Hydrogen peroxide is a toxin used by the human immune system to kill infectious organisms, and increasing evidence shows that it is also a common second messenger in eukaryotic signaling. In humans, cytokines, growth factors and toll-like receptors of the innate immune system are thought to signal via hydrogen peroxide. Catalase and glutathione peroxidase have long been viewed as the major enzymes degrading peroxide in cells, however, over the past few years, a distinct, highly abundant family of peroxide- reducing enzymes, peroxiredoxins (Prxs), have moved from relative obscurity to become a major focus of redox biology research. The peroxidase activity of eukaryotic Prxs was overlooked for many years, because those Prxs that are highly expressed in eukaryotes are easily inactivated by peroxide. We have developed expertise in Prx enzymology over more than 15 years of characterizing of Prxs from pathogenic bacteria (e.g. Salmonella typhimurium AhpC). These Prxs are targets for antibiotic development because of the role they play in protecting the bacteria from the human immune system. In 2003, our structural and functional studies on S. typhimurium AhpC led us to discover the structural basis for the sensitivity toward peroxides that is conserved for a subset of Prxs that are highly expressed across all eukarya (this is the basis for the structural hypothesis that underlies the present grant, which dictates that the mobility of proximal secondary structures packing near the active site is a key determinant of the sensitivity of Prxs to overoxidation by peroxides and of the ability of Prxs to act as antioxidants). We further proposed the floodgate hypothesis for how this sensitivity to inactivation would actually be beneficial in organisms where hydrogen peroxide is being used as a signaling molecule, so that the antioxidant properties of the Prxs could be switched off under appropriate conditions to allow for a controlled burst in peroxide levels. Given the importance of Prxs both in pathogen defense and in human cells for combating oxidative stress and for cellular regulation, we propose here to identify the key determinants of sensitivity toward overoxidation and of efficient antioxidant function by investigating the conformational mobility of a few carefully chosen proteins and mutants; relevant rates constants within the catalytic cycle and inactivation pathways for these proteins will also be examined (Aim 1). In Aim 2, we will identify structural features around the highly conserved active site of Prxs which are important for binding and reduction of distinct hydroperoxide substrates. In Aim 3, we will determine whether or not the sensitivity of Prxs toward inactivation by peroxides during turnover (the floodgate) is critical to modulating the levels of H2O2 generated during cell signaling events through cell-based studies of Prx functions.