Insights into the pH-dependent reactivity and substrate induced active-site reorganization of the non- heme iron enzyme CthEgtB.

Ergothioneine biosynthesis relies on the non-heme Fe(II) sulfoxide synthase EgtB, which couples O₂ activation to stereoselective C-S bond formation between trimethylhistidine (TMH; hercynine) and L-cysteine (L-Cys). Here, we utilize circular dichroism (CD), differential scanning fluorescence, light scattering, and stopped-flow spectroscopy to define how metal loading, substrate addition, oxygen availability, and pH shape the active site and catalysis of tetrameric EgtB from Chloracidobacterium thermophilum (Cth). Far-UV CD and thermal unfolding data confirm CthEgtB's stable, predominantly alpha helical secondary structure under all conditions studied, while Fe(II) and sequential substrate binding help to stabilize the enzyme. Visible CD demonstrates a distinct ligand-field reorganization upon sequential addition of Fe(II), L-Cys, and TMH to apo-CthEgtB. Subsequent exposure to O₂ results in a shift of the ligand-field and charge-transfer features with associated appearance of a high energy feature consistent with the formation of sulfoxide product. The pH- and temperature-dependent kinetics of the reaction were monitored via stopped-flow absorption spectroscopy and show max activity at pH 7.0 and rates that scale with O₂ concentration, indicating tightly coupled O₂ binding and activation. Mass spectrometry confirms oxygen dependent formation of the hercynylcysteine sulfoxide product. Finally, we provide clear evidence that retention of a 6x N-terminal histidine-tag decreases the activity and stability of CthEgtB, confirming that tagged constructs do not fully reproduce native behavior. Together, these results support a unified mechanism in which Fe(II), substrate binding, protonation environment around the active site, and O₂ availability poise the CthEgtB active site for efficient oxygen activation and sulfoxide formation.