|Cysteine Dioxygenase (CDO) catalyzes the oxidation of L-cysteine to L-cysteine sulfinic acid utilizing iron as a cofactor. Adjacent to the iron center is a protein-derived thioether crosslink formed by a cysteine (Cys93) and a tyrosine (Tyr157) residue. The crosslinked isoform of CDO increases catalytic activity ~5-fold compared to non-crosslinked CDO. Tyr157 forms a hydrogen-bonding network with His155 and Ser153 which has been proposed to participate in cysteine oxidation. Substitutions of His155 were made in CDO to evaluate the importance of the hydrogen-bonding network in both cysteine oxidation and crosslink formation. All substitutions resulted in minimal catalytic activity compared to wild-type CDO. Even though H155Q CDO showed minimal catalytic activity, the variant was able to generate the crosslink as effectively as wild-type CDO, while crosslink formation was limited with the H155A and H155E CDO variants. All substitutions of His155 altered the microenvironment of the metal coordination center of CDO. The different effects of the His155 CDO variants on crosslink formation and cysteine oxidation suggest the hydrogen-bonding network plays dual roles due to the distinct chemical steps utilized in these processes. Located ~8 Å away from the iron center is a conserved Cys (Cys164) residue at the opening to the active site. Cys164 does not participate in any intramolecular disulfide bonds and exists as a free thiol. Several bacterial CDO homologs contain either an Arg or a Met residue at a comparable position as Cys164 in mammals. Therefore, it has been speculated that Cys164 and amino acids at comparable positions could contribute to the substrate specificity of mammalian and bacterial CDO. Other studies have shown that Cys164 is involved in a disulfide bond with a free Cys in three-dimensional structures which suggested that Cys164 may serve as a redox switch in CDO. However, this hypothesis has not been adequately evaluated. Multiple variants of Cys164 in CDO were constructed to evaluate the role of Cys164 in catalysis, crosslink formation, and oxidative regulation. All Cys164 CDO variants displayed diminished activity compared to wild-type CDO. Crosslink formation studies showed that C164A CDO was the only Cys164 variant that could generate the fully crosslinked species at increased L-cysteine substrate concentrations similar to wild-type CDO. In addition, wild-type, C164A, non-crosslinked wild-type, and non-crosslinked C164A CDO did not appear to be modified by hydrogen peroxide or L-cysteine. Although Cys164 is not in the active site, these studies suggest that Cys164 likely plays a key role in L-cysteine substrate oxidation. Since Cys164 is located at the opening of the active site, it may regulate accessibility of the L-cysteine substrate to the active site. Both of these studies evaluated the mechanistic roles of conserved residues in CDO. The hydrogen-bonding network appears to stabilize iron-oxo intermediates through hydrogen-bonding interactions during catalysis. The Cys164 residue may act as a gate-keeper by regulating accessibility of the L-cysteine substrate during catalysis and crosslink formation. These studies also provided a foundation for future studies regarding the elucidation of the L-cysteine oxidation and crosslink formation mechanisms.