Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.