PROJECT SUMMARY/ABSTRACT Oxygenase and oxidase enzymes must coordinate the delivery of four protons and four electrons to O2 in order to prevent the formation of harmful, partially reduced reactive oxygen species. The risks posed by reactive intermediates are so great that aerobic organisms need mechanisms to protect oxygen- utilizing enzymes from inactivation when primary electron/proton transfer mechanisms are disrupted. Radical transfer pathways that deliver strongly oxidizing holes from frustrated reactive intermediates in enzyme active sites to the protein surface for reaction with intracellular antioxidants can provide such protection. These radical transfer pathways are likely constructed from chains of Trp, Tyr, Cys, and possibly Met residues. This research program will focus on the cytochromes P450 (P450), the 2-oxo- glutarate dependent nonheme iron oxygenases (2OG-Fe), and the multicopper oxidases (MCOs). The cytochromes P450 are members of a superfamily of heme oxygenases involved in xenobiotic metabolic and biosynthetic pathways. In mammals these functions include drug metabolism, conversion of lipophilic molecules to more polar products for enhanced elimination, steroid biosynthesis, and eicosanoid synthesis and degradation. Cytochromes P450 also are responsible for 66% of enzymatic activation of carcinogens. Elucidating the mechanisms by which P450s avoid inactivation in the presence of diverse substrates can contribute to defining therapeutic drug efficacies and mitigating the risks of adverse drug-drug interactions. Drugs for cancer treatment and prevention have been designed to target P450s through competitive inhibition and mechanism based irreversible inhibition. To understand the biological response of P450s to these compounds, it is essential to delineate the mechanisms that the enzymes use to protect themselves against degradation. Enzymes from the 2OG-Fe superfamily use 2-oxoglutarate as a 2-electron donating co-substrate, Fe2+ as a cofactor, and O2 to effect the hydroxylation of organic substrates. The 60-70 human 2OG-Fe enzymes exhibit a wide array of biological functions including collagen biosynthesis, lysyl hydroxylation of RNA splicing proteins, DNA repair, RNA modification, chromatin regulation, epidermal growth factor- like domain modification, hypoxia sensing, and fatty acid metabolism. Enzymatic turnover also is accompanied in some cases by generation of reactive oxygen species. Elucidation of ROS generating pathways will provide deeper insight into the functioning and mis-functioning of these enzymes. The blood and intestinal human enzymes ceruloplasmin and hephaestin are MCOs involved in iron oxidation. Oxygen reduction occurs at a trinuclear copper center (TNC) and a fourth electron is provided by a distant type 1 copper center. A Trp or Trp/Tyr adjacent to the active site may transiently provide the fourth electron. The proposed studies will elucidate the role of the TNC proximal Trp/Tyr residues.