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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：1)制备用于光谱电化学测量的光学透明功能电极；2)开发新的光谱电化学技术；3)在这些发展的基础上，研究与金属蛋白结构变化相关的生物功能。金属蛋白电化学功能电极的制备我们成功地制备了肌红蛋白和铁氧化还原蛋白的光学透明的In_2O_3功能化电极。In_2O_3电极的表面亲水性对肌红蛋白的伏安分离有很大的影响，电极表面的水层可以阻止肌红蛋白的强吸附，防止蛋白质的变性，而且表面的水更有利于参与氧化还原反应（水分子作为肌红蛋白的第六配体）。另一方面，在带正电荷的多肽修饰电极上，例如带负电荷的铁，更多的毒物显示出清晰的伏安图。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。利用功能化的光学透明电极，制备了原位紫外可见、近红外、圆二色和磁性圆二色电化学电池。此外，通过改进仪器，数据积累和自动测量成为可能，获得可靠和可重复的高信噪比数据。利用本研究开发的技术，细胞色素c、肌红蛋白、研究了铁氧化还蛋白及其突变分子在电子转移过程中与生物学功能相关的结构变化动力学，并以耐高温小球藻铁氧化还蛋白为介质，监测了铁氧化还蛋白- nadp ^+还原酶（FNR）存在下的NADPH生成等生物电化学反应。少