Nitration of solvent-exposed tyrosine 74 on cytochrome c triggers heme iron-methionine 80 bond disruption. Nuclear magnetic resonance and optical spectroscopy studies

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.     

The Molten Globule of β2-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

The acid transition of β₂-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH 4 (25 °C). The pH 4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH 4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH 4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pK_a value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH 4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH 4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state → MG → burst-phase intermediate → native state.     

Myoglobin functions in the heart

The physiological role of myoglobin (Mb) within the heart depends on its oxygenation state. The myocardium exhibits a broad oxygen partial pressure (pO₂) spectrum with a transmural gradient from the epicardial to the subendocardial layer, ranging from arterial values to an average of 19.3 mm Hg down to 0 mm Hg. The function of Mb as an O₂ storage depot is well appreciated, especially during systolic compression. In addition, Mb controls myocardial nitric oxide (NO) homeostasis and thus modulates mitochondrial respiration under physiological and pathological conditions. We recently discovered the role of Mb as a myocardial O₂ sensor; in its oxygenated state Mb scavenges NO, protecting the heart from the deleterious effects of excessive NO. Under hypoxia, however, deoxygenated Mb changes its role from an NO scavenger to an NO producer. The NO produced protects the cell from short phases of hypoxia and from myocardial ischemia/reperfusion injury. In this review we summarize the traditional and novel aspects of Mb and its (patho)physiological role in the heart.     

Design,solid phase synthesis and evaluation of cationic ferrocenoyl peptide bioconjugates as potential antioxidant enzyme mimics

Synthetic C-terminal amidated cationic ferrocenoyl peptide bioconjugates Fc-Orn-Orn-Orn (1) and Fc-Tyr-Orn-Orn-Orn (2) were rationally designed as superoxide dismutase (SOD) mimics based on the structure of the iron SOD from Escherichia coli. Ferrocenoyl peptide bioconjugates 1, 2 and ferrrocenecarboxylic acid (4) were subsequently evaluated as SOD mimics and as inhibitors of peroxynitrite-mediated tyrosine nitration. Due to their cationic character, ferrocenoyl peptide bioconjugates 1 and 2 exerted an acceptable SOD activity (EC₅₀ = 575 μM and 310 μM, respectively) in comparison with 4 (EC₅₀ = 1.4 mM). The C-terminal amidated cationic peptide Ac-Tyr-Orn-Orn-Orn (3), designed as marker of peroxynitrite, was used to evaluate the inhibitory activity of 1 and 4 towards peroxynitrite-mediated tyrosine nitration. Both compounds proved to inhibit the nitration especially the cationic ferrocenoyl peptide bioconjugates 1. The ferrocene moiety of conjugate 2 displayed a strong inhibitory activity of peroxynitrite-mediated nitration of the neighboring tyrosine.     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.     

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the k_cat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.     

Structural analysis and molecular modeling of the RvH2-e functional unit of Rapana venosa hemocyanin

Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex structure. To provide details on the stability of the protein, one functional unit, RvH2-e, was compared with the native molecule and the structural subunits, RvH1 and RvH2, via pH–T diagrams, typical phase portraits for stability and denaturation reversibility. By analyzing the T transition curves of RvH2-e at different pH values, several parameters of the thermodynamic functions were obtained. Increasing the temperature from 25 °C to 55 °C, the reversibility of the molecule of protein also increases, opening a reversibility window within the range of pH 4.0–8.0. On analyzing the pH transition curves, the start of the acid denaturation (below pH 6) and alkaline denaturation (above pH 9) was determined to be between 20 °C and 35 °C. For this range, the thermodynamic functions ΔH° and ΔG° for a standard temperature of 25 °C were calculated.     

Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H₂O₂/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30–100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.     

The fluorescent monomeric protein Kusabira Orange. Pressure effect on its structure and stability

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network.     

pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

Formation of spider silk from its constituent proteins—spidroins—involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild‐type (wt) E. australis NT at pH 7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5–6 Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH 7 and pH 6 and indicate that the biological function of NT depends on conversion between the two conformations.     

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4–6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30 W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.     

Characterization of lactase-conjugated magnetic nanoparticles

Conjugation of lactase to magnetic nanoparticles is of interest in biosensor and ingredient processing applications that require high enzyme concentration and catalyst separation from the reaction stream. However, little is known about the effects of these materials on the physicochemical attributes of conjugated lactase. Lactase (Aspergillus oryzae) was covalently attached by carbodiimide chemistry to carboxylic-acid functionalized magnetic particles having a hydrodynamic radius of 18 nm. The resulting enzyme–nanoparticle conjugates were characterized with regard to particle size, zeta potential, enzyme kinetics, temperature and pH stability, catalyst recovery, and secondary structure changes. Following attachment, the materials retained colloidal stability and individual particle characteristics with a zeta potential of ?33 mV compared to ?46 mV for the native particle. The conjugated enzyme showed no changes in secondary structure and exhibited significant catalytic activity with a catalytic efficiency of 2.8 × 10³ M^?1 s^?1 compared to 2.5 × 10³ M^?1 s^?1 for the native enzyme. Relative to the free enzyme, the conjugated enzyme was recovered for repeated use with 78% activity retained after five cycles. This work demonstrates that carboxylic-acid functionalized magnetic nanoparticles can be utilized as a means of producing a simple and effective conjugated-lactase system that achieves both particle and enzyme stability.     

The 1.6 Mb chromosome carrying the avirulence gene AvrPik in Magnaporthe oryzae isolate 84R-62B is a chimera containing chromosome 1 sequences

A genetic map was constructed previously from a cross between Magnaporthe oryzae isolates 84R-62B and Y93-245c-2, and genetic markers closely linked to the cultivar-specific avirulence (Avr) gene, AvrPik, were assigned to a 1.6 Mb small chromosome of 84R-62B that is absent from Y93-245c-2. In the present study, the 1.6 Mb chromosome was characterized by using contour-clamped homogeneous electric fields (CHEF) electrophoresis and hybridization analysis. CHEF electrophoresis analysis showed that the 1.6 Mb chromosome was inherited in Mendelian fashion, and co-segregated with AvrPik. Southern hybridization analysis revealed that the 1.6 Mb chromosome carried sequences only distributed to the supernumerary chromosome in M. oryzae isolates, as well as sequences corresponding to those in the supercontig 17 of chromosome 1 in the M. grisea database. Thus, we conclude that the Mendelian 1.6 Mb chromosome is a chimera containing sequences from chromosome 1 and from supernumerary chromosomes in M. oryzae.     

Modification of lactoferrin by peroxynitrite reduces its antibacterial activity and changes protein structure

Lactoferrin (LF) is a multifunctional protein that plays important physiological roles as one of the most concentrated proteins in many human and other mammalian fluids and tissues. In particular, LF provides antibacterial properties to human milk, saliva, and tear fluid. LF also protects against stress-induced lipid peroxidation at inflammation sites through its iron-binding ability. Previous studies have shown that LF can be efficiently nitrated via biologically relevant mediators such as peroxynitrite (ONOO⁻), which are also present at high intracellular concentrations during inflammation and nitrosative stress. Here, we examine changes in antibacterial properties and structure of LF following ONOO⁻ treatment. The reaction induces nitration of tyrosine and tryptophan residues, which are commonly used as biomarker molecules for several diseases. Treatment with ONOO⁻ at a 10/1 M ratio of ONOO⁻ to tyrosine inhibited all antibacterial activity exhibited by native LF. Secondary structural changes in LF were assessed using circular dichroism spectroscopy. Nitration products with and without the addition of Fe³⁺ show significant reduction in alpha-helical properties, suggesting partial protein unfolding. Iron-binding capacity of LF was also reduced after treatment with ONOO⁻, suggesting a decreased ability of LF to protect against cellular damage. LC-MS/MS spectrometry was used to identify LF peptide fragments nitrated by ONOO⁻, including tyrosine residue Y92 located in the iron-binding region. These results suggest that posttranslational modification of LF by ONOO⁻ could be an important pathway to exacerbate infection, for example, in inflamed tissues and to reduce the ability of LF to act as an immune responder and decrease oxidative damage.     

The Impact of Nitration on the Structure and Immunogenicity of the Major Birch Pollen Allergen Bet v 1.0101

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of T_H1-priming cytokines, thus favoring a T_H2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.     

Redox properties of tyrosine and related molecules

Redox reactions of tyrosine play key roles in many biological processes, including water oxidation and DNA synthesis. We first review the redox properties of tyrosine (and other phenols) in small molecules and related polypeptides, then report work on (H20)/(Y48)-modified Pseudomonas aeruginosa azurin. The crystal structure of this protein (1.18 Å resolution) shows that H20 is strongly hydrogen bonded to Y48 (2.7–2.8 Å tyrosine-O to histidine-N distance). A firm conclusion is that proper tuning of the tyrosine potential by a proton-accepting base is critical for biological redox functions.     

Peroxynitrite treatment in vitro disables catalytic activity of recombinant p38 MAPK

Protein tyrosine nitration is a post-translational modification occurring under conditions of oxidative stress in a number of diseases. The causative agent of tyrosine nitration is the potent prooxidant peroxynitrite that results from the interaction of nitric oxide and superoxide. We have previously demonstrated existence of nitrotyrosine in placenta from pregnancies complicated by preeclampsia, which suggested the possibility of the existence of nitrated proteins. Nitration of various proteins has been demonstrated to more commonly result in loss of protein function. Potential nitration of p38 MAPK, a critical signaling molecule has been suggested and also tentatively identified in certain in vivo systems. In this study we demonstrate for the first time nitration of recombinant p38 MAPK in vitro and an associated loss of its catalytic activity. LC-MS data identified tyrosine residues Y132, Y245 and Y258 to be nitrated. Nitration of these specific residues was deduced from the 45.0-Da change in mass that these residues exhibited that was consistent with the loss of a proton and addition of the nitro group.