Oxygenation Promoted by Protein‐Bound Iron Centers in Biological Transformations

This dissertation focuses on the functional and structural characterization of a histidine-ligated heme hydroxylase (TyrH), a nonheme thiol dioxygenases (cysteamine dioxygenase, ADO), and two nonheme extradiol dioxygenases (3-hydroxyanthranilate-3,4-dioxygenase, HAO and DOPA dioxygenase, DDO). TyrH and DDO catalyze two adjacent steps in the biosynthetic pathways of antibiotics in Streptomyces, in which the former utilizes peroxide to para-hydroxylate tyrosine and the product is further dioxygenated and dearomatized by DDO with molecular oxygen. HAO performs a similar chemistry as DDO and is responsible for the production of quinolinic acid in kynurenine pathway. While ADO regulates thiol metabolism and preserves oxygen homeostasis in animals and plants by oxidizing cysteamine and N-terminal cysteine peptides to sulfinic acids using oxygen. The works presented here integrated absorption and EPR spectroscopies, X-ray crystallography, and other biochemical techniques to investigate the ternary structure, active site architecture, substrate binding, and catalytic activity of these iron-containing enzymes, with the purpose of understanding their catalytic mechanisms and structure-function correlations. In particular, the study of TyrH discovered the dual reactivity of C-H and C-F bond activation which was elucidated by the dual substrate binding orientations and a ferric hydroperoxo intermediate observed in de novo crystal structures. In HAO, a complete catalytic cycle, including a long-sought seven‐membered lactone intermediate and an unexpected isomerization process, was visualized by in crystallo reactions coupled with single-crystal spectroscopies to understand the fine-tuned product profiling at a metabolic junction. For ADO, the finding of a novel Cys-Tyr cofactor probed by incorporation of noncanonical amino acid solved a long-standing puzzle, and the monodentate coordination of thiol substrates unveiled by nitric oxide filled the gap of substrate specificity in thiol dioxygenases. Finally, the de novo crystal structures, substrate binding mode and thermophilic nature revealed in DDO expanded the Type I extradiol dioxygenase family with an unprecedented structural topology.