Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of cytochrome P450 2B1.
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.
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
E S
ES
Roberts
H l
Hl
Lin
J R
JR
Crowley
J L
JL
Vuletich
Y
Y
Osawa
P F
PF
Hollenberg
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000187:drug effects* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008401:Gas Chromatography-Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D051381:Rats; D020318:Rats, Long-Evans; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D003374:Coumarins; D009566:Nitrates; C056033:7-ethoxy-4-trifluoromethylcoumarin; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D009249:NADP; D020122:tert-Butylhydroperoxide; D019362:Cytochrome P-450 CYP2B1