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ELECTROCHEMICAL STUDIES OF E COLI SULFITE REDUCTASE

大肠杆菌亚硫酸还原酶的电化学研究

基本信息

批准号：
2024466
负责人：
MICHEL D RYAN
金额：
$ 11.16万
依托单位：
MARQUETTE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2001-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2024466
关键词：
Escherichia coli bacterial proteins electrochemistry electron transport enzyme activity enzyme substrate complex nitrites oxidation reduction reaction sulfite reductase

项目摘要

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.

描述：亚硝酸盐同化还原为氨（亚硫酸盐还原为氨）。硫化氢）是生产中固定N和S的主要来源。蛋白整个还原过程涉及6电子还原为亚硝酸盐转化为氨。尽管这种酶的重要性，还原机制步骤的直接证据很少。将本研究的目的是：1）表征电子转移血红素蛋白亚基E的三种氧化态的性质。杆菌亚硫酸盐还原酶在不存在底物的情况下，和2）在存在底物阐明了亚硝酸盐转化的还原方案使用直接和介导的电化学还原反应转化为氨，氧化此外，电化学方法，直接和介导，将用于表征E.大肠杆菌亚硫酸盐还原酶（超氧化态）。这项研究特别感兴趣的是与氧化还原反应相关的构象变化的存在其显著影响底物的结合（构象门控）。电化学方法如循环阶梯法和方波伏安法非常适合研究反应。为了减少扩散的影响，并增加分析信号，酶将通过抗体/抗原反应附着到电极表面。这这种方法已被证明可以产生高的酶活性，更快的均相/非均相反应。通过明智选择介质，配体和pH值，亚硝酸盐还原的各个步骤将被确定。