功识别了大部分芳香信号。此外，其中许多是通过核奥沃豪塞效应实验和二维核磁共振进行位点特异性分配的。在此任务的基础上，部分阐明了一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点的氧化还原电位。建立了基于核磁共振和电化学实验的细胞色素c_3 32个微观氧化还原电位的估计方法，成功估计了32个氧化还原电位。此外，根据血红素甲基信号的分配，微观电势被归因于晶体结构中的特定主题。这将为进一步从物理化学和结构因素方面阐明电子转移机制提供坚实的基础。

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)