Chloroperoxidase may provide the key to understanding the functions of and relationships among a wide variety of hemoproteins. It is unique in its ability to catalyze reactions characteristic of all of the major types of heme enzymes. Not only does it catalyze the peroxide supported chlorination of organic substrates, but it also catalyzes reactions characteristic of catalases, peroxidases and cytochrome P-450 mono- oxygenases. Although chloroperoxidase is not spectacularly efficient in any of these alternate roles, its ability to use the same active site to catalyze a diverse set of reactions may provide a powerful tool to study each process and the specific characteristics which favor it over others. Reactions of all types of oxidative heme enzymes are believed to utilize an oxyferryl intermediate as an oxidizing agent. However, the oxidation heme enzymes are believed to utilize an oxyferryl intermediate as an oxidizing agent. However, the oxidation reactions in peroxidase reactions seem to occur by single electron reactions of the intermediate while catalase reactions seem to favor two electron steps and mono-oxygenases may involve different mechanisms with different substrates. Thus, this project is designed to characterize the specific intermediates, both enzymatic and non enzymatic, which are involved in each of the reactions catalyzed by chloroperoxidase. Two general approaches will be used. (1.) Competition Studies: Effects which results from having substrates for more than one of the chloroperoxidase reactions present in the same solution (e.g. effects of peroxidatic substrates on the catalatic evolution of oxygen or the effects of halogenation substrates on the oxidation of peroxidatic substrates). These studies should provide understanding as to the factors that favor one process over another. (2.) Trapping Agent Studies: These procedures will utilize hindered compounds which can react with small molecules, but cannot enter enzyme active sites and thus do not react with enzymatic intermediates. Compounds which react with free oxidized halogen species and free radicals but not with enzyme bound species will be used. Thus, non-competitive inhibition of an enzymatic reaction by these trapping agents will provide evidence for kinetically important non-enzymatic intermediates in the reaction process. The detectors of free halogen species will differentiate between direct halogen transfer from enzyme to substrate and formation of a non-enzymatic halogenating species in halogenation reactions catalyzed by chloroperoxidase. Detection of free radicals will help identify processes which operate by single electron transfer processes. The trapping agents characterized in this study should be valuable tools for studying a variety of oxidative enzymes in the future.