NADH reduction of nitroaromatics as a probe for residual ferric form high-spin in a cytochrome P450

Biochim Biophys Acta Proteins Proteom. 2018 Jan;1866(1):126-133. doi: 10.1016/j.bbapap.2017.04.003. Epub 2017 May 1.


The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Keywords: Electron paramagnetic resonance; Heme protein; Substrate recognition.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetophenones / chemistry*
  • Acetophenones / metabolism
  • Amino Acid Motifs
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Biocatalysis
  • Camphor / chemistry*
  • Camphor / metabolism
  • Camphor 5-Monooxygenase / chemistry*
  • Camphor 5-Monooxygenase / genetics
  • Camphor 5-Monooxygenase / metabolism
  • Cloning, Molecular
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Ferric Compounds / chemistry*
  • Gene Expression
  • Heme / chemistry*
  • Heme / metabolism
  • Kinetics
  • Models, Molecular
  • NAD / chemistry*
  • NAD / metabolism
  • Oxidation-Reduction
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity


  • Acetophenones
  • Bacterial Proteins
  • Ferric Compounds
  • Recombinant Proteins
  • NAD
  • 4-nitroacetophenone
  • Heme
  • Camphor
  • Camphor 5-Monooxygenase