Abstract
Ergothioneine (EGT) is a sulfur-containing antioxidant synthesized by select microorganisms and accumulats in mammals through dedicated transport systems, motivating interest in the enzymes that control its biosynthesis. The defining step is catalyzed by EgtB, a non-heme Fe(II)-dependent sulfoxide synthase that couples O₂ activation to stereoselective C–S bond formation between trimethylhistidine (TMH) and L-cysteine to generate hercynylcysteine sulfoxide. This dissertation establishes a stability-to-function framework for the Type II EgtB homolog from Chloracidobacterium thermophilum (CthEgtB), a tetrameric enzyme with oxygen-triggered reactivity. First, I define the apo-state stability landscape of CthEgtB across pH and temperature using SEC, DLS, circular dichroism, and intrinsic tryptophan fluorescence. These studies show that CthEgtB favors the tetramer near neutral to mildly basic pH, dissociates under acidic conditions, and is most structurally stable near pH 7.0. Direct comparison of tagged and untagged constructs shows that an N-terminal 6×His tag perturbs stability and unfolding behavior. Next, I connect structural competence to catalytic performance through anaerobic Fe(II) reconstitution, visible circular dichroism, and stopped-flow kinetics. Visible CD reveals ligand-field reorganization upon Fe(II) loading and substrate binding, while stopped-flow measurements under defined O₂ conditions show that oxygen availability directly controls sulfoxide formation, with rates and amplitudes scaling with dissolved O₂. Catalysis and thermal resilience are both optimized near pH 7.0, while His-tag retention reduces activity to ~61% of the native construct. Finally, a Mössbauer-focused case study provides electronic-structure context for future iron-state assignments in CthEgtB. Together, these results support a model in which CthEgtB function emerges from coupled tetramer stability, active-site priming, protonation environment, and oxygen gating.