Chem. Res. Toxicol. 1998, 11, 1067-1074
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Peroxynitrite-Mediated Nitration of Tyrosine and Inactivation of the Catalytic Activity of Cytochrome P450 2B1 Elizabeth S. Roberts,†,‡,§ Hsia-lien Lin,†,‡ Jan R. Crowley,| Jennifer L. Vuletich,† Yoichi Osawa,† and Paul F. Hollenberg*,† Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109-0632, and Department of Medicine, Mass Spectrometry Research Facility, Washington University, St. Louis, Missouri 63110 Received May 5, 1998
The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentrationdependent 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 µM, respectively. After incubation of P450 2B1 with 300 µM 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 antinitrotyrosine 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 Tricinesodium 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.
Introduction Peroxynitrite, which can be formed by the rapid reaction of nitric oxide and superoxide (1, 2), appears to be an important species generated in activated macrophages, endothelium, neutrophiles, and Kupffer cells (36). At physiological pH, peroxynitrite can decompose in a few seconds to yield reactive intermediates such as the hydroxyl radical, nitronium ion, and nitrogen dioxide (1, 2). Peroxynitrite can modify proteins via the formation of protein carbonyls and by oxidation of tryptophan, tyrosine, cysteine, and methionine residues (7). In addition, it can damage lipids and cause protein fragmentation and aggregation (8). One stable product of peroxynitrite attack on free or protein-associated tyrosine is the addition of a nitro group to position 3 of tyrosine to form 3-nitrotyrosine (1, 7, 9, 10). The peroxynitrite-mediated nitration of tyrosine residues has been demonstrated in bovine Cu,Zn superoxide dismutase, bovine serum albu* To whom correspondence should be addressed: Department of Pharmacology, MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632. Telephone: (734) 764-8166. Fax: (734) 763-4450. E-mail:
[email protected]. † The University of Michigan. ‡ The first two authors contributed equally to this paper. § Present address: Department of Chemistry and Biochemistry, University of Detroit Mercy, Detroit, MI 48219-0900. | Washington University.
min (BSA1), human surfactant protein, Escherichia coli glutamine synthetase, bovine prostacyclin synthase, and human Mn superoxide dismutase in vitro (1, 7, 8, 1113). Thus, the formation of nitrotyrosine may be a useful marker for peroxynitrite generation in biological systems (1, 7, 9, 10). Significantly elevated levels of nitrotyrosine have been identified in several pathophysiological conditions associated with chronic inflammation, atherosclerotic lesions, acute lung injury, and neurodegenerative diseases (13-18). Although both nitric oxide synthase and prostacyclin synthase have a heme-thiolate prosthetic group, the direct interaction of peroxynitrite with the heme moiety has been demonstrated in the former (19, 20) but not in the latter (12). The cytochrome P450 (P450) enzymes, a family of heme-containing monooxygenases, are involved in the metabolism of a wide variety of xenobiotics, including drugs and carcinogens, as well as endobiotics (21, 22). The P450 enzymes exhibit catalytic diversity which reflects both the structural diversity of the P450 proteins 1 Abbreviations: BSA, bovine serum albumin; P450, cytochrome P450; P450 2B1, major form of P450 from liver microsomes purified from phenobarbital-treated rats; reductase, NADPH-cytochrome P450 reductase; EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; DLPC, dilaurylL-R-phosphatidylcholine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
S0893-228x(98)00099-X CCC: $15.00 © 1998 American Chemical Society Published on Web 08/13/1998
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and the ability of individual P450 enzymes to metabolize structurally distinct compounds. Although the catalytic mechanisms of all the P450s appear to be similar, little information concerning the critical amino acid residues involved in substrate binding and catalysis is known for mammalian P450s. Studies on tetranitromethanetreated P450 isozymes have suggested that the Tyr380 of P450 2B4 is a catalytically essential amino acid residue at the active center of the enzyme and that Tyr243 and Tyr271 of P450 1A2 are functionally involved in the interaction with NADPH-cytochrome P450 reductase (reductase) (23, 24). In this study, we investigated the effect of peroxynitrite on P450 2B1 purified from rat liver microsomes. The activity of peroxynitrite-treated P450 2B1 in the deethylation of 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) was determined in the presence of tert-butylhydroperoxide or NADPH and reductase. The nitrotyrosines were detected by Western blot, HPLC, and GC/MS analysis. Furthermore, the location of the nitrated tyrosine was determined by Lys C digestion followed by amino acid sequence analysis of peptides separated by Tricine-SDSPAGE.
Materials and Methods Materials. Peroxynitrite was from Alexis Corp. (San Diego, CA) and Cayman Chemicals (Ann Arbor, MI) and was stored in small aliquots at -70 °C. Before each use, the peroxynitrite concentration was determined from its UV spectrum (λmax ) 302 nm, 302 ) 1670 M-1 cm-1) (2, 10). NADPH and dilauroyl-L-Rphosphatidylcholine (DLPC) were from Sigma Chemical Co. (St. Louis, MO). EFC was from Molecular Probes, Inc. (Eugene, OR), and 7-hydroxy-4-(trifluoromethyl)coumarin was from Enzyme Systems Products (Dublin, CA). tert-Butylhydroperoxide was from Aldrich Chemical Co. (Milwaukee, WI). Rabbit polyclonal antibody to nitrotyrosine and goat anti-rabbit IgG horseradish peroxidase conjugate were from Upstate Biotechnology, Inc. (Lake Placid, NY), and Bio-Rad (Hercules, CA), respectively. Lys C was from Wako (Richmond, VA). Rabbit polyclonal antibody to P450 2B1 was prepared as described (25). Enzymes. P450 2B1 was purified as described by Saito and Strobel (26) from microsomes prepared from the livers of Long Evans rats (150-175 g; Harlan Sprague Dawley, Inc., Indianapolis, IN). Rat reductase was expressed in E. coli and purified as previously described (27, 28). Activity. The hydroperoxide-supported deethylation of EFC by 2B1 was determined after a 3 min incubation in reaction mixtures containing 100 pmol of 2B1, 10 µg of DLPC, and 31640 µM peroxynitrite in 100 mM potassium phosphate buffer (pH 7.7) in a total volume of 200 µL. Assay buffer (800 µL) containing tert-butylhydroperoxide and EFC, at final concentrations of 25 mM and 100 µM, respectively, in 100 mM potassium phosphate buffer (pH 7.7) was added to the original 200 µL of solution to determine the 2B1 activity remaining. The deethylation was allowed to proceed for 3 min at 30 °C before the addition of 300 µL of cold acetonitrile to stop the reaction. The amount of the product, 7-hydroxy-4-(trifluoromethyl)coumarin, was determined spectrofluorometrically on an SLM-Aminco SPF-500C spectrofluorometer with excitation and emission at 410 and 510 nm, respectively, as described (29). The NADPH- and reductase-supported deethylation of EFC by 2B1 was determined in reaction mixtures containing 100 pmol of 2B1, 10 µg of DLPC, and 2.5-320 µM peroxynitrite in 200 µL of 100 mM potassium phosphate buffer (pH 7.7). After exposure of 2B1 to peroxynitrite, 100 pmol of reductase was added to each mixture, and activity was determined as described previously using an assay mixture containing 100 µM EFC and 0.1 mM NADPH (29). For the control, we used either a solution containing the decomposed form of peroxynitrite provided by
Roberts et al. Alexis Corp. or a decomposed form of peroxynitrite solution generated by adding peroxynitrite to 100 mM potassium phosphate buffer (pH 7.7) for 5 min prior to the addition of P450 2B1 (10). Spectral Analysis. P450 2B1 (250 pmol), control or treated with 300 µM peroxynitrite, in 200 µL of 100 mM potassium phosphate buffer (pH 7.7) was analyzed by absorption spectroscopy. The mixtures were diluted to 1000 µL with 100 mM potassium phosphate buffer and spectra recorded from 400 to 500 nm against a buffer blank on an Aminco-DW2 spectrophotometer modified with the OLIS-DW2 operating system. Electrophoresis and Western Blotting. SDS-PAGE analysis was performed on a 12% polyacrylamide gel according to the method of Laemmli (30). Tricine-SDS-PAGE analysis was performed according to the method of Scha¨gger and von Jagow (31) with modifications (32). For detection of nitrotyrosine-containing peptides, the blots were blocked with 1% BSA in Tween-Tris-buffered saline overnight and incubated with a 1:20000 dilution of rabbit polyclonal anti-nitrotyrosine antibody. For detection of 2B1, blots were incubated with a 1:10000 dilution of rabbit polyclonal anti-2B1 antibody. The immunocomplexed membranes were probed with a 1:10000 dilution of goat anti-rabbit horseradish peroxidase-conjugated secondary antibody, and the immunoreactive bands were detected using enhanced chemiluminescence (Pierce, Rockford, IL). The nitrated BSA standard was prepared according to the method of Ye et al. (33). The extent of nitration of BSA was determined by measuring the absorbance at 430 nm ( ) 4500 M-1 cm-1 at pH >9). The incubation mixtures for the Lys C digestion contained 4 nmol of P450, either control or 300 µM peroxynitrite-treated, in 325 µL of potassium phosphate buffer (pH 7.7). Urea (120 mg) was added to denature the protein prior to heating at 60 °C for 15 min. Lys C (5 µg) was added to each tube, and the digestion was allowed to proceed overnight at 30 °C. The digested samples were subjected to Tricine-SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. The peptides were analyzed for nitrotyrosine by incubation of the membranes with anti-nitrotyrosine antibody or stained for total protein with 0.1% Coomassie Blue/45% CH3OH/10% CH3COOH/45% H2O. The bands corresponding to the nitrated peptides were excised from the Coomassie Blue-stained blot, and the amino acid sequences of the membrane-bound peptides were determined in the Protein and Carbohydrate Structure Facility at The University of Michigan (Ann Arbor, MI) by automated gas phase sequencing on a model 494 Protein Sequencer (PE Applied Biosystems, Foster City, CA). HPLC. The apoprotein and the heme moieties of P450 were separated by HPLC analysis of reaction mixtures containing 400 pmol of P450 2B1, 20 µg of DLPC, and either control, 20 µM, or 150 µM peroxynitrite, in 100 mM potassium phosphate buffer (pH 7.7) in a total volume of 365 µL. The mixtures were analyzed by HPLC on a Waters HPLC system with a 490E detector, 501 pumps, and Maxima software. The solvent system was buffer A (0.1% trifluoroacetic acid) and buffer B (95% CH3CN/5.0% H2O/0.1% trifluoroacetic acid). The apoprotein and heme were resolved on a POROS R/H column (4.6 mm × 100 mm) (Perseptive Biosystems, Cambridge, MA) with a linear gradient from 20 to 75% B over the course of 18 min at a flow rate of 1.0 mL/min. The samples were monitored at 214, 280, 365, and 405 nm. GC/MS. The GC/MS analysis and quantitation of nitrotyrosine were performed following incubation of mixtures containing 2.5 nmol of P450, control or treated with 200 µM peroxynitrite, in 250 µL of potassium phosphate buffer (pH 7.7). The protein was precipitated with cold acetone, and the pellet was washed three times. The analysis was performed in the Washington University Department of Medicine Mass Spectrometry Research Facility (St. Louis, MO) according to the described procedure (34). Briefly, control and peroxynitritetreated 2B1 were acid hydrolyzed after adding 25 pmol of [13C6]nitrotyrosine and 25 nmol of [13C6]tyrosine as internal stan-
Effect of Peroxynitrite on P450 2B1
Figure 1. Effect of peroxynitrite on NADPH- and reductase(b) or tert-butylhydroperoxide-supported (O) deethylation of EFC by P450 2B1. Experimental conditions were as described in Materials and Methods. Peroxynitrite-treated samples were compared to control samples (as 100%). Data are the mean ( SE from triplicate determinations.
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Figure 2. Absolute spectra of control (s) and 300 µM peroxynitrite-treated (- -) P450 2B1. Experimental conditions were as described in Materials and Methods.
dards. The hydrolyzed mixture was dried under a stream of N2. The dried hydrolysate was reconstituted in 1 mL of 50 mM ammonium acetate (pH 6.4) and applied to a strong anionexchange resin (AGI-X8 resin, Bio-Rad). The resin was washed with 6 mL of water, and the nitrotyrosine was eluted with 2 mL of 1 M acetic acid. The acetic acid was evaporated under a stream of N2. The sample was reconstituted in 100 µL of 50 mM ammonium acetate (pH 6.4), and 10 µL of a 10 mM solution of sodium dithionite was added to reduce the nitro group to an amino group. The samples were then taken to dryness for derivatization. To derivatize, the samples were first esterified by heating at 65 °C for 1 h with 1-propanol/HCl (3:1). After being dried, the samples were heated for 5 min at 65 °C with heptafluorobutyric anhydride/ethyl acetate (1:3) to derivatize the amino and hydroxyl groups. The samples were then analyzed by GC/MS in the negative-ion chemical ionization mode. The amount of nitrotyrosine and tyrosine in the samples was corrected on the basis of the recovery of the 13C-labeled internal standards.
Results P450 2B1 was incubated with various concentrations of peroxynitrite, and the 2B1-dependent activity was then determined by the addition of either tert-butylhydroperoxide or NADPH and reductase to support the metabolism of EFC (Figure 1). The concentrations of peroxynitrite that resulted in 50% inhibition of the NADPH- and reductase-supported or peroxide-supported activity were 39 and 210 µM, respectively. The control and peroxynitrite-treated proteins were then analyzed by UV/visible spectroscopy. As shown in Figure 2, the absolute spectrum for the heme absorbance is not altered after incubation with 300 µM peroxynitrite. In most cases, the identification and quantitation of nitrotyrosine in proteins can be accomplished by UV/ visible spectroscopy at either acidic or basic pH (9, 10). Since P450 contains a heme prosthetic group that interferes with measurement of the nitrotyrosine absorbance in the 350-450 nm region, other techniques were employed to detect nitrotyrosine. A HPLC method was used to separate the apoprotein from the heme moiety under acidic conditions. The elution profiles monitored at 214 and 365 nm from the control sample are shown in Figure 3A. The heme elutes at approximately 6 min with a 405 nm:365 nm ratio of >1 (data not shown). The unmodified protein elutes at approximately 12 min with absorbances
Figure 3. HPLC separation of the apoprotein and heme after treatment of P450 2B1 with control (A), 20 µM peroxynitrite (B), or 150 µM peroxynitrite (C). Experimental conditions were as described in Materials and Methods. The peaks representing the nitrotyrosine-containing P450 2B1 are indicated with asterisks.
at 214 and 280 nm and no absorbance at 365 nm. Panels B and C show the elution profiles for the samples treated with 20 and 150 µM peroxynitrite, respectively. The protein peak with the major absorbance at 214 nm also exhibits an absorbance at 365 nm following exposure to peroxynitrite. Under the acidic conditions of this HPLC method, nitrotyrosine-containing proteins would be expected to absorb at 365 nm (10). The absorbance at 365 nm increases with increasing concentrations of peroxynitrite. Since this peak has a 405 nm:365 nm ratio of
