Nitrile hydratases are metalloenzymes that contain trivalent Fe/or Co ions in their active site, coordinated in an N2S3 ligand environment. The sulfur atoms are post-translationally modified and have three different oxidation states (αCys, Cys-SOH and Cys-SOO-) while backbone amines account for the nitrogen ligands and are deprotonated. Theoretical calculations assume that the αCys ligand is a thiolate, making it a strong π-donor ligand and a good nucleophile. The axial thiolate forms a hydrogen bond with a nearby Ser/Thr residue, which is conserved in all NHases. A critical outstanding mechanistic question is whether the formation of a transient disulfide bond is essential for catalytic turnover. Such a transient disulfide bond was proposed based on QM/MM calculations between the αCys sulfur and the sulfur of the proposed sulfenic acid cyclic intermediate. However, more recent theoretical studies are self-inconsistent in its conclusions regarding transient disulfide formation. To examine the role of the axial cysteine ligand in catalysis, the axial cysteine was mutated to Ala, Met, Ser, and His in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and to Ala and Ser in Fe-type NHase Rhodococcus equi. (ReNHase), thus, eliminating possible disulfide bond formation during catalysis. ICP-MS demonstrated reduced metal ion content for each PtNHase mutant: &lt;5% (αCys108Ala), ~44% (αCys108Met), ~50% (αCys108Ser) and ~55% (αCys108His) and catalytic activity also decreased to ~0.3%, ~0.8%, ~1.6%, and ~6.7%, respectively, towards acrylonitrile. The X-ray crystal structure of αCys108Ala confirmed the mutation and the decreased active site metal content but also, surprisingly, a fully formed active site. Therefore, the axial thiolate is important to properly tune the electronic properties of the active site metal ion but is not essential to catalysis. The mechanistic implications for NHases will be discussed.

Elucidating the role of the axial cysteine residue in NHase catalysis and the enzyme maturation

ACS Spring 2022

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