JBIC Journal of Biological Inorganic Chemistry

, Volume 24, Issue 7, pp 1105–1113 | Cite as

Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy

  • K. P. Wasantha Lankathilaka
  • Natalia Stein
  • Richard C. HolzEmail author
  • Brian BennettEmail author
Original Paper


Nitrile hydratase (NHase) is a non-heme iron-containing enzyme that has applications in commodity chemical synthesis, pharmaceutical intermediate synthesis, and reclamation of nitrile-(bromoxynil) contaminated land. Mechanistic study of the enzyme has been complicated by the expression of multiple overlapping Fe(III) EPR signals. The individual signals were recently assigned to distinct chemical species with the assistance of DFT calculations. Here, the origins and evolution of the EPR signals from cells overexpressing the enzyme were investigated, with the aims of optimizing the preparation of homogeneous samples of NHase for study and investigating the application of E. coli overexpressing the enzyme for “green” chemistry. It was revealed that nitrile hydratase forms two sets of inactive complexes in vivo over time. One is due to reversible complexation with endogenous carboxylic acids, while the second is due to irreversibly inactivating oxidation of an essential cysteine sulfenic acid. It was shown that the homogeneity of preparations can be improved by employing an anaerobic protocol. The ability of the substrates acrylonitrile and acetonitrile to be taken up by cells and hydrated to the corresponding amides by NHase was demonstrated by EPR identification of the product complexes of NHase in intact cells. The inhibitors butyric acid and butane boronic acid were also taken up by E. coli and formed complexes with NHase in vivo, indicating that care must be taken with environmental variables when attempting microbially assisted synthesis and reclamation.

Graphic abstract


EPR Nitrile hydratase Nitrile hydration Non-heme iron Post-translational modification 



Butane boronic acid


Electron–electron double resonance-detected nuclear magnetic resonance


Electron-nuclear double resonance


Electron paramagnetic resonance


Nitrile hydratase


Active nitrile hydratase and its EPR signal


Butyrate (carboxylate) complex of active nitrile hydratase and its EPR signal


Inactive nitrile hydratase in which the cysteine sulfenic acid has been oxidized to sulfinic acid, and its EPR signal


Butyrate (carboxylate) complex of inactive nitrile hydratase in which the cysteine sulfenic acid has been oxidized to sulfinic acid, and its EPR signal



This study was supported by the National Science Foundation (CHE-1462201, BB; CHE-1412443, RCH; CHE-1808711, RCH & BB; CHE-1532168, BB & RCH), the Todd Wehr Foundation, and Bruker Biospin.


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Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2019

Authors and Affiliations

  1. 1.Department of PhysicsMarquette UniversityMilwaukeeUSA
  2. 2.Department of ChemistryMarquette UniversityMilwaukeeUSA
  3. 3.Department of BiophysicsMedical College of WisconsinMilwaukeeUSA
  4. 4.Department of ChemistryColorado School of MinesGoldenUSA

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