Project Summary Within Nature, key cellular processes – e.g. transcription, reproduction, and production of small molecule metabolites - are carried out by enzymes containing non-heme iron cofactors. One class of these enzymes, the Fe- and 2-oxogluterate- (2OG) dependent enzymes, are well-known for their versatility and ability to catalyze different reactions within the same active site. This project aims to investigate the reactivity of a less characterized subclass of Fe/2OG-dependent enzymes-the halogenases. The halogenases, like all known Fe/2OG enzymes, utilize the common oxidizing ferryl intermediate, [FeIV=O]2+, to abstract a hydrogen atom from the substrate and then insert a halide ion or exogenous anion. The orientation of the halide ion relative to the ferryl intermediate and substrate is not well understood; however, this orientation has been implicated in the outcome of the product. Recent work has indicated that the unstable ferryl intermediate can be structurally mimicked by the stable vanadyl ion, [VIV≡O]2+. When this is incorporated into a hydroxylase Fe/2OG enzyme active site, it allows for prolonged study. Incorporation of vanadyl in Fe/2OG halogenases offers a unique opportunity to investigate the position of the substrate in relation to the metal and the cosubstrates involved in reactivity. These studies will utilize advanced spectroscopic methods alongside integration of non-canonical amino acids within the halogenase active site. Unnatural amino acid coordination to the metal site may provide the ability to alter, and potentially tune, reactivity and product formation in the native Fe/2OG-bound moiety, while introducing unique spectroscopic comparisons in the vanadyl-bound complex. The knowledge gained by this proposal will lead to a fundamental understanding of factors that dictate the reaction outcome within these halogenase active sites and how to harness selective reactivity for drug design and synthesis.