Macomber Lee, Rensing Christopher, Imlay James A
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
J Bacteriol. 2007 Mar;189(5):1616-26. doi: 10.1128/JB.01357-06. Epub 2006 Dec 22.
Because copper catalyzes the conversion of H(2)O(2) to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H(2)O(2) than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H(2)O(2) damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H(2)O(2) resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H(2)O(2)-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H(2)O(2) stress by inducing iron sequestration proteins, H(2)O(2)-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.
由于铜在体外可催化过氧化氢转化为羟基自由基，因此有人提出氧化性DNA损伤可能是铜毒性的一个重要组成部分。消除铜输出基因copA、cueO和cusCFBA，会使大肠杆菌对铜诱导的生长抑制敏感，从而提供了可检验这一假说的强制条件。当细胞在添加铜的培养基中生长时，细胞内铜含量增加了20倍。然而，与未添加铜生长的细胞相比，负载铜的突变体实际上对过氧化氢杀伤的敏感性更低。细胞死亡动力学表明，细胞内过量的铜消除了铁介导的氧化杀伤作用，而未产生铜介导的成分。诱变测量和定量PCR分析证实，铜降低了过氧化氢损伤DNA的速率。电子顺磁共振（EPR）自旋捕获表明，铜依赖性的过氧化氢抗性不是由抑制芬顿反应引起的，因为添加铜的细胞表现出大量的羟基自由基形成。然而，铜EPR光谱表明，大多数可被过氧化氢氧化的铜位于周质中；因此，大多数铜介导的羟基自由基形成发生在这个区室，远离DNA。事实上，虽然大肠杆菌通过诱导铁螯合蛋白来应对过氧化氢胁迫，但过氧化氢胁迫的细胞不会诱导控制铜水平的蛋白。这些观察结果无法解释铜如何抑制铁介导的损伤。然而，很明显铜在体内不会催化显著的氧化性DNA损伤；因此，铜毒性必定是通过不同的机制发生的。
