Energetics of Metal and Substrates Binding to the 2-His-1-Carboxylate Binding Motif in Nonheme Iron(II) Enzymes

Nonheme iron(II) oxygenases within a common 2-His-1-carboxylate binding motif catalyze a wide range of oxidation reactions involved in biological functions like DNA repair and secondary metabolic processes. The mechanism of O2 activation catalyzed by this enzyme family has been examined by spectroscopic, crystallographic, and computational studies, where it is clear the iron(II) center works with substrate, and cosubstrate to activate O2 by forming a highly oxidizing iron species (iron(IV)-oxo). From a thermodynamic perspective, substrate and/or co-substrate binding organizes the active site for O2 activation, and understanding the interactions among metal, substrate, cosubstrate, and enzyme provides insight into the intramolecular contacts that guide the reaction catalyzed by the enzymes. This dissertation is focused on elucidating the interactions between metal, substrate, and co-substrate in a representative enzyme subfamily of nonheme iron(II) oxygenases, namely the 2-oxoglutarate dependent dioxygenases. Specifically, we investigated the thermodynamic properties of divalent metal ions binding to taurine-dependent dioxygenase (TauD), using Mn2+, Fe2+, and Co2+ ions. Additionally, the thermodynamics associated with substrate and co-substrate binding to Fe·TauD and iron(II)-ethylene forming enzyme (Fe·EFE) were explored using calorimetry and other biophysical techniques