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

基本信息

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

项目摘要

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i）制备用于光谱电化学测量的光学透明功能电极，ii）开发新的光谱电化学技术，以及iii）在这些进展的基础上，研究与金属蛋白结构变化相关的生物学功能。金属蛋白电化学功能电极的研究成功制备了肌红蛋白和铁氧还蛋白的光学透明In_2O_3功能化电极。 In_2O_3电极对肌红蛋白伏安分离的影响很大，电极表面的水层可以防止肌红蛋白的强烈吸附，从而排除蛋白质的变性，而且表面水也有利于参与氧化还原反应（水另一方面，在带正电的多肽修饰电极上，例如带负电的铁蛋白，显示出清晰的结果。诸如电荷、亲水性和蛋白质与电极之间的相互作用强度等表面功能被发现很重要，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性电极表面。开发新的光谱电化学技术使用功能化光学制备了透明电极、原位紫外-可见、近红外(NIR)、圆二色性(CD)和磁圆二色性(MCD)光谱电化学池。此外，通过修改仪器，获得可靠的数据积累和自动测量，并成为具有高信噪比的可重复数据。金属蛋白的光谱电化学测量使用本研究中开发的技术，细胞色素 c、肌红蛋白、铁氧还蛋白和对它们的突变分子进行了检查，以原位研究与这些金属蛋白的生物功能相关的电子转移过程中结构变化的动态，并监测生物电化学反应，例如 NADPH 的产生在铁氧还蛋白-NADP^+-还原酶 (FNR) 存在下，使用耐热小球藻铁氧还蛋白作为介体。