CthEgtB is a tetrameric, non-heme iron-dependent sulfoxide synthase that catalyzes the formation of a carbon–sulfur bond between trimethyl histidine (TMH) and L-cysteine (L-Cys), a key step in ergothioneine (EGT) biosynthesis. Despite the growing interest in this enzyme’s mechanism and potential biotechnological applications, many questions remain about how pH, temperature, oxygen (O₂) concentration, and affinity tag retention affect its reactivity. Notably, no published data explore CthEgtB activity under strictly anaerobic conditions paired with a quantitative addition of O₂, even though O₂ is essential for the sulfoxide product formation. In the present work, we employed far-UV circular dichroism (CD) spectroscopy and thermal unfolding studies to elucidate how Fe(II) and substrate binding influence CthEgtB’s secondary structure and stability. We observed that neither Fe(II) nor substrate binding significantly alters the enzyme’s secondary structure. However, both factors enhance thermal stability, supporting a model in which sequential substrate binding progressively stabilizes CthEgtB and boosts its catalytic efficiency. Our data also show that enzyme activity is highly sensitive to pH and temperature, implicating the protonation states of essential residues and substrates, as well as the overall protein stability, as key determinants of reaction rates. Although previous research has examined a 6xHis-tagged variant (6xHisCthEgtB) for kinetic studies, primarily under high reductant conditions, our direct comparison of 6xHisCthEgtB and the untagged CthEgtB reveals distinct differences in reaction rates attributable to the affinity tag. Furthermore, this work underscores the necessity of precise O₂ control to enable reliable kinetic modeling, given the enzyme’s O₂-dependent sulfoxide formation. Together, these findings provide new insights into how CthEgtB carries out its sulfoxide synthase activity and how crucial factors such as substrate binding, pH, temperature, and O₂ availability intersect to regulate its reactivity. These results also offer a valuable framework for understanding other type II sulfoxide synthases and their broader roles in thiol-based redox biochemistry.

Kassidy Rodriguez

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