A metallopeptide model for the active site of cobalt nitrile hydratase reveals coupled metal and cysteine oxidation during active-site maturation

Nitrile hydratases (NHases, EC 4.2.1.84) are metalloenzymes that catalyze the hydration of nitriles to amides. They contain a unique active site in which a low-spin Fe(III) or Co(III) ion is coordinated by two backbone amide nitrogens and three cysteine-derived sulfur ligands, two of which are post-translationally modified to sulfenic and sulfinic acids. Although this unusual coordination environment is essential for catalysis, the mechanism by which metal insertion and cysteine oxidation occur during active-site maturation remains poorly understood. To examine these processes, an eight-residue peptide (VCTLCSCY) derived from the conserved Co-type NHase metal-binding motif was synthesized and used to prepare Co-type NHase biomimetic peptide complexes. Spectroscopic characterization using UV–visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy revealed high- and low-spin Co(II) signals with approximately equal relative integrated EPR intensities under anaerobic conditions. Spectroscopic and computational analyses favor a distorted tetrahedral N 2 S 2 model for the high-spin species and a five-coordinate, distorted square-pyramidal model for the low-spin species. Exposure to dioxygen results in the loss of the Co(II) EPR signals consistent with oxidation of the metal center to an EPR-silent low-spin Co(III) species. Mass spectrometry reveals signals consistent with incorporation of up to three oxygen atoms into the peptide after air exposure. Together, these results show that the conserved NHase metal-binding sequence supports distinct Co(II) precursor states and that exposure to dioxygen is accompanied by loss of the Co(II) EPR signals along with peptide oxygenation.