New inhibitors of iron-containing nitrile hydratases

There is growing evidence in the literature emphasizing the significance of the posttranslational modification of cysteine thiols to sulfenic acids (S OH), which have been found in a number of proteins. Crystallographic and ma ss spectrometric evidence has shown the presence of this group in an inacti ve form of the industrially important enzyme nitrile hydratase (NHase). Thi s oxidized cysteine is unique in that it forms part of the coordination sph ere of the low-spin iron III at the active site of the enzyme. The presence of this unstable sulfenic group in the active form of NHase is the subject of some controversy. To try to detect this function in NHase, we have stud ied the inhibitory effect on nitrile hydration of reagents known to react w ith sulfenic acids. Two NHases were studied, namely, Rhodococcus rhodochrou s R312 NHase and Comamonas testosteroni NI1 NHase, and the reagents used we re meta-chlorocarbonyldicyano-phenylhydrazone (m-CICP), 7-chloro-4-nitroben zo-2-oxa-1,3-diazole (NBD-Cl), and 2-nitro-5-thiocyanato-benzoic acid (NTBA ). Following this approach we report three novel inhibitors of NHases. In a ddition, we report thiocyanate reagents that can be used to monitor NHase a ctivity spectroscopically.