Document Type

Article

Publication Date

3-2026

Publisher

Elsevier

Source Publication

Journal of Inorganic Biochemistry

Source ISSN

0162-0134

Abstract

The metal binding motif of nitrile hydratases (NHases, EC 4.2.1.84) is highly conserved (CXXCSCX) in the alpha subunit. The conserved axial cysteine ligand in both the Co-type and Fe-type NHases was mutated to Ala in Co-type NHases Pseudonocardia thermophila JCM 3095 (PtNHase) and to Ala and Ser in the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase). Kinetic and spectroscopic data on both the Co- and Fe-type NHases provide definitive mechanistic evidence that the formation of a proposed transient disulfide bond intermediate is not essential for catalysis. X-ray crystallographic data for PtNHase and the αC108A PtNHase mutant expressed in the presence and absence of Co2+ and/or the required activator (ɛ) protein indicate that the ε protein is required for metal insertion and the post-translational modification of the active site equatorial Cys residues. The axial αC108 ligand clearly plays a role in the post-translational modification of the NHase active site as the absence of αC108 impedes active site maturation, including metal ion insertion, and consequently subunit swapping. It is also possible, perhaps probable, that the correct post translational modification requires the proper incorporation of metal, which is disrupted by the mutation to the αC108 residue. These data further support the hypothesis that the proposed subunit swapping mechanism is different in Co- vs. Fe-type NHases likely due to the very different ɛ proteins used for each type of NHase.

Comments

Accepted version. Journal of Inorganic Biochemistry, Vol. 280 (2026): 113293. DOI. © 2026 Elsevier. Used with permission.

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