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，氧攻击酶铁 细胞防御系统通过阻断、逆转或绕过合成物来发挥作用， 受伤然而，关键的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 压力排除了硫酸盐作为硫源的使用，我们将研究为什么硫氧还蛋白和 谷氧还蛋白作为对过氧化氢的细胞反应的一部分被强烈诱导。 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 金属酶中心受损。这个模型将确定硫和 氧化还原态和活性氧。 两种致力于厌氧代谢的酶-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中心氧化 当细胞被充气时发生的事件。这将包括对反应类型的巧妙利用， 通常是有害的。Aim 2的目标是测试这个惊人的想法。这一假设导致 当缺氧恢复时，细胞如何无缝地恢复无氧代谢的概念。 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- 但潜在的事件和生理影响尚不清楚。我们的数据表明 羰基化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）酶。全球质谱将 通过名字来识别它们。我们还将测试蛋氨酸亚砜是不成比例的 还原酶可以修复的芬顿产物。新颖之处在于甲硫氨酸可以被氧化， 二次电子跳跃事件，而不是直接攻击。 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 反应我们希望解释我们的发现，OxyR激活本身损害细胞 健康，到了禁止在醋酸盐上生长的程度。这并不奇怪，压力反应 应该付出代价，但我们还没有认识到为什么任何OxyR驱动的适应会 如此深刻的影响。 氧化应激的紧急主题是氧物种与铁反应的趋势 中心，和细胞，以回应与防御战术层。我们的四个目标将建立在 通过解决持续存在的问题来了解这些知识，总体目标是 氧化应激是详细的，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.