Enzymatic Reduction of 3-Nitrotyrosine Generates Superoxide

Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good. Barry Halliwell , Kaicun Zhao , Matthew Whiteman. Free Radical Research 1999...
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Chem. Res. Toxicol. 1998, 11, 495-502

495

Enzymatic Reduction of 3-Nitrotyrosine Generates Superoxide Arkadi G. Krainev, Todd D. Williams,† and Diana J. Bigelow* Department of Biochemistry and Mass Spectrometry Laboratory, The University of Kansas, Lawrence, Kansas 66045-2106 Received November 4, 1997

Spin-trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) was used to demonstrate that 3-nitrotyrosine (nitrotyrosine) promotes the formation of substantial amounts of reactive oxygen species (O2•- and •OH), when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor. Spin adduct formation is strongly inhibited by the presence of superoxide dismutase (SOD); spin adduct formation requires aerobic conditions. Nitration of leucine enkephalin, a tyrosine-containing pentapeptide, results in a similar generation of O2•and •OH species. Both nitrotyrosine and nitrated leucine enkephalin stimulate acetylated ferricytochrome c reduction in the presence of NAD(H)-cytochrome c reductase with typical Michaelis-Menten kinetics and Km’s of 104 ( 14 and 0.78 ( 0.11 µM, respectively. No stimulation of acetylated ferricytochrome c reduction is observed in the presence of SOD. Catalase and the metal chelators DTPA and deferoxamine mesylate do not influence observed stimulation of acetylated ferricytochrome c reduction by nitrotyrosine. Nitration of two tyrosines (of four) within the sequence of the 6.5-kDa globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2•- generation implying steric restrictions for BPTIreductase interactions. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferricytochrome c. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine may be enzymatically reduced to the corresponding nitro anion radical (ArNO2•-) which is then oxidized by molecular oxygen to yield O2•- and regenerate ArNO2. Thus, once formed in vivo, nitrotyrosine may act to promote oxidative stress by means of repetitive redox cycling.

Introduction Increased levels of nitric oxide (•NO)1 and superoxide anion (O2•-) have been implicated in numerous inflammation-, alcohol-, or age-related disorders (1-3). It has been shown (4, 5) that peroxynitrite (ONOO-), a highly reactive oxidant species, can be formed endogenously by the interaction of •NO and O2•- and reacts readily with tyrosine residues of proteins to form 3-nitrotyrosine (nitrotyrosine). In fact, under physiological conditions, this nitration probably involves an additional intermediate (ONO2CO2-), resulting from the rapid reaction of ONOO- with carbon dioxide (CO2); this intermediate has been shown to react with tyrosine even more effectively than ONOO- itself (6). Tyrosine nitration of manganese * Correspondence should be addressed to Dr. Diana J. Bigelow. Phone: (913)-864-3831. Fax: (913)-864-5321. E-mail: dbigelow@ falcon.cc.ukans.edu. † Mass Spectrometry Laboratory. 1 Abbreviations: ArNO , nitroaromatic compound; BPTI, bovine 2 pancreas trypsin inhibitor; CAD, collision-activated decomposition; DMPO, 2,2-dimethyl-3,4-dihydro-2H-pyrrole 1-oxide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; EPR, electron paramagnetic resonance; ESI, electrospray ionization; HEPES, N-(2hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); HPLC, highperformance liquid chromatography; LDL, low-density lipoprotein; LE, leucine enkephalin; MS, mass spectrometry; NAD(H), nicotinamide adenine dinucleotide, reduced form; nitrotyrosine, 3-nitrotyrosine; NLE, nitrated leucine enkephalin; •NO, nitric oxide; ONOO-, peroxynitrite; O2•-, superoxide anion; ROS, reactive oxygen species; SOD, superoxide dismutase; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNM, tetranitromethane.

superoxide dismutase (SOD) in vivo has been shown to result in the specific loss of SOD activity during chronic rejection of human renal allografts (7); inactivation of a number of different enzymes also has been specifically linked to nitrotyrosine formation (reviewed in ref 8). In addition to inducing changes in local protein charge and/ or structure [the pK for nitrotyrosine is 6.9 (9)], the formation of nitrotyrosine has been demonstrated to prevent tyrosine phosphorylation, thus impairing many physiologically important signal transduction and regulatory pathways (10, 11). Recent progress in methods of immunochemical nitrotyrosine detection has prompted a number of studies linking disease to peroxynitrite production. Thus nitrotyrosine is now widely used as a marker of conditions of oxidative stress (12). For example, formation of nitrotyrosine has been observed due to aging in SERCA2a isoform of the skeletal muscle Ca2+-ATPase (3) and in many other (for review, see ref 7) pathophysiological conditions, including atherosclerosis, reperfusion injury, and amyotropic lateral sclerosis. Moreover, a recent study (13) has shown that some proteins containing nitrotyrosine are promptly degraded by human plasma, implying a specific enzymatic process for the recognition and removal of nitrotyrosine. However, despite the number of reports of nitrotyrosine formation in vivo, few specific protein targets have been identified, and the functional effects of nitrotyrosine in biological systems are not well-understood. To date, only one early report

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496 Chem. Res. Toxicol., Vol. 11, No. 5, 1998 Scheme 1

has been made on this subject which suggests that nitrotyrosine in the presence of Cu2+ and ascorbate promotes oxidative stress in vitro which leads to DNA damage (14). On the other hand, a number of nitroaromatic compounds (ArNO2), used as antibiotics, can be reduced by cellular reductases to the corresponding nitro anion radicals (ArNO2•-); the toxicity of these compounds is based on the ability of the ArNO2•- species to reduce molecular oxygen to O2•- (Scheme 1; for review, see ref 15). An important feature of this mechanism is the continuous regeneration of the nitroaromatic compound in its parent state; thus ArNO2 may act as a catalyst, undergoing numerous redox cycles, each time yielding superoxide radical O2•-. However, the possibility of this redox transformation for nitrotyrosine formed in proteins and its role to further promote oxidative stress under physiological conditions has been overlooked and deserves further study. Here we report that nitrotyrosine promotes the formation of substantial amounts of reactive oxygen species (O2•- and •OH), when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor. Nitration of leucine enkephalin, a tyrosine-containing pentapeptide and neurotransmitter, results in a similar enhancement of O2•- generation. However, the nitration of the tightly packed globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2•- generation under the same conditions. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine, depending on its accessibility in the local environment, may be enzymatically reduced to the corresponding nitro anion radical yielding O2•-, further contributing to oxidative stress in vivo.

Experimental Procedures Caution: Tetranitromethane (TNM) is a hazardous chemical and should be handled carefully; see NIOSH Pocket Guide to Chemical Hazards (DHHS/NIOSH 90-117, 1990, p 210). Chemicals. Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu, Lot 64H58251) and bovine pancreas trypsin inhibitor (T-0256, Lot 104F8035) were obtained from Sigma (St. Louis, MO) and purified by HPLC as described below. HPLC grade organic solvents were from Fisher (Medford, MA). TNM from Aldrich (Milwaukee, WI) was purified immediately before use by extracting four times with a 20-fold excess of deionized water. TPCK-treated trypsin and lima bean trypsin inhibitor were from Worthington Biochemical Corp. (Freehold, NJ). All other enzymes and chemicals of research grade were used as supplied by Sigma. Solutions were prepared using deionized water (WaterPro PS, LABCONCO, Kansas City, MO). Potassium phosphate (10 mM) (pH 7.6) buffer containing 100 mM KCl and treated with Chelex 100 (Bio-Rad; 5 g/100 mL for 2 h) was used throughout unless stated otherwise. Nitration of Tyr Residues. Tyrosine nitration was done as described (16) with the following modifications. Leucine enkephalin (0.1 mg) or BPTI (0.4 mg) in 1 mL of 1 M NaCl, 0.5

Krainev et al. mM CaCl2, and 0.1 M TRIS (pH 7.9) was mixed with 134 µmol of freshly purified TNM [100 µL of 15% (v:v) ethanol stock solution] and incubated at 25 °C for 1 h with gentle mixing. Then the reaction mixture was brought to 100 µL using a LABCONCO centrivap concentrator followed by HPLC separation. The concentration of nitrated tyrosine was measured specrophotometrically in 6 M guanidine chloride, 1 mM EDTA, and 25 mM HEPES (pH 10) using 428 ) 4200 M -1 cm-1 (17). HPLC Purification of Nitrated Products. Purification of leucine enkephalin and BPTI in their native and nitrated forms was done using an ISCO (Lincoln, NE) HPLC setup (2360 gradient programmer, 2350 HPLC pump and V4 UV/vis detector) equipped with a 4.6- × 250-mm ISCO Spherisorb 5-µm ODS-2 C18 column. Protein or peptide was diluted to 200 µL with 0.1% TFA in water (mobile phase A), injected, and eluted at 1 mL/min for 10 min with a linear gradient (0-25%) of mobile phase B [0.1% TFA in 2/2/1 (v:v:v) acetonitrile/2-propanol/water] followed by another linear gradient (25-35% mobile phase B in 20 min) and isocratic elution (35% B for 15 min). Peaks were monitored by absorbance at 285 nm, collected into plastic microcentrifuge tubes, concentrated 10 times with centrivap concentrator (see above), and identified by mass spectrometry. Leucine enkephalin eluted at 15 min, nitrated leucine enkephalin (NLE) at 17.5 min, BPTI at 38 min, and a major nitrated BPTI product at 35 min. Tryptic Digestion of Nitrated BPTI. Fraction of doublenitrated BPTI purified by HPLC as described above was dried on a centrivap concentrator and reconstituted in 50 mM ammonium bicarbonate (pH 7.8) to 0.78 mg/mL. To reduce BPTI’s three disulfide bonds and prevent cysteine residues from being reoxidized, solution was incubated at 60 °C with 3.6 mM dithiothreitol for 40 min followed by addition of 7.2 mM iodoacetamide and another incubation at 25 °C for 1 h. The resulting mixture was mixed with trypsin (at 1/50 ratio, or 16 µg/mL) and maintained at 25 °C. At desired times aliquots were removed, mixed with lima bean trypsin inhibitor (final concentration 8 µg/mL), and subjected to acetylated ferricytochrome c assay. Tryptic fragments were separated by HPLC using an ISCO setup described above with the following modifications. Isocratic elution (flow rate 0.75 mL/min) for 10 min with 100% mobile phase A (0.1% TFA) was followed by a linear gradient (0-95% within 50 min) of mobile phase B [0.1% TFA in 2/1 (v: v) of acetonitrile/water] and isocratic elution (95% B for 20 min). Peaks were monitored at 214 nm and, to identify peptides containing nitrotyrosine, at 360 nm. Nitrotyrosine-containing peptides were collected and analyzed by mass spectrometry. Acetylated Ferricytochrome c Assay for Superoxide. Production of superoxide was assayed at 25 °C by spectrophotometry of acetylated ferricytochrome c (18, 19) using 550 ) 21 000 M -1 cm-1. The extent of acetylation of commercially available horse heart acetylated cytochrome c (C-4186, Lot 87H9156 from Sigma) was 58.7 ( 4.5% as determined by TNBS assay (20). Typical conditions were as follows: 0.02 mg/mL NAD(H)-cytochrome c reductase, 1 mM NAD(H), 20 mM glucose6-phosphate, 0.02 mg/mL glucose-6-phosphate dehydrogenase, and 60 µM cytochrome c in 10 mM potassium phosphate (pH 7.6) buffer containing 100 mM KCl. The reaction was initiated by addition of NAD(H)-cytochrome c reductase, and kinetic traces were recorded using a Beckman DU 7500 instrument (registration 550 nm, reference 700 nm). Reduction rates were obtained from the slope (linear portion from 0.5 to 2 min) of kinetic curves. Essentially the same results were obtained when assay mixture lacked glucose-6-phosphate and glucose-6phosphate dehydrogenase; however, kinetic curves showed a considerably shorter linear portion due to earlier depletion of NAD(H). Mass Spectrometry. Electrospray ionization (ESI) spectra were acquired on an AUTOSPEC-Q (VG Analytical Ltd., Manchester, U.K.) equipped with the Mark III ESI source. This version has the “pepper pot” counter electrode and hexapole transfer optics and was operated at 4 kV. Collected concentrated HPLC peaks were diluted with H2O, and peptides were

Enzymatic Reduction of Nitrotyrosine

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Figure 1. 3-Nitrotyrosine stimulates acetylated ferricytochrome c reduction: addition of free (a) 3-nitrotyrosine or (b) nitrated pentapeptide LE. The incubation mixture contained 0.02 mg/mL NAD(H)-cytochrome c reductase, 0.2 mM NAD(H), 20 mM glucose6-phosphate, 0.02 mg/mL glucose-6-phosphate dehydrogenase, and 50 µM acetylated ferricytochrome c in 10 mM potassium phosphate (pH 7.6) buffer with 100 mM KCl. Various concentrations (0-0.5 mM) of nitro compounds were added in the same amount (10% of total volume) of 1/3 (v:v) acetonitrile/water mixture. Additional measurements were performed in the presence of 1 mM DTPA (2) or 1 mM deferoxamine mesylate (9). Data represent means of 2-3 separate measurements ((SD). Dotted lines represent nonlinear Levenberg-Marquardt least-squares fits to the Michaelis-Menten equation performed using Origin 4.1-32 bit software from Microcal (Northampton, MA). trapped and desalted prior to ESI by washing the peaks onto a 1-mm × 2-cm trapping column filled with Zorbax C18 resin (SBC18, 5 µm, 300 Å; Rockland Technologies, Newport, DE) with 0.1% acetic acid at 250 µL/min. Retained peptides were eluted into the ESI source with 70/30 (v:v) methanol/0.1% acetic acid at 10 µL/min through a 130-µm i.d. stainless steel needle and nebulized with a coaxial N2 gas flow of 10 L/h. Collisionactivated decomposition (CAD) experiments were performed with precursor ions attenuated 50% with argon in the collision quadrupole. The collision energy used was 48 eV (laboratory frame of reference). The analyzer quadrupole was tuned to 1.5 amu full width at half-height. The scan rate was 1 s/100 amu, and 10 scans were integrated during the elution of the peptide peak off the trap. CAD data were collected on about 100 pmol of sample. ESI-MS analysis of the HPLC peaks eluted at 15 and 17.5 min from the nitration of LE provided singly charged ions of m/z 556 and 601, respectively, consistent with native LE and NLE (calculated masses of MH+ ion are 556.3 and 601.3, respectively). The ESI spectrum of unreacted BPTI contained abundant 4, 5, and 6 positively charged ions which, upon charge deconvolution, establish the average mass as 6511.85 ( 0.43. The major nitration product of BPTI produced abundant ions of 3, 4, and 5 positive charges which deconvolute to 6602.12 ( 0.18. The mass difference of 90.3 indicates a double nitration of BPTI. Computer Analysis of Peptide Fragments. Mass search and HPLC index calculations were done using GPMAW software (version 2.1.0) from Lighthouse Data (Odense SV, Denmark). EPR Measurements. Purification of DMPO and spin trapping were done essentially as described earlier (21, 22). Spectral simulations and double integration of EPR spectra were done using software supplied by Bruker-Franzen Analytic GmbH (Bremen, Germany).

Results Reduction of Free Nitrotyrosine. If 3-nitrotyrosine acts similarly to other nitroaromatic compounds as described above (Scheme 1), the generation of superoxide would be expected in the cell where cellular reductases

are abundant. Therefore we monitored O2•- production from nitrotyrosine in the presence of a typical flavincontaining cellular reductive enzyme NAD(H)-cytochrome c reductase in two ways: (1) indirectly, by measuring the SOD-inhibitable reduction (18, 19) of acetylated ferricytochrome c, and (2) directly, by spintrapping with the 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) spin trap (23). For the first assay a model system was used consisting of NAD(H)-cytochrome c reductase; NAD(H), as a reductive cofactor; glucose-6-phosphate with glucose-6-phosphate dehydrogenase, as a means to regenerate NAD(H); and acetylated ferricytochrome c. Reduction of ferricytochrome c can be mediated enzymatically by NAD(H)-cytochrome c reductase as well as nonenzymatically, directly by superoxide (18). Acetylation of 60% of lysine residues of horse heart ferricytochrome c results in the decreased ability to be reduced by reductases but does not affect its ability to be reduced by O2•- (19). Thus the stimulation of the enzymatically mediated reduction of acetylated ferricytochrome c in this model system by nitrotyrosine can be taken as an indication of superoxide generation. Figure 1a shows an increase in the acetylated ferricytochrome c reduction rate observed with increasing nitrotyrosine concentrations. This concentration dependence shows Michaelis-Menten-like behavior suggesting interaction of nitrotyrosine with the active site of NAD(H)-cytochrome c reductase. The apparent Km for nitrotyrosine is equal to 104 ( 14 µM, a value that is typical for the enzymatic reduction of aromatic compounds (24). In control experiments, 250 µM unmodified L-tyrosine does not change the rate of acetylated ferricytochrome c reduction. The presence of the metal chelators DTPA and deferoxamine mesylate does not considerably influence acetylated ferricytochrome c reduction (Figure 1a). We also find that nitrotyrosine does not stimulate the

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Table 1. Influence of Nitrotyrosine Compounds and SOD on Rates of Acetylated Ferricytochrome c Reduction (%)a

control +250 µM nitrotyrosine +7 µM NLE

-SOD

+SOD (100 units)b

100 183 ( 4 191 ( 12

67 ( 6 57 ( 5 61 ( 2

a Model system used was the same as in Figure 1. 100% corresponds to 0.405 ( 0.035 µM hemoprotein reduced/min. b Further addition of SOD did not further inhibit acetylated ferricytochrome c reduction rate, indicating that the SOD content was adequate to inhibit all O2•--dependent reduction.

rate of acetylated ferricytochrome c reduction when all O2•- present in the reaction mixture was scavenged by SOD confirming that the observed stimulation is entirely a result of the generation of O2•- (Table 1). In the absence of nitrotyrosine, the rate of acetylated ferricytochrome c reduction is also decreased by SOD consistent with the presence of an additional pathway for reduction of this hemoprotein through the generation of O2•- from molecular oxygen mediated by NAD(H)-cytochrome c reductase itself. Addition of up to 500 units/mL catalase does not influence the nitrotyrosine-stimulated increase in the rate of acetylated ferricytochrome c reduction (data not shown). As a direct test of the nitrotyrosine-dependent production of superoxide, we performed the following spintrapping experiments. In the presence of DMPO, the same model system (acetylated ferricytochrome c omitted) yields a complex EPR spectrum (Figure 2a). This spectrum can be simulated (Figure 2b) as a composite of two individual spin adduct species, assigned as the hydroxyl radical DMPO/•OH (Figure 2c) and the superoxide radical DMPO/O2•- (Figure 2d) spin adducts on the basis of their hyperfine coupling constants (25). The ratio of DMPO/O2•--to-DMPO/•OH adduct formation, estimated from double integration of the corresponding spectra, is about 2:1 at 500 µM nitrotyrosine (Figure 3a). Taking into account the relative rate constants for the formation of DMPO/O2•- [10 M-1 s-1 at pH 7.8, 25 °C (26)] and DMPO/•OH [near-diffusionlimited rate constant of 2 × 109 M-1 s-1 under the same conditions (26)] and assuming similar decay rates of these spin adducts [equal to 1 nmol/min(mg of protein) (27)], a steady-state concentration of O2•- can be estimated as 50 pM, which is 4 × 108 times greater than that of •OH (1.25 × 10-19 M). This ratio of [O2•-]/[•OH] is likely to be an underestimate since this estimation does not take into account the two possible pathways described below that increase the DMPO/•OH content at the expense of DMPO/ O2•-. Thus, superoxide can be identified as a primary free-radical species in this model system. The •OH spin adduct detected probably originates from O2•- as a result of (i) superoxide’s ability to spontaneously dismutate into hydrogen peroxide to produce •OH in the presence of even trace amounts of transition metals via the Fenton reaction and (ii) the ability of DMPO/O2•- spin adducts to convert into DMPO/•OH under similar experimental conditions (28, 29). The following control experiments help clarify the mechanism of DMPO/•OH adduct formation. In agreement with the results from the acetylated ferricytochrome c assay, the production of O2•- in spintrapping experiments also depends on the concentration of nitrotyrosine (Figure 3a). In the absence of nitroty-

Figure 2. EPR spectra of DMPO spin adducts observed (a) in a model system containing 5 mg/mL NAD(H)-cytochrome c reductase, 1 mM NAD(H), 100 mM glucose-6-phosphate, 1 mg/ mL glucose-6-phosphate dehydrogenase, and 100 mM DMPO in 10 mM potassium phosphate (pH 7.6) buffer with 100 mM KCl; 500 µM nitrotyrosine was added in a small amount (10% of the total volume of 40 µL) of 1/3 (v:v) acetonitrile/water mixture. Spectrometer settings were as follows: gain 2 × 105, modulation amplitude 1.0 G; 50 scans over 20 min at room temperature were accumulated. Simulated spectra: composite spectrum (b) of DMPO/•OH (c) and DMPO/O2•- (d). Simulation parameters: (c) aN ) 14.84 G, aH ) 14.88 G, superhyperfine splittings as in ref 22, peak-to-peak line width 0.8 G; (d) aN ) 14.1 G, aH ) 11.3 G, aHγ ) 1.32 G, peak-to-peak line width 1.4 G; relative contribution of Lorenzian and Gaussian line shape was one-to-two in both spectra c and d.

rosine we observe a considerable decrease in the intensity of the EPR spectrum (compare Figure 4a, 4b), thus confirming the involvement of nitrotyrosine in the production of reactive oxygen species (ROS). The remaining spectral signal is also comprised of DMPO/O2•- and DMPO/•OH species (at 25% and 50% levels, respectively, as compared with the complete system in the presence of 500 µM nitrotyrosine), most likely due to reductasemediated O2•- production. The addition of SOD to the complete system also decreases spin adduct formation (Figure 4c); DMPO/O2•- spin adduct formation is completely abolished, but the formation of some DMPO/•OH spin adduct is always observed. To find the origin of the DMPO/•OH spin adduct, the •OH f •CH conversion test was performed: if 10 vol % 3 DMSO was present in the complete reaction system, the distinct six-line DMPO/•CH3 spectrum [aN ) 16.1 G, aH ) 23.0 G (25)] appeared concomitant with the profound decrease in the intensity of the DMPO/•OH spectrum (Figure 4d). At such DMSO concentrations the majority of •OH species generated in the bulk solution are trapped by DMSO accompanied by release of •CH3 radicals (21). This control experiment shows that •OH species are indeed formed in our model system. However, the formation of some DMPO/•OH spin adduct is still observed, suggesting that DMPO/O2•- f DMPO/•OH conversion may also take place. Finally, no spin adduct formation is observed under anaerobic conditions (Figure 4e), confirming that the formation of all ROS observed in these experiments requires molecular oxygen.

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Figure 3. Dependence of DMPO/•OH (b) and DMPO/O2•- (0) spin adduct concentrations on the concentration of (a) 3-nitrotyrosine and (b) nitrated LE. Experimental conditions were the same as in Figure 2. EPR spectra were deconvoluted (see Figure 2), and individual spectra of DMPO/•OH and DMPO/O2•- were double-integrated. To calculate concentrations of spin adducts, double integrals were compared to values obtained with 4-hydroxy-TEMPO standard. Data presented are means of 2-3 independent determinations ((SD).

Figure 4. EPR spectra of DMPO spin adducts observed in the same model system as described in Figure 2 and the effect of additions/deletions: complete system (a), nitrotyrosine omitted (b), 50 units of SOD added (c), in the presence of 10 vol % DMSO (d), and in anaerobic conditions (e). Instrument settings were the same as in Figure 2. Anaerobic conditions (e) were developed enzymatically by the addition of 10 mg/mL glucose, 1 mg/mL glucose oxidase, and 0.1 mg/mL catalase.

Enzymatic Reduction of Peptide- or ProteinBound Nitrotyrosine. We have extended these studies to ask if reductive enzymes are also capable of reducing nitrotyrosine within a peptide or a protein. The in vitro nitration of tyrosine in a number of proteins employing tetranitromethane (TNM) has been previously reported (for review, see ref 30). We have adopted this procedure for the pentapeptide leucine enkephalin (Tyr-Gly-GlyPhe-Leu), which contains a single tyrosine, and for the 6.5-kDa globular bovine pancreatic trypsin inhibitor protein (BPTI), which contains four Tyr residues. In the case of leucine enkephalin, reaction with TNM results

in nitrated peptide with 95% yield. This nitrated leucine enkephalin (NLE) shows an absorbance spectrum having a λmax at 420 nm (pH 10), which is characteristic of nitrotyrosine (data not shown). The specificity of nitration to the tyrosine residue of LE was confirmed by CAD-MS analysis of the peptides. The CAD spectrum of LE for this study is virtually identical to that obtained on a similar instrument after FAB ionization (31). The CAD spectrum of LE is rich in sequence-specific ions including full series that demonstrate the sequence from both the N- and C-termini: A, B, and Y ions (32), respectively. The CAD spectrum from m/z 601 is readily rationalized as (nitro-Tyr-Gly-Gly-PheLeu) because all N-terminal-contaning ions (A and B ions) have been shifted by 45 amu and all C-terminal ions (Y + 2H) are detected at the same mass as for native LE. There was no evidence of nitration on Phe in the CAD spectum from m/z 601, which would be isobaric with NLE but would result in mass shifted for Phe-containing fragments. In the acetylated ferricytochrome c assay, the rate of hemoprotein reduction shows essentially the same Michaelis-Menten-like dependence on NLE concentration as is observed for nitrotyrosine, and with the same extent of rate stimulation (Figure 1b). However, nitrated tyrosine within the leucine enkephalin peptide shows a higher affinity for enzymatic reduction (with a Km of 0.78 ( 0.11 µM). As with nitrotyrosine, in control experiments native leucine enkephalin does not change the rate of cytochrome c reduction, and SOD also abolishes the NLEinduced stimulation of O2•- production (Table 1). In spin-trapping experiments, the same model system used previously but containing NLE also yields composite spectra originating from both DMPO/•OH and DMPO/ O2•- spin adducts. Nitrated leucine enkephalin exhibits similar properties to nitrotyrosine, e.g., formation of spin adducts: (i) decreased considerably in the presence of SOD (similar decrease to 25% of saturating DMPO/O2•level and to 50% of saturating DMPO/•OH level), (ii)

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requires molecular oxygen, and (iii) shows a dependence on NLE concentrations (Figure 3b). In agreement with results from the acetylated ferricytochrome c reduction assay shown above (Figure 1 and Table 1), the spintrapping assay also demonstrates that NLE has a higher affinity for enzymatic reduction than does nitrotyrosine (compare Figure 3a,b). However, the decreased levels of spin adducts observed with NLE as compared to nitrotyrosine are probably due to inhibition of cytochrome c reductase by trifluoroacetate present in the NLE preparation after HPLC purification. In contrast the saturating levels in the hemoprotein reduction rates (Figure 1) are not different for nitrotyrosine compared with NLE which might be explained by the fact that the spin-trapping assay lasts about 10 times longer than the acetylated ferricytochrome c assay, thus increasing the probability of trifluoroacetate inhibition of the reductase. Indeed, when the spin-trapping assay was run for only 3 min the same amplitudes of EPR spectra were observed in the presence of nitrotyrosine and NLE, although in this case the low signal-to-noise ratio obstructed the qualitative analysis of [DMPO/O2•-]/ [DMPO/•OH] ratios. Nevertheless, the spin-trapping experiment confirms the NLE-dependent formation of ROS in this model system and, based on the ratio of [DMPO/O2•-]/[DMPO/•OH] (about 1.5 at 60 µM NLE, Figure 3b), again identifies superoxide as a primary freeradical species. We have examined BPTI as a protein model for these experiments because it contains four tyrosine residues and its three-dimensional organization is well-described from both solution and crystal structures (33). Two tyrosine residues (Tyr-23 and Tyr-35) are buried inside the BPTI globule and are largely involved in intramolecular hydrogen bond formation, while side chains of Tyr-10 and Tyr-21 residues are solvent-exposed and participate in intermolecular interactions. The BPTI molecule is tightly packed; only four water molecules are included in two interior cavities, and the bigger cavity containing three water molecules is situated close to Tyr10. Therefore, only two of the four tyrosine residues of BPTI (Tyr-10 and Tyr-21) are good candidates for nitration by TNM. Nitration of BPTI with TNM confirms the prediction that not all of the four tyrosine residues are equally accessible. The major product of BPTI nitration is comprised of doubly nitrated BPTI (identified by MS as described in Experimental Procedures); its yield is estimated to be about 70% of the initial BPTI based on the absorbance of nitrotyrosine. We therefore subjected the doubly nitrated BPTI to exhaustive (24 h at 37 °C) tryptic digestion in order to identify the sites of nitration. Only two peptide fractions obtained from doubly nitrated BPTI show absorbance at 360 nm when separated by HPLC, i.e., those that elute at 38.5 and 69 min. The former fraction analyzed by ESI-MS exhibits abundant triply and doubly charged ions (at 411 and 617 amu) as indicated by isotopomer separation of 0.33 and 0.5, respectively, and corresponds to a mass of 1232.04 ( 0.02. This mass is consistent with the singly nitrated incomplete tryptic fragment Ile18-Ile-Arg-Tyr-Phe-Tyr-Asn-AlaLys26 within the BPTI sequence (calculated mass of 1231.65). Single nitration of this fragment is consistent with the solvent exposure of the Tyr-21 residue. Although the second fraction did not provide reliable MS data, the elution time of this fraction is consistent with

Krainev et al.

Figure 5. Stimulation of acetylated ferricytochrome c reduction by tryptic digest of nitrated BPTI. Nitrated BPTI was incubated with trypsin as described in Experimental Procedures; aliquots containing 32 µg of protein were taken at times shown in the plot, mixed with trypsin inhibitor, and subjected to acetylated ferricytochrome c reduction assay as described in Figure 1.

the high HPLC index calculated (see Experimental Procedures) for the N-terminal BPTI fragment containing Tyr-10. In contrast to free nitrotyrosine and NLE, nitrated BPTI does not stimulate superoxide production (using up to 40 µM nitrated BPTI, i.e., 80 µM in nitrotyrosine concentration) as assayed by both acetylated ferricytochrome c and spin-trapping assays. This observation suggests that the nitrated tyrosine side chains on the bulky BPTI molecule may not be accessible to the active site of the NAD(H)-cytochrome c reductase. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferricytochrome c in a timedependent manner (Figure 5). This in turn may be explained by the trypsin-induced release of BPTI peptides containing nitrotyrosine in a conformation that is compatible with the flavoenzyme active site.

Discussion Summary of Results. These in vitro experiments that model biological systems containing reductive flavoenzymes indicate that free nitrotyrosine as well as nitrotyrosine within the sequence of a peptide such as leucine enkephalin may induce production of ROS, such as O2•- and •OH. Spin-trapping experiments indicate the substantial formation of both O2•- and •OH; based on the known rate constants for their spin adduct formation, it can be concluded that superoxide is the primary radical species produced. In agreement with an earlier proposed mechanism (15), we suggest that O2•- production involves (Scheme 1) enzymatic reduction of the nitrotyrosine molecule to the corresponding nitro anion radical (ArNO2•-) which is then oxidized by molecular oxygen to yield O2•- with the concomitant regeneration of the nitrotyrosine. Thus, the present study suggests that, once formed in vivo, nitrotyrosine may act to promote oxidative stress by means of repetitive redox cycling in the presence of reductive flavoenzymes. Enzymatic Reduction of Nitrotyrosine. A number of nitroaromatic compounds with a wide range of one-

Enzymatic Reduction of Nitrotyrosine

electron reduction potentials (from -0.257 to -0.486 V) have been reported to be reduced by flavoenzymes in the presence of corresponding electron donors (15). The reduction rates of these compounds depend on the ArNO2 concentration and can be described by Michaelis-Menten kinetics. In the present work, we have examined the enzymatic reduction of the nitroaromatic compound nitrotyrosine in both its free and peptide-bound form. Based on the one-electron reduction potential of nitrotyrosine [-0.362 V for o-nitrophenol (34)] which is well within the range of other flavoenzyme-reducible compounds, the possibility of enzymatic reduction is expected; the following considerations support this model. First, the acetylated ferricytochrome c assay employed here shows a Michaelis-Menten-like dependence of the hemoprotein reduction rate on nitrotyrosine and NLE concentrations (Figure 1), implying reduction of these compounds at the NAD(H)-cytochrome c reductase active site. Second, the Km values obtained for nitrotyrosine and NLE are typical for the enzymatic reduction of aromatic molecules (24). And, finally, the MichaelisMenten-like dependence of O2•- and •OH production on nitrotyrosine concentration is also confirmed in spintrapping experiments (Figure 3). The appearance of DMPO/•OH spin adducts during this enzymatic reduction is consistent with both (1) a previous study showing that flavoenzyme-catalyzed reduction of molecular oxygen to O2•- radicals is accompanied by the abundant formation of •OH species in the presence of transition metals (35) and (2) the ability of DMPO/O2•to convert spontaneously into DMPO/•OH spin adducts (28, 29). The persistence of DMPO/•OH spin adducts in the presence of SOD (Figure 4c) suggests a local environment that is inaccessible to the large SOD protein but accessible to DMPO and where transition metals are trapped which facilitate the formation of superoxidederived •OH species. As follows from the crystal structure data, a number of flavin-dependent reductases are comprised of a flavin-binding and a pyridine nucleotidebinding domain separated by the cleft filled with flavin (36). Dimensions of this cleft are consistent with the above hypothesis. Similar localized production of •OH species catalyzed by surface-chelated metal ions has been previously observed in free-radical chemistry (reviewed in refs 37 and 38). Physiological Significance. Increased levels of nitric oxide have been implicated in numerous inflammatory processes. Besides its function as a neurotransmitter and a modulator of many cellular regulatory mechanisms, •NO possesses a high chemical reactivity, which results in the increased level of in vivo tyrosine nitration (7, 8). For example, increased levels of free nitrotyrosine have been found in blood serum and synovial fluid from patients with inflammatory rheumatoid arthritis (39), where in the presence of abundant soluble and membrane-bound blood oxidoreductases (40) nitrotyrosine may potentially add to oxidative damage. Specifically oxidized low-density lipoprotein (LDL) particles, containing elevated levels of nitrotyrosine, have been found in atherosclerosis patients (41) suggesting that the levels of free nitrotyrosine may be significant in the development of LDL oxidation-mediated pathologic conditions. However, our results with nitrated BPTI indicate that the accessibility to reductive flavoenzymes may be a key issue for nitrotyrosine within the protein structure. Lack

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of O2•- generation by nitrated BPTI can be rationalized by the bulkiness of BPTI which probably does not allow even surface Tyr residues to enter the reductase active site. Thus BPTI may be an extreme case of nitrotyrosine residues within proteins; results obtained with the NLE pentapeptide (Figure 1) and the tryptic digest of nitrated BPTI (Figure 5) suggest that some proteins bearing nitrated Tyr residues within a more unstructured domain or peptides resulting from proteolytic degradation of nitrated proteins may similarly result in the generation of ROS in vivo. In addition, NLE exhibits a higher affinity than free nitrotyrosine to enzymatic reduction yielding O2•- (Figure 1), suggesting that this important neurotransmitter peptide (42) is highly susceptible to reduction and may further interfere with normal physiological functions by promoting conditions of oxidative stress in the brain.

Conclusion Many nitrophenol compounds homologous to nitrotyrosine have been shown to be reduced by flavoenzymes to the corresponding ArNO2•- species (43, 44). The current work shows that nitrotyrosine in both the free and peptide-bound state is also subject to enzymatic reduction according to the previously proposed (15) mechanism (Scheme 1). Thus, nitrotyrosine formed in living cells as a result of increased •NO production also may exert additional toxicological effects by both (i) producing ROS species by the above-mentioned mechanism and (ii) facilitating the further formation of ONOOreactive species from •NO by increasing supply of O2•-. Further studies on this topic will contribute to a better mechanistic understanding of nitrotyrosine-related oxidative stress and provide clues to its prevention.

Acknowledgment. This work was supported by the National Institute of Aging (RO1 AG12275 and PO1 AG12993). The EPR spectrometer was purchased with an NSF instrument grant, BIR 9214315.

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