The objectives of the research are to explore the catalytic mechanisms of enzymic reactions in which the exceptional reactivity of organic free radicals are employed. Reactions of free radicals are of fundamental importance in biology. This research focuses on the means by which organisms naturally exploit the reactivity of radicals to carry out difficult steps in metabolism and bisynthesis. Thiamine-pyrophosphate (TPP), the cofactor form of thiamine, is essential in carbohydrate metabolism. The thiazole moeity of TPP has a well known polar chemistry. TPP is, however, also capbable of stablizing free radicals, and such free radicals are intermediates in the catalytic cycles of the microbial pyruvate metabolizing enzymes, pyruvate:ferredoxin oxidoreductase (PFOR) and pyruvate oxidase (POX). Electronic structures of these radical intermediates and the mechanisms of their transformations to the end products are not well understood. The proposed research will examine the thiamine based radical intermediates through freeze trapping and spectroscopic determination of their electronic structures. Electronic structure calculations will be used to examine the ernergetics of radical structures. A proposed hydroxyethylidine-TPP-radical adduct with the acetyl group acceptors, coenzyme-A for PFOR and phosphate for POX, will be investigated experimentally and computationally. The radical-based chemistry of TPP is incompletely understood, and the proposed research will provide new insight into this important aspect of the chemistry of TPP. PFOR is found in anaerobic microbes from all three kingdoms. The low potential electrons generated in the PFOR reaction in pathogens such as parasitic protozoa are believed to activate the antimicrobial drug, metronidazole. Ethanolamine ammonia-lyase (EAL), from enteric bacteria, catalyzes an adenosylcobalamin (AdoCbl)- dependent conversion of ethanolamine to acetaldehyde and ammonia. The S'-deoxyadenosyl radical generated from AdoCbl is the radical initiator in this free radical based rearrangment/elimination reaction. The proposed research will use transient-phase kinetics in conjunction with kinetic isotope and temperature effects to examine the stepwise transformations among the free radical intermeidates with the substrate, ethanolamine. The transient-phase kinetic approach with isotopes will provide new insight into the mechanism of the hydrogen atom abstraction steps in the catalytic cycle.