Studies on Group 5 Diiron Monooxygenases: Insight into the Mechanism of Regulation of the Diiron Center Pablo Sobrado, Department of Biochemistry, Virginia Tech Many enzymes harbor a carboxylate-bridged diiron center in their active sites that, upon reduction by NADH, reacts with molecular oxygen to form a high-valent diiron(IV)oxo species - a very powerful oxidizing agent capable of catalyzing the cleavage of a C-H bond. The outcome of this reaction is enzyme specific and includes desaturation of fatty acids, hydroxylation of hydrocarbons, and formation of tyrosine radicals. Despite a wealth of mechanistic and structural studies on diiron-containing enzymes, our understanding of how the protein environment tunes the reactivity of the diiron center remains superficial. This project focuses on the biochemical, biophysical and structural characterization tetrahydrofuran monoooxygenases. This enzyme is newly identified member of the group 5 diiron-containing bacterial multicomponent monooxygenases (BMM) and present an ideal system for structure-function studies due to lower subunit composition compared to other BMM. A variety of techniques will be used to characterize the kinetic mechanism and the role of protein-protein interactions in catalysis. Structural determination of the various components will be performed to gain insight into the mechanism by which the protein environment modulates the substrate selectivity and chemical reactivity. Notable outcomes from these studies include: Protein expression and crystallization of the regulatory component, ThmC. Expression and characterization of the oxidoreductase component, ThmD. Construction of a dual expression plasmid for the production of recombinant oxidoreductase component. ThmC Wavelength, nm Absorbance ThmD