Study on Bioloigical Functions Related to Dynamic Structural Change of Metalloproteins Using Newly Developed Spectroelectrochemical Techniques

利用新开发的光谱电化学技术研究与金属蛋白动态结构变化相关的生物功能

• 批准号: 04453082
• 负责人: TANIGUCHI Isao
• 金额: $ 4.61万
• 依托单位: Kumamoto Uiversity
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04453082/
• 关键词: Spectroelectrochemistry; Metalloprotein; Functional Modified Electrode; Optically Transparent Electrode; Circular Dichroism; Stopped-Flow Measurement; Site-Directed Mutagenesis; Dynamics of Structural Change; マイクロ電極集合体モデル; ラマン分光法; 合成ポリペプチド; 構造変

The aims of the present study are to i) prepare of optically transparent functional electrodes for Spectroelectrochemical measurements, ii) develop of new spectroelectrochemical techniques, and iii) on the basis of these developments, study on biological functions related to structural change of metalloproteins.Preparation of functional electrodes for metalloprotein electrochemistryWe have succeeded to prepare optically transparent In_2O_3 functionalized electrodes for myoglobins and ferredoxins. The surface hydrophilicity of an In_2O_3 electrode affected very much the perk separation of the voltammogram of myoglobin, The water layr of the electrode surface may prevent from the strong adsorption of myoglobin to exclude denaturation of the protein, and also surface water would preferably take part in the redox reaction (water molecule acts as the sixth ligand of metmyoglobin). On the other hand, at positively charged polypeptide modified electrodes, for example, negatively charged ferre … More doxins showed clear voltammograms. Such surface functions as charge, hydrophilicity and strength of interaction between protein and electrode, are found to be important, but importantly the required degrees of these functions depend on the proteins and functional electrode surfaces of interest.Development of new spectroelectrochemical techniquesUsing functionalized optically transparent electrodes, in situ UV-visible, Near Infratred (NIR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroelectrochemical cells were prepared. Also, by modification of the instruments, data accumulation and automatic measurements became possible to obtain reliable and reproducible data with high S/N ratios.Spectroelectrochemical measurements of metallopreteinsUsing the developed techniques in the present study, redox reactions of cytochrome c, myoglobins, ferredoxins and their mutated molecules were examined to study in situ the dynamics of structural change during electron transfer related to biological functions of these metalloprtoteins and to monitor the bioelectrochemical reactions such as NADPH production in the presence of ferredoxin-NADP^+-reductase (FNR) using thermostable chlorella ferredoxin as a mediator. Less

本研究的目的是：1)制备用于光谱电化学测量的光学透明功能电极；2)发展新的光谱电化学技术；3)在此基础上，研究与金属蛋白结构变化有关的生物学功能。制备金属蛋白电化学功能电极。In_2O_3电极的表面亲水性对肌红蛋白伏安曲线的最佳分离有很大影响，电极表面的水层可能会阻止肌红蛋白的强吸附以排除蛋白质的变性，而且地表水最好参与氧化还原反应(水分子作为高铁肌红蛋白的第六配体)。另一方面，在带正电的多肽修饰电极上，例如带负电的Ferre…更多的多辛显示了清晰的伏安图。电荷、亲水性和蛋白质与电极之间相互作用的强度等表面功能是重要的，但重要的是这些功能所需的程度取决于感兴趣的蛋白质和功能电极表面。发展新的光谱电化学技术利用功能化的光学透明电极，原位紫外可见、近红外(NIR)、圆二色(CD)和磁性圆二色(MCD)光谱电化学池。此外，通过对仪器的改进，数据积累和自动测量成为可能，获得可靠和重复性高的高S/N比的数据。金属还原蛋白的光谱电化学测量利用本研究中发展的技术，检测了细胞色素c、肌红蛋白、铁氧还蛋白及其突变分子的氧化还原反应，以原位研究这些金属蛋白在电子转移过程中与生物功能相关的结构变化的动力学，并使用热稳定性铁氧还蛋白作为介体，监测在铁氧还蛋白-NADP^+-还原酶存在下的生物电化学反应。较少

项目成果

TOMINAGA,Masato: "Effect of Surface Hydrophylicity of an Indium Oxide Electrode on Direct Electro Transfer of Myoglobin" Chemistry Letters. 1993. 1771-1774 (1993)

富名正人：“氧化铟电极的表面亲水性对肌红蛋白直接电子转移的影响”化学快报。

谷口 功: "金属タンパク質に対するプローブとしての電気化学的手法" ぶんせき. 1994. 860-861 (1994)

Isao Taniguchi：“作为金属蛋白探针的电化学方法”Bunseki 1994。860-861 (1994)

谷口 功: "化学工業(特集号:高機能化する電極とその応用)「分子修飾による電極の機能制御」 43(4)" 化学工業社, 80(279-285) (1992)

Isao Taniguchi：“化学工业（特刊：高功能电极及其应用）通过分子修饰控制电极的功能43（4）” Kagaku Kogyosha，80（279-285）（1992）

I.Taniguchi: "Preparation and Application of Functional Modified Electrodes (in Japanese)" Chemical Industry. 43. 279-285 (1992)

I.Taniguchi：“功能性修饰电极的制备与应用（日文）”化学工业。

B.C.King, F.M.Hawkridge and I.Taniguchi: "Electrochemistry of Myoglobin" Rev. Polarography. 38. 7-10 (1992)

B.C.King、F.M.Hawkridge 和 I.Taniguchi：“肌红蛋白的电化学”Rev.极谱法。