Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy

Authors

Wasantha Lankathilaka Karunagala Pathiranage, Marquette University
Natalie Gumataotao, Loyola University Chicago
Adam T. Fiedler, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow
Brian Bennett, Marquette UniversityFollow

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. Fe^III-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.

Comments

Accepted version. Biochemistry, Vol. 60, No. 49 (November 2021): 3771-3782. DOI. © 2021 American Chemical Society. Used with permission.

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