Inactivation of NADP+-dependent Isocitrate Dehydrogenase by Peroxynitrite

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 μm SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.

The in vivo formation of this compound has recently been demonstrated in endothelial cells, Kupffer cells, neutrophils, neurons, macrophages, and other cellular systems (5)(6)(7)(8)(9). Peroxynitrite is a relatively stable species, but its protonated form decays with a rate constant of 1.3 s Ϫ1 at 25°C (10). Peroxynitrite reacts with a diverse array of other biological target molecules, including cysteine, tyrosine, methionine, and tryptophan residues of proteins (11)(12)(13). Peroxynitrite has been demonstrated to readily oxidize or nitrate various enzymes such as metalloproteinase-1 inhibitor, alcohol dehydrogenase, aconitase, xanthine oxidase, cytosolic glyceraldehyde-3-phosphate dehydrogenase, glutamine synthetase, creatine kinase, and succinate dehydrogenase (14 -21). Antioxidant enzymes, which provide a substantial defense against damage induced by reactive oxygen species (ROS), could be susceptible to the damaging effect of peroxynitrite. It has been shown that glutathione peroxidase, manganese SOD (Mn-SOD), and glutathione reductase are inactivated by peroxynitrite (22)(23)(24). It is implied that the inactivation of antioxidant enzymes by peroxynitrite may lead to the perturbation of the cellular antioxidant defense system and subsequently exacerbate the harmful effect of peroxynitrite as well as ROS. The isocitrate dehydrogenases (ICDHs, EC1.1.1.41 and EC1. 1.1.42) catalyze oxidative decarboxylation of isocitrate to ␣-ketoglutarate and require either NAD ϩ or NADP ϩ , producing NADH and NADPH, respectively (25). NADPH is an essential reducing equivalent for the regeneration of reduced glutathione (GSH) by glutathione reductase and for the activity of NADPHdependent thioredoxin system (26,27), both are important in the protection of cells from oxidative damage. Therefore, ICDH may play an antioxidant role during oxidative stress. We recently reported that ICDH is involved in the supply of NADPH needed for GSH production against cytosolic and mitochondrial oxidative damage (28,29). Hence, the damage of ICDH may result in the perturbation of the balance between oxidants and antioxidants and subsequently lead to a pro-oxidant condition.
Because cysteine residues serve as an essential role in the catalytic function of ICDH (30,31), the highly reactive sulfhydryl groups in ICDH could be potential targets of peroxynitrite. In this study we show that peroxynitrite modifies tyrosine and thiol groups of ICDH, thus forming nitrotyrosine and S-nitrosothiol adducts with a concomitant loss of its activity in vitro and in vivo.
Cell Culture-U937, a human histiocytic lymphoma cell line was purchased from the American Type Culture Collection and maintained in RPMI 1640 and Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate, respectively. These cells were incubated in a humidified atmosphere of 5% CO 2 and 95% air at 37°C.
Animals and Treatment-Young adult male Sprague-Dawley rats weighing 200 -250 g were randomly separated into two groups: (a) rats fed ethanol-containing liquid diets for 30 days (n ϭ 5); (b) rats pair-fed isocaloric liquid diets without ethanol for 30 days (n ϭ 5). They were housed in individual cages under conditions of constant temperature (22°C) and humidity. The calorie distribution of liquid diets component is as follows: 16% as protein, 36% as fat, 13% as carbohydrate, and 33% as either ethanol or additional carbohydrate in the isocaloric liquid diets. Ethanol was incorporated into the liquid diets containing all required nutrients, and liquid diets were the only source of fluid and food provided, as recommended by the manufacturer. Rats given isocaloric liquid diets without ethanol were pair-fed daily on an isoenergetic basis with the corresponding littermates fed the ethanol-containing diets. Before sacrifice, the rats were fasted overnight but had free access to drinking water for 14 -16 h.
Preparation of Liver Mitochondrial Fraction-Rats were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (1 mg/kg body weight), and the liver was removed and dissected for biochemical analysis. For the preparation of mitochondria sample from liver tissue, the tissue portions were homogenized with a Dounce homogenizer in sucrose buffer (0.32 M sucrose, 10 mM Tris-Cl, pH 7.4). The mitochondrial fractions from tissue homogenates were prepared as described below.
Measurement of ICDH Activity-ICDH (6.5 g) was added to 1 ml of Tris buffer, pH 7.4, containing NADP ϩ (2 mM), MgCl 2 (2 mM), and isocitrate (5 mM). Activity of ICDH was measured by the production of NADPH at 340 nm at 25°C (34). One unit of ICDH activity is defined as the amount of enzyme catalyzing the production of 1 mol of NADPH/min. For the determination of ICDH activities in mammalian cells, cells were collected at 1,000 ϫ g for 10 min at 4°C and were washed once with cold PBS. Briefly, cells were homogenized with a Dounce homogenizer in sucrose buffer (0.32 M sucrose, 10 mM Tris-Cl, pH 7.4). Cell homogenates were centrifuged at 1,000 ϫ g for 5 min, and the supernatants further centrifuged at 15,000 ϫ g for 30 min. The resulting supernatants were used as a cytosolic fraction to measure the activity of cytosolic ICDH. The precipitates were washed twice with sucrose buffer to collect mitochondria pellet. The mitochondrial pellets were resuspended in 1ϫ PBS containing 0.1% Triton X-100, disrupted by ultrasonication (4710 Series, Cole-Palmer, Chicago, IL) twice at 40% of maximum setting for 10 s, and centrifuged at 15,000 ϫ g for 30 min. The supernatants were used to measure the activity of mitochondrial ICDH. The protein levels were determined by the method of Bradford using reagents purchased from Bio-Rad.
Reversibility-ICDH was exposed to peroxynitrite or SIN-1 and then passed through Econo-pac 10 DG gel filtration column (Bio-Rad). The enzyme was incubated with 20 mM DTT at 37°C. The percentage of ICDH activity recovered after inactivation was calculated.
Titration of Sulfhydryl Groups and Competitive Labeling-The thiol groups of ICDH were titrated in 50 mM Tris, pH 8.0/0.5, mM EDTA/1 mM DTNB. A molar coefficient of 1.36 ϫ 10 4 M Ϫ1 cm Ϫ1 for the anion thionitrobenzoic acid was used. Peroxynitrite-, SIN-1-treated, and untreated ICDH (50 g) were labeled with 10-fold molar excess of IANBD (150 M) in a total volume of 100 l of buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCl). The reaction was allowed to proceed for 1 h at room temperature in the dark and was quenched by addition of 1 mM cysteine. Cysteine-reacted dye was removed by extensive dialysis against labeling buffer. IANBD-labeled samples were excited at 481 nm, and emission was monitored between 490 and 625 nm. Each recorded spectrum was corrected for background fluorescence of the relevant control.
Immunoblot Analysis-Proteins were separated on 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and subsequently subjected to immunoblot analysis using appropriate antibodies. Immunoreactive antigen was then recognized by using horseradish peroxidase-labeled anti-rabbit IgG and an enhanced chemiluminescence detection kit (Amersham Biosciences).
Immunoprecipitation-Mitochondrial fractions were cleared with Protein-A-Sepharose (Amersham Biosciences) for 1 h at 4°C. Supernatants were then incubated with rabbit polyclonal anti-mitochondrial ICDH (5 g) for 12 h at 4°C followed by protein-A-Sepharose incubation for 1 h at 4°C. Immunoprecipitated proteins were washed, separated by SDS-PAGE, and visualized by staining with Coomassie Blue. For mass spectrometry, the immunoprecipitated proteins were washed and dissolved in 20 l of 0.5% trifluoroacetic acid (v/v).
Determination of Nitrotyrosine-Nitrotyrosine content of ICDH treated with peroxynitrite or SIN-1 was continually monitored from 250 to 500 nm using a Shimadzu UV-visible spectrophotometer. Nitrotyrosine-containing proteins were detected by incubating blots with a mouse monoclonal anti-nitrotyrosine antibody as described above.
Mass Spectrometry-Positive ion electrospray ionization mass spectrometry (ESI-MS) was performed on HP 1100 Series LC/MSD triplequadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA) equipped with an atmospheric pressure ion source. Control and peroxynitrite (10 M, 10 min)-treated ICDH samples were subjected to gel filtration and mixed with 0.1% trifluoroacetic acid. Aliquots of ICDH samples (5 g of protein) were directly infused into the ESI source of the mass spectrometer. For the tryptic digestion and peptide mapping, ICDH samples treated with peroxynitrite were denatured in the 95°C water for 20 min and cleaved with trypsin for 24 h at 37°C, at an enzyme/substrate ratio of 1/10 (w/w). Positive ion mass spectra were acquired for capillary LC/MS analyses. The effluent from octadecyl silica gel (C-18) reverse-phase column (5 mm; 4.6 ϫ 250 mm) (Beckman Coulter Inc., Fullerton, CA) was introduced directly into the ionization needle of the mass spectrometer. Solvent A was 0.1% trifluoroacetic acid in ultra pure water, and solvent B was 0.1% trifluoroacetic acid in acetonitrile. Peptides were eluted using an increasing linear gradient of solvent B from 0 -60% in 60 min, 60 -100% in 40 min, with a flow rate of 1 ml/min. The molecular masses of fractionated peptide fragments were examined by ESI-MS, and the amino acid sequences were assigned using the data obtained.
Structural Analysis-For circular dichroism (CD) spectroscopy, samples of ICDH were desalted on Econo-Pac 10 DG column (Bio-Rad) equilibrated in 20 mM Tris buffer, pH 7.4, and fractions containing the protein were pooled. CD spectra were recorded on a temperature-controlled spectropolarimeter (JASCO, J-810). Spectra were recorded at 25°C in 0.05-cm quartz cells from 190 to 250 nm with protein concentrations of 0.05 mg/ml at a digital resolution of 0.5 nm, with scan speed of 5 nm/min for wavelength above and below 190 nm, respectively. Multiple spectra were recorded for duplicated samples. These spectra were averaged and corrected for baseline contribution from the buffer. Susceptibility to proteolysis was measured by the incubation of ICDH samples (65 g) with 12.5 g of Pronase in 250 l of 25 mM Hepes (pH 8.0)/100 mM NaCl for 1 h at 37°C. After incubation, the aliquots were removed and subjected to a 10% trichloroacetic acid treatment. After centrifugation of precipitated proteins for 10 min at maximum speed in an Eppendorf microcentrifuge, the supernatant was first neutralized with a predetermined volume of 2 M potassium borate, pH 10. The amount of small peptides in the supernatant was then determined as described by Church et al. (35). ANSA (100 M) was incubated with the various forms of ICDH in 25 mM potassium phosphate buffer, pH 7.0/50 mM KCl. The fluorescence emission spectra (excitation, 370 nm) of the different mixtures were monitored on spectrofluorometer. Binding of ANSA to the protein was evidenced by subtracting the emission spectrum of ANSA from that of ANSA in the presence of enzyme.
Measurement of Intracellular ROS-Intracellular peroxide production was measured using the oxidant-sensitive fluorescent probe DCFH-DA with confocal microscopy (39). Cells were grown at 2 ϫ 10 6 cells per 100-mm plate containing slide glass coated with poly-L-lysine and maintained in the growth medium for 24 h. Cells were exposed to 10 M DCFH-DA for 15 min and treated with 100 M SIN-1 for 5 min. Cells on the slide glass were washed with PBS, and a coverglass was put on the slide glass. 2Ј7Ј-Dichlorofluorescein (DCF) fluorescence (excitation, 488 nm; emission, 520 nm) was imaged on a laser confocal scanning microscope (DM/R-TCS, Leica) coupled to a microscope (Leitz DM REB). Hydrogen peroxide oxidizes ferrous (Fe 2ϩ ) to ferric ion (Fe 3ϩ ) selectively in dilute acid, and the resulting ferric ions can be determined using a ferric sensitive dye, xylenol orange, as an indirect measure of hydrogen peroxide concentration. Mitochondrial fractions were added to FOX solution (0.1 mM xylenol orange, 0.25 mM ammonium ferrous sulfate, 100 mM sorbitol, and 25 mM H 2 SO 4 ) and incubated in a room temperature for 30 min, and absorbance was measured at 560 nm. Hydrogen peroxide was used to draw standard curve as described (40).
Measurements of O 2 . /NO Formation and SOD/iNOS Activity-Superoxide was measured using lucigenin-enhanced chemiluminescence (41). Nitrite, a stable product of NO oxidation, was determined by the Griess reaction (42). The activity of iNOS was determined by the rate of conversion of [L-3 H]arginine to [L-3 H]citrulline as described (43). The iNOS protein was assessed immunochemically in Western blots by using polyclonal anti-iNOS antibodies. SOD activity was assayed spectrophotometrically using a pyrogallol assay (44).
Replicates-Unless otherwise indicated, each result described in the paper is representative of at least three separate experiments.

Inactivation of ICDH by
Peroxynitrite-Incubation of mitochondrial ICDH with synthesized peroxynitrite or SIN-1 at pH 7.4 at 37°C resulted in a time-and concentration-dependent loss of enzyme activity as shown in Fig. 1 (A and B). Previously decomposed peroxynitrite does not inactivate the enzyme (data not shown), indicating that the decomposition products of peroxynitrite are not responsible for the inactivation of enzyme.  SIN-1 is a nitric oxide and superoxide anion donor and thus considered a peroxynitrite releasing compound (45). Under the conditions chosen, incubation with 0.1 mM peroxynitrite or 0.1 mM SIN-1 resulted in ϳ90 or 65% inhibition. The mouse liver cytosolic ICDH was expressed as a fusion protein to GST, and it was purified as a recombinant protein, which was almost pure as estimated by SDS-PAGE (data not shown). The inhibition of recombinant cytosolic ICDH by peroxynitrite or SIN-1 demonstrated basically the same pattern, as shown in Fig. 1C.
We evaluated whether the presence of product would protect the active site of ICDH from peroxynitrite-and SIN-1-mediated inactivation. The inactivation of enzyme by peroxynitrite or SIN-1 was partially prevented in the presence of NADPH in a dose-dependent manner as shown in Fig. 1D. This observation strongly suggests that the inactivation with peroxynitrite or SIN-1 takes place in the vicinity of the active site of ICDH. To gain insight into the mechanism by which peroxynitrite or SIN-1 inactivates ICDH, the protective effect of several molecules that react with either peroxynitrite or secondary oxidants with hydroxyl radical-like reactivity from peroxynitrite decomposition was evaluated. Table I shows that hydroxyl radical scavengers such as mannitol, benzoate, and Me 2 SO did not protect ICDH from inactivation, suggesting that hydroxyl radicals have little effect, if any, on the inactivation of enzyme. On the other hand, molecules known to inhibit peroxynitrite-dependent oxidation reactions such as DTT, GSH, cysteine, 2-mercaptoethanol, penicillamine, methionine, selenomethionine, selenocysteine, ebselen, and human serum albumin afforded significant protection, indicating that the direct reactions of the anion were more relevant in inactivating the enzyme. We also investigated the protective action of SOD against oxidative damage brought about by peroxynitrite and SIN-1. SOD did not protect ICDH from inactivation induced by peroxynitrite. When SIN-1 is used as a peroxynitrite donor, inactivation of ICDH was not pronounced in the presence of SOD. This result supports the proposal that a loss of ICDH activity under the conditions employed here occurred mainly due to the oxidation by peroxynitrite, but not by nitric oxide.
Identification of Modified Residues-To test the reversibility of ICDH modification, ICDH was incubated with 100 M per-oxynitrite or 100 M SIN-1 at 37°C until the remaining enzyme activity reached to ϳ45%. When both peroxynitrite-and SIN-1-treated ICDH were incubated with 20 mM DTT, a portion of the enzyme activity was restored in a time-dependent manner ( Fig. 2A), which is consistent with a mechanism involving the modification of cysteine residues in ICDH. We also performed competition experiments with DTNB and a cysteine-reactive fluorescent probe IANBD to support the proposal that cysteine residues are the major targets for the modification of ICDH by peroxynitrite. When ICDH was allowed to react simultaneously with various concentrations of peroxynitrite or SIN-1 and DTNB or IANBD, the dose-dependent depletion of DTNB-accessible thiols (data not shown) and the dose-dependent decrease in the IANBD fluorescence were observed. The representative spectra of IANBD fluorescence of peroxynitritetreated ICDH are shown in Fig. 2B. Immunoblot analysis of peroxynitrite-and SIN-1-treated ICDH using anti-nitrosocysteine antibody revealed that the dose-dependent increase of S-nitrosocysteine, confirming the involvement of S-nitrososylation of cysteine residues in ICDH (Fig. 2C). In previous studies, peroxynitrite has been shown to modify tyrosine residues on a variety of proteins. 3-Nitrotyrosine has been used as a biomarker of nitrative pathology cause by peroxynitrite (46). The nitrotyrosine content of ICDH as a function of added peroxynitrite or SIN-1 was determined using a spectrophotometric assay based on its pH-dependent absorbance and immunoblot analysis with anti-nitrotyrosine antibody. After a reaction with peroxynitrite or SIN-1, the spectrum of ICDH at pH 12 exhibited a new absorption peak at 428 nm that is characteristic of nitrotyrosine, whereas the spectrum of ICDH was unaffected without the treatment of peroxynitrite (Fig. 3A) or SIN-1 (Fig.  3B). This finding indicates that the tyrosine residue of the enzyme can be nitrated by peroxynitrite or SIN-1. Western blot analysis using the anti-nitrotyrosine antibody indicated that treating ICDH with peroxynitrite resulted in a dose-dependent formation of nitrotyrosine (Fig. 3C). To further confirm whether peroxynitrite modified the cysteine and tyrosine residues, ICDH was treated with 10 M peroxynitrite for 10 min at 37°C, and subjected to ESI-MS. Molecular masses of unmodified and peroxynitrite-modified ICDH samples were 47,785 and 47,888 Da, respectively (Fig. 4A). The increased value corresponds to the incorporation of two NO molecules (58 Da) and one NO 2 molecule (45 Da) to the parental protein. This result strongly suggests that two cysteine residues and one tyrosine residue of the enzyme are targets of modification by peroxynitrite. When the peroxynitrite-inactivated ICDH was treated with 20 mM DTT to restore the original activity and was subjected to ESI-MS, the molecular mass of the ICDH sample was 47,830 Da (47,785 ϩ 45 Da), indicating that the nitrotyrosine residue was not reversible with DTT (Fig. 4B). The results indicate that nitration of tyrosine does not appear to play a critical role in ICDH inactivation by peroxynitrite. To localize the modification sites, untreated and peroxynitrite-treated ICDH samples were digested with trypsin and then fractionated by reverse-phase HPLC using a C-18 column. When the reverse-phase HPLC elution profile of peptides generated by tryptic digestion of peroxynitrite-treated ICDH was compared with that of native ICDH, two new peaks (peaks 1 and 2) were observed (Fig. 4C). The observed molecular weights obtained by ESI-MS of these fractions allowed the complete identification of modification sites in ICDH. The peptide eluting at 21 min (peak 1) had a molecular mass of 3,238 Da, corresponding Structural Changes in Modified ICDH-To examine the secondary structure of the ICDH species after modification with peroxynitrite or SIN-1, far UV-CD spectra of non-treated and SIN-1-and peroxynitrite-treated ICDH were recorded and analyzed for specific elements of secondary structure. The CD spectrum of ICDH is very similar to that of the protein after modification with peroxynitrite or SIN-1, suggesting that the interaction of peroxynitrite species with ICDH does not appreciably change the secondary structure of the protein (Fig. 5A). Protein damage by ROS usually results in enhanced proteolytic susceptibility due to protein unfolding and increased accessibility of peptide bonds to proteases (47). To test whether the damage induced the alteration in the susceptibility of ICDH to proteolysis, control and peroxynitrite-and SIN-1-treated enzyme were digested with indicated amounts of Pronase, and then the liberated primary amines were then quantitated by reaction with o-phthaldehyde (35). The results indicate that peroxynitrite-and SIN-1-treated ICDH appeared to be significantly more susceptible than the native protein to limited proteolysis by Pronase (Fig. 5B). To reveal increases in flexibility of a partial unfolding of peroxynitrite-and SIN-1-induced ICDH, the binding of the fluorescent probe ANSA was used to detect the accessibility of the hydrophobic regions on the protein. When ICDH was exposed to peroxynitrite or SIN-1, it bound the hydrophobic probe ANSA more efficiently than does the native protein. The representative result with peroxynitrite is shown in Fig. 5C. Intensity of ANSA fluorescence was increased, and the blue shift of maximum emission was seen in the peroxynitrite-treated ICDH.
Inactivation of ICDH by NO in Intact Cells-Because peroxynitrite and SIN-1 readily inactivates ICDH in vitro, we examined ICDH activity in U937, a histiocytic lymphoma cell line, after treatment with 100 M SIN-1 bolus as a source of peroxynitrite. Time-dependent decrease of both cytosolic and mitochondrial ICDH activity in SIN-1-treated cells was observed (Fig. 6, A and B). However, the activity of glucose-6phosphate dehydrogenase, the other major NADPH-generating enzyme in cytosol, was not affected by SIN-1 (Fig. 6A). To test whether SIN-1 affects the level of protein, Western blot analysis of the proteins from SIN-1-treated cells was carried out with anti-ICDH antibodies. The protein level examined by immunoblotting using anti-ICDH antibodies was unchanged (data not shown), suggesting that the decreased ICDH activity was due to chemical modification of the enzyme. To identify the modified residues of ICDH in cells, mitochondrial ICDH from control and SIN-1-treated U937 cells were purified by immunoprecipitation (Fig. 7A). Western blot analysis of purified mitochondrial ICDH with anti-nitrosocysteine and anti-nitrotyrosine showed immunoreactive bands in mitochondrial ICDH from SIN-1-treated U937 cells but not in mitochondrial ICDH from control cells (Fig. 7B). To further confirm immunoblotting results, the purified mitochondrial ICDH samples were subjected to ESI-MS. Molecular mass of mitochondrial ICDH from control and SIN-1-treated cells were 46,885 and 46988 Da, respectively (Fig. 7C). The increased mass (103 Da) corresponds to the incorporation of two NO molecules and one NO 2 molecule to the parent protein. These findings are consistent with the in vitro results regarding tyrosine and cysteine residues of the enzyme are targets of peroxynitrite. To evaluate the effect of ICDH inactivation by peroxynitrite on cellular redox state, we measured the cellular levels of NADPH and GSH as well as intracellular ROS generation. In SIN-1-treated cells, the ratio of [NADPH]/[NADPH ϩ NADP ϩ ] was significantly reduced (Fig. 8A), indicating that much less NADPH is present in SIN-1-treated cells compared with control cells. One important parameter of GSH metabolism is the ratio of [GSSG]/[total GSH (GSHt)], which may reflect the efficiency of GSH turn- over. The ratio of [GSSG]/[GSHt] in SIN-1-treated cells was significantly higher than that of the control (Fig. 8B), indicating that GSSG in SIN-1-treated cells was not reduced as efficiently as it was in the control cells. The effect of ICDH inactivation by peroxynitrite on ROS generation was demonstrated by the relative intensity of DCF with confocal microscopy. DCF fluorescence intensity increased markedly in SIN-1-treated cells compared with the control cells (Fig. 8C).

Inactivation of Mitochondrial ICDH by Peroxynitrite in Liver from Ethanol-fed Rats-The concentration of NO and O 2
. in the liver was 1.9-and 3.1-fold, respectively, higher in the ethanolfed rats than in their pair-fed control. The accumulation of NO and O 2 . in liver from the ethanol-fed animals was associated with decreased activity of Cu,Zn-SOD and Mn-SOD and increased activity and protein expression of iNOS (Fig. 9). To evaluate the inactivation of ICDH by peroxynitrite in vivo, we examined mitochondrial ICDH activity of liver from ethanolfed rats. The enzymatic activity of liver mitochondrial ICDH from ethanol-fed rats was significantly (p Ͻ 0.01) lower than that from control rats (Fig. 10A). Mitochondrial fractions from both control and ethanol-fed rat livers were subjected to immunoprecipitation with mitochondrial ICDH antibody followed by separation by SDS-PAGE. Western blot analysis of purified mitochondrial ICDH with anti-nitrosocysteine and anti-nitrotyrosine IgG showed immunoreactive bands in liver mitochondrial ICDH from ethanol-fed rats but not in that from control rats (Fig. 10B). To evaluate the effect of mitochondrial ICDH inactivation by peroxynitrite on redox status of liver mitochon-dria, we measured the level of NADPH and GSH recycling as well as mitochondrial ROS generation. The peroxide level of mitochondria increased markedly in ethanol-fed rats compared with that in control rats (Fig. 10C). In liver mitochondria from ethanol-fed rats, the ratio of [NADPH]/[NADPH ϩ NADP ϩ ] was significantly reduced and the ratio of [GSSG]/[GSHt] was significantly increased compared with that from control rats (Fig. 10D), indicating that much less NADPH is present, and the GSH recycling is much less efficient in liver mitochondria from ethanol-fed rats compared with that from control rats.

DISCUSSION
Cysteine-containing proteins are susceptible to modification by reactive nitrogen species (48). It has been established that ICDH contains reduced cysteinyl residues that are important for enzyme activity (30,31). Here we present evidence indicating that ICDH can be inhibited by peroxynitrite through direct modification by its cysteine residues. The inactivation of ICDH was protected by thiols, and the inactivated ICDH was reversed by a reduction with DTT. These findings confirmed that peroxynitrite targeted the cysteine residues. The strong correlation between the decrease in IANBD-and DTNB-accessible thiol content and the loss of activity of ICDH suggests that modifications of other protein residues are not responsible for the loss of enzyme activity. Using ESI-MS, we were able to identify the peroxynitrite-modified species of ICDH, thereby providing direct evidence of its modification. When treated with peroxynitrite, modified species of ICDH are seen, their masses differing by multiples of the mass of NO. These data unequivocally establish that S-nitrosation of ICDH occurs at cysteine residues, Cys 305 and Cys 387 , through the formation of S-nitrosothiol adduct. It has been reported that Cys 387 of pig heart mitochondrial ICDH is close to the coenzyme binding site (49). Peroxynitrite was also capable of nitrating the tyrosine of ICDH at Tyr 280 , as demonstrated by Western blot analysis and by mass spectrometry studies. However, our results demonstrate that nitration of tyrosine has little effect on the activity of ICDH. Although peroxynitrite has been known to be a powerful modifier of proteins, including oxidation and nitration of cysteine, methionine, tyrosine, and tryptophan residues, our data indicate that S-nitrosylation of cysteine and nitration of tyrosine are the predominant modifications in ICDH.
There are several lines of evidence obtained from the present study indicating that peroxynitrite-treated ICDH-induced structural alterations. This finding is reflected in the changes in protease susceptibility and in binding of ANSA. However, the CD spectrum and, therefore, the secondary structure content of ICDH was not altered by the treatment with peroxynitrite or SIN-1, which suggests that only subtle, not drastic, conformational changes may occur in modified protein. The increase and susceptibility of oxidatively modified proteins by ROS to proteases is considered to be a form of secondary defense (47). Peroxynitrite-and SIN-1-modified ICDH are more susceptible to proteolytic degradation, indicating that damaged ICDH is turned over more rapidly. Among the techniques aimed at following conformational changes of proteins, binding of the fluorescent probe ANSA has been used to detect the accessibility of the hydrophobic regions on protein upon in- creases in flexibility or partial unfolding. Binding can be easily monitored, because it is accompanied by an increase in fluorescence and a blue shift of the emission maximum associated with the transfer of the ANSA from a hydrophilic to a hydrophobic environment (50). As revealed by an increase in ANSA fluorescence at 490 nm, ICDH modified with peroxynitrite binds ANSA.
NADPH is an essential cofactor for the regeneration of GSH, the most abundant low molecular mass thiol in most organisms, by glutathione reductase in addition to its critical role for the activity of NADPH-dependent thioredoxin system (26,27). The oxidized form of thioredoxin, with a disulfide bridge between the half-cystines, can be reduced by NADPH in the presence of a flavoprotein, thioredoxin reductase (51). Reduced thioredoxin may provide reducing equivalents to at least two enzymes, thioredoxin peroxidases, which remove hydrogen peroxide using hydrogen provided by the NADPH-dependent thioredoxin system (26,27), and methionine sulfoxide reductase, which can reactivate damaged proteins at their methionine residues (51), presumably, involved in the defense against oxidative stress. NADPH is also required for the formation of active catalase tetramers, where each catalase monomer contains one NADPH binding site necessary for its enzymatic activity (52). Glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the pentose phosphate pathway, has long been regarded as the major enzyme to generate NADPH.
In fact, the role of glucose-6-phosphate dehydrogenase in the cell response to oxidative stress is well established in yeast, in human erythrocytes, and in the mouse embryonic stem cells (52)(53)(54). However, two other NADP ϩ -linked dehydrogenases, malic enzyme and cytosolic ICDH, are also responsible for the generation of cytosolic NADPH (55). Earlier study indicated that cytosolic ICDH in the rat liver was 16-and 18-fold more active in producing NADPH than glucose-6-phosphate dehydrogenase and malic enzymes, respectively (56), suggesting an important role of cytosolic ICDH in the production of NADPH and eventually for the cellular defense against oxidative stress. Recently, cytosolic ICDH that is preferentially expressed in bovine corneal epithelium has been identified. The role of this enzyme in contributing to corneal transparency is likely attributed to its protective effect against UV radiation (57). We also demonstrated that the control of cytosolic redox balance and oxidative damage is one of the primary functions of cytosolic ICDH (28). Because glucose-6-phosphate dehydrogenase, the other major NADPH-generating enzyme in cytosol, was not affected by peroxynitrite the significantly less efficient GSH turnover within cells after treatment with a peroxynitrite donor was likely a consequence of inactivation of cytosolic ICDH by peroxynitrite.
Mitochondria are the major source of O 2 . , and NO diffusion or formation may result in peroxynitrite formation. Peroxynitrite may react with mitochondrial components causing impairment FIG. 10. Inactivation of liver mitochondrial ICDH inactivation in ethanol-fed rats. A, activities of liver mitochondrial ICDH of ethanol-fed rats and control rats were measured as described under "Experimental Procedures." Data are presented as means Ϯ S.D. of five separate experiments. B, liver mitochondrial ICDH was purified from control and ethanol-fed rats using immunoprecipitation with antimitochondrial ICDH antibody as described. Purified ICDH was probed with anti-nitrosocysteine and anti-nitrotyrosine antibodies. C, ROS generation in liver mitochondrial from control and ethanol-fed rats. Production of total peroxides in mitochondria was determined by the method described under "Experimental Procedures." Data are presented as means Ϯ S.D. of five separate experiments. D, ratios of NADPH versus total NADP pool and GSSG versus total GSH pool in liver mitochondrial from control and ethanol-fed rats. Data are presented as means Ϯ S.D. of five separate experiments. of mitochondrial functions. Mitochondrial ICDH is a key enzyme in cellular defense against oxidative damage by supplying NADPH in the mitochondria, needed for the regeneration of mitochondrial GSH or thioredoxin. Elevation of mitochondrial NADPH and GSH by mitochondrial ICDH in turn suppressed the oxidative stress and concomitant ROS-mediated damage. It is well established that mitochondrial dysfunction is directly and indirectly involved in a variety of pathological states caused by genetic mutations as well as exogenous compounds or agents (58). Mitochondrial GSH becomes critically important against ROS-mediated damage, because it not only functions as a potent antioxidant but is also required for the activities of mitochondrial glutathione peroxidase and mitochondrial phospholipid hydroperoxide glutathione peroxidase (59), which removes mitochondrial peroxides. NADPH is a major source of reducing equivalents and cofactor for mitochondrial thioredoxin peroxidase family/peroxiredoxin family, including peroxiredoxin III/protein SP-22 (60 -62) and peroxiredoxin V/AOEB166 (63). Therefore, any mitochondrial NADPH producer, if present, becomes critically important for cellular defense against ROS-mediated damage. In this regard, the inactivation of mitochondrial ICDH by peroxynitrite may result in the disruption in regulating the mitochondrial redox balance by providing NADPH.
Modification of ICDH by peroxynitrite will likely have biological and medicinal significance. For a variety of human diseases, high levels of nitrotyrosine are found, which strongly implicates peroxynitrite as a pathophysiological agent (64). We observed a decrease in mitochondrial ICDH activity and the modulation of the cellular redox status in liver from the ethanol-fed rats. S-Nitrosocysteine and nitrotyrosine adducts of mitochondrial ICDH purified by immunoprecipitation were also detected. It has been reported that chronic ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide (65). The present results show that the accumulation of NO and O 2 . in liver from the ethanol-fed rats was associated with significant reduction of Mn-SOD and Cu,Zn-SOD activities and increased iNOS activity and induction of iNOS protein. The effects of peroxynitrite are aggravated when the intracellular level of glutathione is decreased (66,67). It has been proposed that GSH depletion in hepatic mitochondria is considered the most important sensitizing mechanism in the pathogenesis of alcoholic liver injury (66). In this context, the observed modification of ICDH by peroxynitrite leading to disturbances of integrity in the antioxidant defense mechanisms through the decrease in the generation of NADPH, an essential cofactor of GSH recycling. This may lead to pathological conditions associated with generation of ROS and reactive nitrogen species. Therefore, it is tempting to speculate that inactivation of ICDH by peroxynitrite is, presumably, at least in part responsible for the perturbation of cellular redox status and oxidative damage in chronic alcoholism. The possibility that inactivation of ICDH by peroxynitrite in neurodegenerative diseases, diabetes, and chronic inflammation is worthy of further consideration.
In conclusion, the peroxynitrite-mediated damage to both cytosolic and mitochondrial ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.