Insight into the Maturation Process of the Nitrile Hydratase Active Site

Abstract

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