In order to gain insight into the catalytic mechanism of Fe-type nitrile hydratases (NHase), the pH and temperature dependence of the kinetic parameters kcat, Km, and kcat/Km along with the solvent isotope effect were examined for the Fe-type NHase from Comamonas testosteroni Ni1 (CtNHase). CtNHase was found to exhibit a bell-shaped curve for plots of relative activity vs pH over pH values 4−10 for the hydration of acrylonitrile and was found to display maximal activity at pH ∼7.2. Fits of these data provided a pKES1 value of 6.1 ± 0.1, a pKES2 value of 9.1 ± 0.2 (k′cat = 10.1 ± 0.3 s−1), a pKE1 value of 6.2 ± 0.1, and a pKE2 value of 9.2 ± 0.1 (k′cat/K′m of 2.0 ± 0.2 s−1 mM−1). Proton inventory studies indicate that two protons are transferred in the rate-limiting step of the reaction at pH 7.2. Since CtNHase is stable to 25 °C, an Arrhenius plot was constructed by plotting ln(kcat) vs 1/T, providing an Ea of 33.3 ± 1.5 kJ/mol. ΔH° of ionization values were also determined, thus helping to identify the ionizing groups exhibiting the pKES1 and pKES2 values. Based on ΔH°ion data, pKES1 is assigned to βTyr68 while pKES2 is assigned to βArg52, βArg157, or αSer116 (NHases are α2β2 heterotetramers). Given the strong similarities in the kinetic data obtained for both Co- and Fe-type NHase enzymes, both types of NHase enzymes likely hydrate nitriles in a similar fashion.