The Physiology of Oxidative Stress in Escherichia coli

我们已经了解到，大多数氧化毒性产生时，氧物种攻击酶铁中心 细胞防御系统通过阻断、逆转或绕过所造成的损伤来发挥作用。然而关键 观察结果仍然无法解释。在目标1中，我们将研究为什么超氧化物应激排除了使用 硫酸盐作为硫源，我们将研究为什么硫氧还蛋白和谷氧还蛋白被强烈诱导为 是细胞对过氧化氢反应的一部分大量的工作使我们提出细胞内 半胱氨酸和氧化还原酶有助于修复受损的金属酶中心。这个模型可以识别出一个关键点 硫氧化还原状态和ROS之间的联系。 两种酶专门用于厌氧代谢-丙酮酸：甲酸裂解酶激活酶和 乙醇脱氢酶-已被认为是失活的铁为中心的氧化事件时，细胞 是充气的。这将包括对通常有害的反应类型的巧妙利用。的目标 第二个目标是验证这个惊人的想法。这一假设引出了细胞如何无缝恢复的概念 缺氧恢复时的无氧代谢。 蛋白质羰基化（Aim 3）长期以来一直被用作氧化应激的方便标志物，但 潜在事件和生理影响尚不清楚。我们的数据表明，羰基化是集中在 相对较少的蛋白质，而不是完整的蛋白质组，我们怀疑这些蛋白质是单核铁（II） 内切酶全球质谱仪将通过名称识别它们。我们还将测试蛋氨酸 亚砜是还原酶可以修复不成比例的芬顿产物。新颖之处在于蛋氨酸可以 被氧化的二次电子跳跃事件，而不是通过直接攻击。 最后，在目标4中，我们将采用转录组学方法来完全定义OxyR过氧化物反应。我们 我希望解释我们的发现，OxyR的激活本身会损害细胞的适应性，到了禁止 在乙酸盐上生长。压力反应应该付出代价，这并不奇怪，但我们还没有认识到， 为什么OxyR驱动的适应会产生如此深远的影响。 氧化应激的紧急主题是氧物种与铁中心反应的趋势， 和细胞的反应与防御策略层。我们的四个目标将建立在这些知识的基础上， 解决持续存在的问题，总体目标是收集详细的氧化应激图片， 定量的，统一的。

We have learned that most oxidative toxicity arises when oxygen species attack enzymic iron centers and that cellular defenses work by blocking, reversing, or by-passing the resultant injuries. Yet key observations remain unexplained. In Aim 1 we will investigate why superoxide stress precludes the use of sulfate as a sulfur source, and we will examine why thioredoxins and glutaredoxins are strongly induced as part of the cellular reaction to hydrogen peroxide. Extensive work has led us to the proposal that intracellular cysteine and redoxins help to repair damaged metalloenzyme centers. This model would identify a key connection between sulfur redox state and ROS. Two enzymes dedicated to anaerobic metabolism—pyruvate:formate lyase activating enzyme and alcohol dehydrogenase—have been suggested to be inactivated by iron-centered oxidation events when cells are aerated. This would comprise a clever exploitation of reaction types that are usually harmful. The goal of Aim 2 is to test this striking idea. This hypothesis leads to notions of how the cell might seamlessly restore anaerobic metabolism when anoxia is restored. Protein carbonylation (Aim 3) has long been used as a convenient marker of oxidative stress—but the underlying events and physiological impact are unclear. Our data indicate that carbonylation is focused upon relatively few proteins rather than the full proteome, and we suspect that these proteins are mononuclear Fe(II) enzymes. Global mass spectrometry will identify them by name. We will also test the idea that methionine sulfoxide is a disproportionate Fenton product that reductases can repair. The novelty is that methionine may be oxidized by a secondary electron-hopping event, rather than by direct attack. Finally, in Aim 4 we will take a transcriptomic approach to fully define the OxyR peroxide response. We hope to explain our discovery that OxyR activation per se compromises cells fitness, to the point of prohibiting growth on acetate. It is not surprising that a stress response should exert a price, but we do not yet recognize why any OxyR-driven adaptation would have such a profound effect. The emergent theme of oxidative stress is the tendency of oxygen species to react with iron centers, and of cells to respond with layers of defensive tactics. Our four Aims will build upon this knowledge by tackling persistent questions, with the overall goal of assembling a picture of oxidative stress that is detailed, quantitative, and unified.