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

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）在这些发展的基础上，研究了金属蛋白质的结构变化及其生物学功能.金属蛋白质电化学功能电极的制备肌红蛋白和铁氧还蛋白的3个功能化电极。In_2O_3电极表面的亲水性对肌红蛋白伏安图的峰分离有很大的影响。电极表面的水层可以阻止肌红蛋白的强吸附，从而排除蛋白质的变性，而且表面水分子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，带负电荷的铁离子可以被修饰。 关于我们 多辛显示清晰的伏安图。这些表面功能如电荷、亲水性和蛋白质与电极之间的相互作用强度，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。制备了圆二色（CD）和磁性圆二色（MCD）光谱电化学池。此外，通过对仪器的修改，数据积累和自动测量成为可能，以获得可靠和可重复的数据与高S/N比。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，铁氧还蛋白及其突变分子被检测以原位研究与生物功能相关的电子转移过程中的结构变化动力学并监测生物电化学反应，如在铁氧还蛋白-NADP ^+-还原酶（FNR）存在下，使用热稳定的小球藻铁氧还蛋白作为介体的NADPH产生。少

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