ELECTROCHEMICAL STUDIES OF E COLI SULFITE REDUCTASE

描述：亚硝酸盐的同化还原为氨(亚硫酸盐到氨 硫化氢)是固定氮的主要来源，S用于生产 蛋白。整个还原过程包括6个电子还原为 亚硝酸盐转化为氨。尽管这种酶很重要，但还有 几乎没有直接证据表明减排机制中的步骤。会是 这项拟议工作的目的是：1)表征电子转移 大肠杆菌血红素蛋白亚单位三种氧化态的性质 亚硫酸盐还原酶在没有底物的情况下，以及2)在存在的情况下 底物阐明亚硝酸盐转化的还原方案 利用直接和中介的电化学还原方法对氨进行还原 氧化。此外，电化学方法，无论是直接的还是中介的， 将被用来表征大肠杆菌亚硫酸盐的阳离子自由基 还原酶(过氧化状态)。这项研究特别感兴趣的是 与氧化还原反应有关的构象变化的存在 它们显著影响底物的结合(构象门控)。 循环阶梯和方波伏安法等电化学方法 非常适合研究这些反应。为了减少 扩散效应和增加分析信号时，酶会被 通过抗体/抗原反应连接到电极表面。这 已经证明这种方法可以产生高酶活性并使这项研究成为可能 更快的均相/非均相反应。通过明智地选择 介体、配体和pH，亚硝酸盐还原的各个步骤 将会被确定。

DESCRIPTION: The assimilatory reduction of nitrite to ammonia (sulfite to hydrogen sulfide) is the major source of fixed N and S for the production of protein. The overall reduction process involves the 6-electron reduction to nitrite to ammonia. In spite of the importance of this enzyme, there is little direct evidence for the steps in the reduction mechanism. It will be the aim of this proposed work to: 1) characterize the electron transfer properties of the three oxidation states of the hemoprotein subunit E. coli sulfite reductase in the absence of substrate, and 2) in the presence of substrate elucidate the reduction scheme for the transformation of nitrite to ammonia using direct and mediated electrochemical reduction and oxidation. In addition, electrochemical methods, both direct and mediated, will be used to characterize the pi-cation radical of E. coli sulfite reductase (superoxidized state). Of particular interest to this study is the presence of conformational changes associated with the redox reactions which significantly affect the binding of substrate (conformational gating). Electrochemical methods such as cyclic staircase and square-wave voltammetry are ideally suited to investigate the reactions. In order to reduce the effects of diffusion and increase the analytical signal, the enzyme will be attached to the electrode surface via an antibody/antigen reaction. This approach has been shown to yield high enzyme activity and enable the study of faster homogeneous/heterogeneous reactions. By judicious choice of mediators, ligands and pH, the individual steps in the reduction of nitrite will be determined.