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Two conserved second-sphere βArg (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 βR52A, βR52K, βR157A, βR157K and ReNHase βR61A) were successfully expressed and purified. Apart from the PtNHase βR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase βR 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 βR 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 calcu- lations 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 βR residues in the proposed subunit swapping process and post-translational modification of the α-sub- unit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.