Peroxynitrite-Mediated Nitration of Tyrosine and Inactivation of the Catalytic Activity of Cytochrome P450 2B1

Abstract

The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration-dependent loss of the NADPH- and reductase-supported or tert-butylhydroperoxide-supported 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 microM, respectively. After incubation of P450 2B1 with 300 microM peroxynitrite, the heme moiety was not altered, but the apoprotein was modified as shown by HPLC and spectral analysis. Western blot analysis of peroxynitrite-treated P450 2B1 demonstrated the presence of an extensive immunoreactivite band after incubating with anti-nitrotyrosine antibody. However, the immunostaining was completely abolished after coincubation of the anti-nitrotyrosine antibody with 10 mM nitrotyrosine. These results indicated that one or more of the tyrosine residues in P450 2B1 were modified to nitrotyrosines. The decrease in the enzymatic activity correlated with the increase in the extent of tyrosine nitration. Further demonstration of tyrosine nitration was confirmed by GC/MS analysis by using 13C-labeled tyrosine and nitrotyrosine as internal standards; approximately 0.97 mol of nitrotyrosine per mole of P450 2B1 was found after treatment with peroxynitrite. The peroxynitrite-treated P450 2B1 was digested with Lys C, and the resulting peptides were separated by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid sequence of the major nitrotyrosine-containing peptide corresponded to a peptide containing amino acid residues 160-225 of P450 2B1, which contains two tyrosine residues. Thus, incubation of P450 2B1 with peroxynitrite resulted in the nitration of tyrosines at either residue 190 or 203 or at both residues of P450 2B1 concomitant with a loss of 2B1-dependent activity.

Substituent Effects on the Stability and Antioxidant Activity of Spirodiazaselenuranes

“…Similar to the previous reports [22,43], the diaryl selenides (15-21) first react with one equivalent of PN to produce the corresponding selenoxides (22-28), which after elimination of one water molecule generate the corresponding spirodiazaselenuranes (29)(30)(31)(32)(33)(34)(35). Subsequent reaction of these spiro compounds with second equivalent of PN leads to the overoxidation of selenium centres producing the corresponding selenurane oxides (46-52).…”

“…It was found that substitution of the aromatic ring with benzylic (29), pyridylmethyl (30)(31), heterocyclic (32)(33) and aliphatic chains (34)(35) do not alter the catalytic mechanism [21]. Diaryl selenide is the active species, which reduces H2O2 to water and is oxidized to the corresponding selenoxide intermediate.…”

“…During inflammation, the production of PN increases, which subsequently, induces DNA damage [30,31], lipid peroxidation in biomembranes and lipoproteins [32,33]. Peroxynitrite can also inactivate a variety of enzymes by nitrating the tyrosine residue generating 3-nitrotyrosine (3-NT) [34][35][36]. Therefore, inhibition of the PN-mediated nitration and oxidation reaction is very essential.…”

“…The aromatic ring on the nitrogen plays an important role in the cyclization by stabilizing the Se-N bonds in the spirodiazaselenurane. In the present study, we have substituted the phenyl/substituted phenyl group with benzylic (29), pyridylmethyl (30)(31), heterocyclic (32)(33) and aliphatic side chains (34)(35) at the nitrogen atoms to understand their influence on the cyclization and stability of the spirodizaselenuranes. Although selenurane oxides with two oxygen atoms at the axial positions are known, such compounds with two nitrogen atoms bonded to selenium are still unknown.…”

Regulation of renal CYP4A expression and 20-HETE synthesis by nitric oxide in pregnant rats

“…These NO-CYP adducts are enzymatically inactive in vitro. Moreover, Roberts et al (32) demonstrated that PN can modify tyrosine residues of CYP2B1 and inactivate CYP2B1-catalyzed reaction. It is likely that NO interacts with the major CYP4A isoforms in female rats, i.e., CYP4A1 and 4A3, in a similar manner.…”