American Chemical Society
A new method to trap catalytic intermediate species was employed with Fe-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase). ReNHase was incubated with substrates in a 23% (w/w) NaCl/H2O eutectic system that remained liquid at −20 °C, thereby permitting the observation of transient species that were present at electron paramagnetic resonance (EPR)-detectable levels in samples frozen while in the steady state. FeIII-EPR signals from the resting enzyme were unaffected by the presence of 23% NaCl, and the catalytic activity was ∼55% that in the absence of NaCl at the optimum pH of 7.5. The reaction of ReNHase in the eutectic system at −20 °C with the substrates acetonitrile or benzonitrile induced significant changes in the EPR spectra. A previously unobserved signal with highly rhombic g-values (g1 = 2.31) was observed during the steady state but did not persist beyond the exhaustion of the substrate, indicating that it arises from a catalytically competent intermediate. Distinct signals due to product complexes provide a detailed mechanism for product release, the rate-limiting step of the reaction. Assignment of the observed EPR signals was facilitated by density functional theory calculations, which provided candidate structures and g-values for various proposed ReNHase intermediates. Collectively, these results provide new insights into the catalytic mechanism of NHase and offer a new approach for isolating and characterizing EPR-active intermediates in metalloenzymes.
Karunagala Pathiranage, Wasantha Lankathilaka; Gumataotao, Natalie; Fiedler, Adam T.; Holz, Richard C.; and Bennett, Brian, "Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy" (2021). Physics Faculty Research and Publications. 182.
Available for download on Tuesday, November 01, 2022