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首页> 外文学位 >Spectroscopic and electrochemical studies of Shewanella oneidensis cytochrome c nitrite reductase, and improving c-heme expression systems.
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Spectroscopic and electrochemical studies of Shewanella oneidensis cytochrome c nitrite reductase, and improving c-heme expression systems.

机译：光谱学和电化学研究Shewanella oneidensis细胞色素c亚硝酸盐还原酶，并改善c-血红素表达系统。

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摘要

In this work the redox properties of cytochrome c nitrite reductase (CcNiR), a decaheme homodimer that was isolated from S. oneidensis, were determined in the presence and absence of the strong-field ligands cyanide and nitrite. Four hemes per CcNiR protomer are hexa-coordinate with tightly bound axial histidines, while the fifth (active site) has one tightly bound lysine and a distal site that can be open, or contain exogenous ligands such as the substrate nitrite. Controlled potential electrolysis in combination with UV/visible absorption (UV-vis) and electron paramagnetic resonance (EPR) spectroscopies allowed for assignment of all heme midpoint potentials under each set of conditions. The studies show that the active-site heme is the first to be reduced under all conditions. The midpoint redox potential of that heme shifts approximately 70mV to the positive upon binding a strong field ligand such as nitrite or cyanide. When controlled potential electrolysis was carried out in the presence of nitrite, a concerted two electron reduction was observed by UV-vis, and a {Fe(NO)}7 reduced product was revealed in EPR. In addition, an asymmetry in ligand binding between active sites was revealed. This information is relevant for the interpretation of planned and ongoing mechanistic studies of CcNiR.;Over-expression, partial purification and characterization of another S. oneidensis multiheme enzyme, known as octaheme tetrathionate reductase (OTR), is also described herein. Though of unknown cellular function, OTR was previously reported to have tetrathionate reductase activity, in addition to nitrite and hydroxylamine reductase activities. The new results indicate that the expression of OTR has no effect on tetrathionate or nitrite reductase activities in the whole cell lysate, and only hydroxylamine reductase activity was substantially elevated in the overexpressing bacteria. OTR was stable in buffered solutions, but substantial activity loss during all attempts at column chromatography was a major obstacle to the complete purification. OTR also proved quite hydrophobic, so possible membrane association should be considered in future attempts to purify this protein.;Finally, this dissertation also reports attempts to improve S. oneidensis' ability to express foreign proteins. Though ideally suited to expressing c-hemes, it proved difficult to express carboxy his-tagged proteins in S. oneidensis because of persistent tag degradation. Attempts to knock out lon protease, a cytoplasmic carboxypeptidase, as well as the result of redirecting ccNiR from the SecA to the possibly more protected signal particle recognition (SRP) secretion pathway, are described.;Iron heme cofactors are single-electron transport moieties that play a crucial role in respiration. While oxygen is the electron acceptor of choice in aerobic atmospheres, microorganisms that live in anaerobic environments utilize other molecules with similarly high reduction potentials. S. oneidensis can utilize numerous terminal electron acceptors, including nitrite, dimethylsulfoxide and even uranium, thanks to a particularly rich array of multi c-heme respiratory proteins. Understanding of how the midpoint potentials and heme arrangements within the proteins influence these exotic respiratory processes is of interest in the fields of bioremediation and fuel development.
机译：在这项工作中，在存在和不存在强磁场配体氰化物和亚硝酸盐的情况下，确定了从色素单胞菌中分离的十色纯同二聚体细胞色素C亚硝酸还原酶（CcNiR）的氧化还原特性。每个CcNiR启动子有四个血红素是六坐标，带有紧密结合的轴向组氨酸，而第五个（活性位点）具有一个紧密结合的赖氨酸和一个远端位点，该位点可以是开放的，或含有外源性配体，例如底物亚硝酸盐。与UV /可见吸收（UV-vis）和电子顺磁共振（EPR）光谱相结合的受控电势电解允许在每组条件下分配所有血红素中点电势。研究表明，在所有条件下，活性部位血红素均会被首先还原。该血红素的中点氧化还原电势在结合强电场配体（例如亚硝酸盐或氰化物）时向正极偏移约70mV。当在亚硝酸盐存在下进行受控电势电解时，通过紫外可见观察到一致的两个电子还原，并且在EPR中显示出{Fe（NO）} 7还原的产物。另外，揭示了活性位点之间的配体结合不对称。该信息与计划的和正在进行的CcNiR机理研究的解释有关。本文还描述了另一种称为S.oneidensis多血红素酶的过表达，部分纯化和表征，该多血红素酶被称为八氢四硫代硫酸盐还原酶（OTR）。尽管其细胞功能未知，但以前据报道OTR除亚硝酸盐和羟胺还原酶活性外，还具有四硫酸酯还原酶活性。新结果表明，OTR的表达对全细胞裂解物中的四硫酸盐或亚硝酸盐还原酶活性没有影响，而在过表达细菌中只有羟胺还原酶活性显着提高。 OTR在缓冲溶液中是稳定的，但是在进行柱层析的所有尝试中，大量的活性损失是完全纯化的主要障碍。 OTR还被证明具有很强的疏水性，因此在将来纯化这种蛋白的尝试中应考虑可能存在的膜缔合。最后，本论文还报道了尝试提高拟南芥表达外源蛋白的能力。尽管非常适合表达c-hemes，但是由于持续的标签降解，事实证明难以在S.oneidensis中表达带有羧基his标签的蛋白。描述了试图敲除lon蛋白酶，胞质羧肽酶的尝试，以及将ccNiR从SecA重定向到可能受到更多保护的信号颗粒识别（SRP）分泌途径的结果。铁血红素辅因子是单电子转运部分，在呼吸中起关键作用。尽管氧气是有氧环境中的首选电子受体，但生活在厌氧环境中的微生物会利用其他具有类似高还原电位的分子。由于特别丰富的多种c-血红素呼吸蛋白，S。oneidensis可以利用许多末端电子受体，包括亚硝酸盐，二甲基亚砜甚至铀。在生物修复和燃料开发领域，了解蛋白质中的中点电位和血红素排列如何影响这些奇异的呼吸过程是很重要的。

著录项

  • 作者

    Stein, Natalia.;

  • 作者单位

    The University of Wisconsin - Milwaukee.;

  • 授予单位 The University of Wisconsin - Milwaukee.;
  • 学科 Chemistry Organic.;Chemistry Inorganic.;Chemistry Biochemistry.;Chemistry Physical.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 165 p.
  • 总页数 165
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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