A novel inhibitor for Fe-type nitrile hydratase: 2-Cyano-2-propyl hydroperoxide

Nitrile hydratase (NHase) is a non-heme iron or non-corrin cobalt enzyme having two post-translationally modified ligand residues, cysteine-sulfinic acid (alphaCys112-SO2H) and -sulfenic acid (alphaCys114SOH). We studied the interaction between Fe-type NHase and isobutyronitrile (iso-BN) which had been reported as a competitive inhibitor with a K-i value of 5 muM. From detailed kinetic studies of the inhibitory effect of iso-BN on Fe-type NHase, we found that authentic iso-BN was hydrated normally and that the impurity present in commercially available iso-BN inhibited NHase activity strongly. The inhibitory compound induced significant changes in the UV-vis absorption spectrum of NHase, suggesting its interaction with the iron center. This compound was purified by using reversed-phase HPLC and identified as 2-cyano-2-propyl hydroperoxide (Cpx) by H-1 and PFG-HMBC NMR spectroscopy, Upon addition of a stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of alphaCys114-SOH to CysSO(2)H. This result suggests that the -SOH structure of alphaCys114 is essential for the catalytic activity. The oxygen atom in CYS-SO2H is confirmed to come from the solvent H2O. The oxidized NHase was found to induce the UV-vis absorption spectral changes by addition of Cpx, suggesting that Cpx strongly interacted with iron(III) in the oxidized NHase to form a stable complex. Thus, Cpx functions as a novel irreversible inhibitor for NHase.