Nitrile 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.