Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy. Biochemistry 2021 Dec 14;60(49):3771-3782

Date

11/30/2021

Pubmed ID

34843221

Pubmed Central ID

PMC8721871

DOI

10.1021/acs.biochem.1c00574

Scopus ID

2-s2.0-85120578868 (requires institutional sign-in at Scopus site)   2 Citations

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.

Author List

Karunagala Pathiranage WL, Gumataotao N, Fiedler AT, Holz RC, Bennett B

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University




MESH terms used to index this publication - Major topics in bold

Acetonitriles
Bacterial Proteins
Biocatalysis
Catalytic Domain
Cold Temperature
Electron Spin Resonance Spectroscopy
Escherichia coli
Gene Expression
Hydro-Lyases
Hydrogen-Ion Concentration
Iron
Kinetics
Nitriles
Protein Subunits
Recombinant Proteins
Rhodococcus equi
Sodium Chloride
Substrate Specificity
Water