Document Type
Article
Publication Date
11-2021
Publisher
American Chemical Society
Source Publication
Biochemistry
Source ISSN
0006-2960
Abstract
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.
Recommended Citation
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.
https://epublications.marquette.edu/physics_fac/182
Comments
Accepted version. Biochemistry, Vol. 60, No. 49 (November 2021): 3771-3782. DOI. © 2021 American Chemical Society. Used with permission.