Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.

Insight into the Maturation Process of the Nitrile Hydratase Active Site.

The metal binding motif of all nitrile hydratases (NHases, EC 4.2.1.84) is highly conserved (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, indicating an S = 2 system with unusually small axial ZFS, D = 0.29 cm-1 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 with electronic and structural similarity to the NHase from Rhodococcus equi TG328-2 (ReNHase). Coupled with MS data, these data support 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.