Insight into the Maturation Process of the Nitrile Hydratase Active Site

Nitrile hydratases (NHases) are metalloenzymes that catalyze stereo and regio-specific hydration of nitriles to their corresponding higher value amides under ambient conditions physiological pH. NHase has acquired substantial interest as a biocatalyst in preparative organic chemistry industries due its advantages over the chemical catalysis such zero byproduct formation, high specificity, yield, recycling ability, mild reaction conditions. The metal ion is coordinated by three cysteine sulfur atoms, two amide nitrogen atoms (from backbone amino acid chain), water molecule. Two active site ligands at equatorial plane have post-translationally oxidized into sulfinic (Cys-SO2H) sulfenic (Cys-SOH) which essential for nitrile hydration. binding motif all (NHases, EC 4.2.1.84) highly conserved within single sequence (CXXCSCX) α-subunit. Accordingly an eight peptide (VCTLCSCY), based on Co-type from Pseudonocardia thermophilia (PtNHase) was synthesized shown coordinate Fe(II) anaerobic Parallel-mode EPR data mononuclear Fe(II)-peptide complex confirmed integer-spin signal g' ~ 9, suggesting S = 2 system (D 0.29 cm-1, E/D 0.14, sD 1.2 GHz, sE 200 MHz). Exposure air yielded transient high-spin most consistent with intermediate/admixed 3/2 spin-state, while extinguished. Prolonged exposure resulted observation signals g 2.04, 2.16, 2.20, formation low-spin Fe(III)-peptide remarkably similar Rhodococcus equi TG328-2 (ReNHase); however, these only account ~8% total spins sample. These indicate progression iron oxidation NHases proceeds reduced oxidized-high-spin followed center, something heretofore not been observed.