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首页> 外文学位 >Mechanisms of bacterial copper detoxification and oxygen reduction in CueO and chemotactic signal amplification by receptor clustering.
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Mechanisms of bacterial copper detoxification and oxygen reduction in CueO and chemotactic signal amplification by receptor clustering.

机译：CueO中细菌铜的解毒和氧还原的机理以及通过受体簇形成的趋化信号放大。

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摘要

CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.;This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.;Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered to be necessary for removing Ag(I) inhibition of copper detoxification by the cue system. These results provide further insights into the role of CueO in copper detoxification and the effect of silver on the detoxification mechanism.
机译：CueO是一种多铜氧化酶，可催化双电子还原成水的双电子，并具有保护大肠杆菌免受铜诱导的毒性的作用。已经对多铜氧化酶中的氧还原机理进行了深入研究，但反应中间体的关键结构尚不清楚。进行了动力学测量，诱变和X射线晶体学研究的结合，以捕获并结构表征CueO中的反应状态。 CueO具有富甲硫氨酸的插入片段和不稳定的铜结合位点，这是仅在参与铜解毒的多铜氧化酶中发现的两个特征。已经研究了这些功能在CueO活性中的作用。在另一项研究中，建立了一个基于簇状受体之间传染性的简单数学模型来解释趋化性敏感性，反应范围和趋化性的其他关键特征;该研究描述了CueO中四个功能状态中成功捕获了三个功能状态。给出了这些反应态的晶体结构。使用通过截止流设备测量的单周转氧还原动力学，捕获了三种不同的完全氧化形式的CueO的光吸收特征：天然中间体，静止的氧化态以及位于它们之间的另一种中间体。停止流研究与电子转移动力学测量相结合，揭示了保守残基E506在天然中间体的质子化或作为反应产物形成的水分子的释放中的作用.Cu（I）和Ag（ I）确定CueO的结合晶体结构，揭示了沿着两个金属离子所使用的富含甲硫氨酸的螺旋的三个结合位点。 CueO中不稳定的，调节性的铜位点显示为Cu（I）重结晶的氧化位点。 Ag（I）被证明是体外所有CueO活性和体内cue系统对铜解毒的有效抑制剂。发现该cus系统对于消除由暗示系统消除Ag（I）抑制铜解毒的作用是必要的。这些结果为CueO在铜排毒中的作用以及银对排毒机理的影响提供了进一步的见解。

著录项

  • 作者

    Singh, Satish Kumar.;

  • 作者单位

    The University of Arizona.;

  • 授予单位 The University of Arizona.;
  • 学科 Chemistry Biochemistry.;Biology Molecular.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 197 p.
  • 总页数 197
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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