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.