An emergent class of enzymes that harness the extreme reactivity of electron-deficient free radical species to carry out some of the most difficult chemical reactions in biology has been recently identified. The regio- and stereo-selectivity achieved by these enzymes defies long- held ideas that radical reactions are non-specific. This class includes the following: ribonucleotide reductases, which catalyze the first unique step in DNA biosynthesis, prostaglandin H-synthase, the target of aspirin and other non-steroidal anti-inflammatory drugs, and the family of B12 coenzyme-dependent enzymes, which catalyze metabolite covalent bond rearrangements. The common primary step in the catalysis is metallocenter- or metal-assisted generation of an electron-deficient organic radical. This initiator radical abstracts a hydrogen atom from the substrate to form a substrate-based radical, opening a new reaction channel that facilitates rearrangement to a product radical. A challenging issue is how the radical is stabilized against recombination with the metal. There are analogies with the charge separation reactions in photosynthetic and respiratory bioenergetic complexes, for which the rudimentary performance principles have been established. However, unlike simple electron-hole separation by electron tunneling among weakly interacting redox sites, metal-radical separations involve large- scale movement of heavy nuclei and relatively strong electronic interactions. Elucidating the basic principles of how protein and cofactors guide radical stabilization and ensuing substrate radical rearrangement will be sustained focuses of the proposed studies. The adenosylcobalamin-dependent systems, and ethanolamine deaminase specifically, have been selected for scrutiny because homolytic cleavage of the cobalt-carbon bond to form the CoII metalloradical and 5'- adenosyl initiator radical can be triggered by a visible laser pulse, allowing the coherent preparation of the radical pair state under catalytic conditions. The radical pair separation and substrate radical rearrangement will be tracked by time-resolved techniques of pulsed-EPR spectroscopy. Dynamic electron spin-spin and electron-nuclear hyperfine interactions will be measured and used to construct a detailed molecular mechanism. The insights and techniques developed will promote identification of transient radical intermediates in other enzyme reactions, indicate designs for programmed site-specific radicals reactions in vivo, and assist therapeutic efforts to combat biologically-destructive free radicals.