Post-translational modification is essential for catalytic activity of nitrile hydratase

Nitrile hydratase from Rhodococcus sp. N-771 is an alpha beta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alpha Cys112 and alpha Cys114 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 alpha beta 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 alpha beta complex did not have the modification of alpha Cys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alpha beta complex correlated with the amount of alpha Cys112-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 alpha Cys114 is modified to Cys-SOH together with the sulfinic acid modification of alpha Cys112. These results suggest that alpha Cys112 and alpha Cys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alpha Cys112-SO2H is responsible for the catalytic activity solely or in combination with alpha Cys114-SOH.