The Physiology of Oxidative Stress in Escherichia coli

我们已经了解到，大多数氧化毒性发生在氧物种攻击酶铁中心时。 而细胞防御通过阻止、逆转或绕过由此产生的损伤来发挥作用。但关键是 观察结果仍未得到解释。在目标1中，我们将调查为什么超氧应激阻止使用 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 细胞对过氧化氢反应的一部分。广泛的研究使我们提出了细胞内 半胱氨酸和氧化还蛋白有助于修复受损的金属酶中心。该模型将标识一个密钥 硫氧化还原状态与ROS的关系。 两种专门用于厌氧代谢的酶-丙酮酸：甲酸裂解酶活化酶和 酒精脱氢酶-已被认为是失活的铁为中心的氧化事件，当细胞 是充气的。这将包括对通常有害的反应类型的巧妙利用。的目标是 目标二是测试这一惊人的想法。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。 长期以来，蛋白质羰化(AIM 3)一直被用作氧化应激的便捷标志--但 潜在的事件和生理影响尚不清楚。我们的数据表明，羰化反应主要集中在 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Fe(II) 酵素。全球质谱仪将通过它们的名字来识别它们。我们还将测试蛋氨酸 亚砜是一种还原酶可以修复的不成比例的芬顿产物。新奇之处在于蛋氨酸可能 被二次电子跃迁事件氧化，而不是被直接攻击。 最后，在目标4中，我们将采用转录转录的方法来完全定义OxyR过氧化氢反应。我们 我希望解释我们的发现，氧合酶激活本身会损害细胞的适应性，以至于不能 在醋酸盐上生长。压力反应应该付出代价并不令人惊讶，但我们还没有意识到 为什么任何由OxyR驱动的适应都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， 以及细胞以层层防御策略做出反应。我们的四个目标将建立在这一知识的基础上 解决持续存在的问题，总体目标是构建一幅详细的氧化应激图景， 量化的，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.