Aerobic organisms exploit O2 to extract large amounts of energy from oxidative metabolism of food, and employ oxidative reactions for a number of important chemical transformations involved in antibiotic biosynthesis, metabolism of xenobiotics, construction of biopolymers, and more. In order to access this chemistry, O2 must be activated, and this is often accomplished via Fe-containing enzymes. Free activated O2 reacts with many components of cells and the damage that ensues contributes to many diseases including diabetes, arthritis, neurodegenerative conditions, cancer, and the symptoms of old age. A crucial biochemical defense against this array of ills is the enzyme superoxide dismutase (SOD), which catalyzes conversion of the parent activated O2 species, superoxide, to O2 + H2O2. The current work focuses on the Escherichia coli SOD that is evolved to use Fe as its catalytic metal ion (FeSOD), with supporting in-vivo experiments on a homologous yeast SOD that employs Mn instead (MnSODSc). The yeast enzyme is highly homologous to the human mitochondrial MnSOD, and thus serves as a model for the human enzyme, while also drawing on extensive genetic and physiological studies of yeast, which cannot be performed on humans. The major thrusts address mechanisms by which the Fe of FeSOD (and by extension the Fe of O2- activation enzymes) interacts with activated O2 without succumbing to it, and means by which the active site of FeSOD tunes the reactivity of Fe (such that it will de-activate superoxide). The efforts on yeast SOD initiate investigations of possible pathological consequences of Fesubstitution into human SOD, via the yeast model, and test a mutation by which this pathology could be prevented or corrected. The major thrust builds directly on past research in the applicant's lab, using E. coli FeSOD variants that have been shown to be trapped in one of the two states employed by FeSOD's catalytic cycle, but to retain otherwise native-like active sites. Because they cannot progress through the catalytic cycle, they are ideal systems in which to generate models of enzyme-substrate and enzyme-product complexes, to learn how the FeSOD active site binds and interacts with its substrate and products. Detailed studies by skilled spectroscopists, as well as mechanistic and thermodynamic studies will provide an exceptionally complete picture of models of the intermediates of SOD turnover, and insights into how Fe enzymes handle their essential but dangerous substrate. Established enzymological and biophysical approaches will be blended with stopped- flow and freeze-quenched methods to extend the lab's ability to treat short-lived complexes. The yeast system will be launched. Initial studies of the reactions between peroxide and Fe-substituted MnSOD will evaluate the potential significance of Fe substitution into SOD, to human oxidative stress. A mutation developed in E. coli and characterized as part of the mechanistic work will also be tested for ability to reverse the possible toxic effects of Fe substitution into yeast MnSOD, thus carrying translating mechanistic insights into potential treatments.