AbstractNitrile hydratase from Rhodococcus sp. N‐771 is an αβ heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, αCys112 and αCys114 are modified to a cysteine sulfinic acid (Cys‐SO2H) and a cysteine sulfenic acid (Cys‐SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The αβ complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI‐LC/MS analysis showed that the anaerobically reconstituted αβ complex did not have the modification of αCys112‐SO2H and aerobic incubation induced the modification. The activity of the reconstituted αβ complex correlated with the amount of αCys112‐SO2H. Furthermore, ESI‐LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that αCys114 is modified to Cys‐SOH together with the sulfinic acid modification of αCys112. These results suggest that αCys112 and αCys114 are spontaneously oxidized to Cys‐SO2H and Cys‐SOH, respectively, and αCys112‐SO2H is responsible for the catalytic activity solely or in combination with αCys114‐SOH.