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首页> 外文学位 >Spectroscopic and electrochemical investigation of multielectron catalysis in sulfite and nitrite reductase enzymes.
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Spectroscopic and electrochemical investigation of multielectron catalysis in sulfite and nitrite reductase enzymes.

机译：在亚硫酸盐和亚硝酸盐还原酶中多电子催化的光谱和电化学研究。

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摘要

Multi-electron multi-proton reactions form the basis of nearly every chemical reaction involved in energy storage and manipulation. Despite their importance, the basic properties of these chemical transformations, such as the details of how electron transfer and proton-coupled redox events that must occur during these reactions are controlled, remain poorly understood. The sulfite and nitrite reductase family of enzymes are responsible for carrying out the six-electron reduction of sulfite to sulfide and nitrite to ammonia, respectively. These enzymes play fundamental roles in microbial metabolism and are either dissimilatory or assimilatory in nature. Multi-electron multi-proton reactions are investigated by the study of the catalytic mechanisms of two enzymes that are structurally different, but carry out similarly complex chemistry: the dimeric multi heme cytochrome c nitrite reductase from Shewanella oneidensis and the monomeric siroheme and [4Fe-4S] cluster containing sulfite reductase from Mycobacterium tuberculosis. Employing protein electrochemistry the properties of electron transfer steps and proton-coupled redox steps that occur throughout the catalytic cycle of cytochrome c nitrite reductase during its reduction of substrate revealed the strategies employed by this enzyme. The results presented indicate the reduction of substrate by the enzyme occurs in a series of one electron steps rather than coupled two-electron transfers. Mutational analysis of active site amino acids reveals their role in governing proton coupled redox events, which likely involves a hydrogen bonding network consisting of these residues and water molecules. Additionally, steady state kinetics assays coupled to site-directed mutagenesis of M. tuberculosis sulfite reductase identify a tyrosine residue adjacent to the active site which partially controls substrate preference, by influencing the electronic environment of the active site siroheme cofactor. Stopped-flow absorbance spectroscopy and rapid freeze quench electron paramagnetic resonance studies provide a first glimpse of a potential reaction intermediate during reduction of sulfite by sulfite reductase. Overall, our fundamental understanding of how sulfite and nitrite reductase enzymes catalyze complex multi-electron multi-proton reactions is advanced, and insight into the different approaches Nature employs to govern such powerful chemistry is revealed.
机译：多电子多质子反应几乎构成了涉及能量存储和操纵的每个化学反应的基础。尽管它们具有重要意义，但对这些化学转化的基本性质，例如如何控制在这些反应期间必须发生的电子转移和质子偶联的氧化还原事件的细节，仍然知之甚少。亚硫酸盐和亚硝酸盐还原酶家族的酶分别负责将亚硫酸盐六价电子还原为硫化物，将亚硝酸盐还原为氨。这些酶在微生物代谢中起基本作用，并且本质上是异化的或同化的。多电子多质子反应是通过研究两种在结构上不同但进行相似的复杂化学反应的酶的机制研究的：来自Shewanella oneidensis的二聚多血红素细胞色素C亚硝酸还原酶和单体Siroheme和[4Fe- [4S]簇含有来自结核分枝杆菌的亚硫酸盐还原酶。利用蛋白质电化学，在细胞色素亚硝酸还原酶还原底物的整个催化循环中发生的电子转移步骤和质子偶联的氧化还原步骤的性质揭示了该酶采用的策略。给出的结果表明酶对底物的还原发生在一系列的一个电子步骤中，而不是偶合的两个电子转移中。活性位点氨基酸的突变分析揭示了它们在控制质子偶联的氧化还原事件中的作用，这可能涉及由这些残基和水分子组成的氢键网络。另外，与结核分枝杆菌亚硫酸还原酶的定点诱变相结合的稳态动力学测定鉴定了与活性位点相邻的酪氨酸残基，该酪氨酸残基通过影响活性位点西罗血红素辅因子的电子环境而部分地控制了底物的偏好。停止流吸收光谱法和快速冷冻猝灭电子顺磁共振研究提供了亚硫酸还原酶还原亚硫酸盐过程中潜在反应中间体的第一印象。总的来说，我们对亚硫酸盐和亚硝酸盐还原酶如何催化复杂的多电子多质子反应的基本理解得到了发展，并且揭示了对自然界用来控制这种强大化学反应的不同方法的见解。

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  • 作者

    Judd, Evan Thomas.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Biochemistry.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 312 p.
  • 总页数 312
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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