Mutation of tyrosine 190 to alanine eliminates the inactivation of cytochrome P450 2B1 by peroxynitrite

Chem Res Toxicol. 2003 Feb;16(2):129-36. doi: 10.1021/tx020040b.


We have previously reported that cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine. Digestion of the peroxynitrite-treated P450 2B1 with Lys C followed by amino acid sequencing of the major nitrotyrosine-containing peptide demonstrated that it spanned residues 160-225. This peptide contains two tyrosine residues at positions 190 and 203. In this study, we mutated Tyr 190 to Ala (Y190A) and Tyr 203 to Ala (Y203A) in wild-type recombinant P450 2B1 (WT) in order to identify the specific residue(s) that is nitrated and to determine whether nitrotyrosine formation is reponsible for the peroxynitrite-mediated inactivation of P450 2B1. All three P450s were expressed in Escherichia coli, purified to homogeneity, and characterized. The catalytic activities for four different substrates of P450 2B1 increased approximately 2-fold for the Y203A mutant, but decreased by about 60% for the Y190A mutant when compared to WT. The addition of peroxynitrite to the P450s resulted in concentration-dependent decreases in the catalytic activities of WT and Y203A, but no loss of the catalytic activities of Y190A. The extent of tyrosine nitration of Y190A by peroxynitrite decreased by approximately 75% as compared with WT or the Y203A protein. Following digestion of the peroxynitrite-modified proteins with Lys C, a major nitrotyrosine-containing peptide was detected from WT and Y203A, but not from Y190A. Collectively, these results indicate that Tyr 190 is the target residue for peroxynitrite-mediated nitration and that nitration of this tyrosine is a responsible for the inactivation of P450 2B1. Modeling studies suggest that Tyr 190 may play a structural role in maintaining the integrity of the protein for maximal activity through hydrogen bonding with Glu 149.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / genetics*
  • Amino Acid Substitution
  • Catalysis
  • Cytochrome P-450 CYP2B1 / genetics*
  • Cytochrome P-450 CYP2B1 / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology*
  • Models, Molecular
  • Nitric Oxide / metabolism
  • Peroxynitrous Acid / pharmacology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Substrate Specificity
  • Tyrosine / analogs & derivatives
  • Tyrosine / genetics*
  • Tyrosine / metabolism


  • Enzyme Inhibitors
  • Recombinant Proteins
  • Peroxynitrous Acid
  • Nitric Oxide
  • Tyrosine
  • Cytochrome P-450 CYP2B1
  • Alanine