Quantification of nitrotyrosine in nitrated proteins

For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to $ {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) $ {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) . A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules.     

Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.     

Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry

Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.     

Determination of nitration degrees for the birch pollen allergen Bet v 1

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration.

DFT calculations of the intermediate and transition state in the oxidation of NO by oxygen in the gas phase

The B3LYP density functional method using the extended basis set 6-311++G(3df) was used to calculate the stationary points along the reaction coordinate 2NO + O₂ → 2NO₂. The results of the calculation were compared with the reported physicochemical characteristics of this reaction. The origin of the barrierless activation of the oxygen molecule and driving force for the spontaneous oxidation of NO were examined.     

Design of an electroactive peptide probe for sensing of a protein

We designed a new electroactive peptide probe that has a molecular recognition function for the sensing of a protein. Ovalbumin (OVA) was the model protein, and when RNRCKGTDVQAW interacted with OVA, it conjugated with a tyrosine-rich peptide (Y₄C). This peptide is electroactive, has a high degree of biocompatibility, and offers the possibility of gene expression. To measure the effect of a number of the tyrosine residues, voltammetric measurements were conducted using a series of tyrosine-rich peptides (Y_nC, n = 3–7) with sensitivities that ranged from 10⁻⁹ to 10⁻⁸ M. The electrode response of Y₅C was the maximum value in the series. However, the peak current did not increase when the number of tyrosine residues was increased in a linear fashion. This may have been due to the micelles that are formed by a tyrosine-rich surfactant peptide. Thus, Y₄C was suitable as an electroactive label for the construction of the peptide probe. The electrode response of Y₄CRNRCKGTDVQAW obtained by a glassy carbon electrode was 100-fold that of tyrosine alone. The measurement of OVA via the peptide probe resulted in a detection on the order of 10⁻¹² M. In contrast, the sensitivity of OVA using RCKGTDVQAWY₄C probe was at the 10⁻¹¹ M level, because the hydrophobic moiety gave it a molecular recognition function. The recoveries of the OVA using Y₄CRNRCKGTDVQAW in a solution containing fetal bovine serum ranged between 98 and 101%. Consequently, the combination of a specific peptide and an electroactive element could be a powerful probe for the sensing of proteins.     

Quantitative WDXS Microanalysis of Bismuth-Based BaBi4Ti4O15 Perovskites Doped with Nb and Fe

 Quantitative electron-probe microanalysis was used to determine the chemical composition of an Fe- and Nb-doped bismuth-based BaBi₄Ti₄O₁₅ perovskite compound. Elemental concentrations of Fe, Nb, Bi, Ba and Ti were accurately measured using wavelength-dispersive X-ray spectroscopy that was optimised for the analysis of a complex oxide matrix containing minor concentrations of dopants. Measurements were performed with a JEOL JXA 840A electron probe microanalyser at 20 and 26 kV, 50 nA beam current, 100 s maximum counting time and 0.3% preset counting deviation (σ_c) using both PET and LiF crystals. K-ratios were quantified by the ZAF and the φ(ρz) PAP matrix-correction procedures. The results showed that dopants incorporate into the BaBi₄Ti₄O₁₅ at Ti^4 + sites according to the Ba_1−4XBi_4 + 4XTi_4−4XFe_4XO₁₅ and Ba_1 + 4XBi_4−4XTi_4−4XNb_4XO₁₅ solid-solution formulae. The majority of the excess charge introduced by the substitution of Ti^4 + with Fe^3 + or Nb^5 + is compensated for the change in the Ba^2 +/Bi^3 + ratio.     

Influence of H<Subscript>2</Subscript>O and SO<Subscript>2</Subscript> on the activity of deposited cobalt oxide catalysts in the processes of reduction of nitrogen(I), (II) oxides with carbon monoxide and C<Subscript>3</Subscript>-C<Subscript>4</Subscript> alkanes

It is shown that palladium–cobalt oxide–cerium catalyst deposited on cordierite catalyzes the reduction of nitrogen(II) oxide with carbon monoxide, and cobalt–iron catalysts in simultaneous reduction of NO + N₂O with C₃-C₄ alkanes retained high activity in the presence of water vapor and sulfur dioxide. The Pd-Co₃O₄/cordierite catalyst exceeds the Pt-Co₃O₄/codierite catalyst in the conversion of NO and CO in the reaction mixture CO + NO + O₂ + H₂O + SO₂. Modification of the Pd-Co₃O₄/cordierite with cerium oxide considerably increases its sulfur resistance.     

Novel Atom‐Economic Nitration of Benzene with a Nitrogen Dioxide–Oxygen System using Cocatalyst of Solid Oxides and Lanthanide(III) Metal Triflates

Benzene is nitrated by a novel atom‐economic nitration procedure with a NO₂‐O₂ system in the presence of a mixture of solid oxides and Ln(OTf)₃. The only by‐product in this novel method is water. The efficiency of NO₂ is much high than 50%, the theoretical efficiency of NO₂ of the known methods. Among the solid oxides and Ln(OTf)₃ studied, HZSM‐5 and Sm(OTf)₃ were the most efficient catalysts in this process. Therewith, the yield of the benzene nitration reached 72.5%, calculated by NO₂.     

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.     

Electrochemical properties of perovskite and A<Subscript>2</Subscript>MO<Subscript>4</Subscript>-type oxides used as cathodes in protonic ceramic half cells

Three selected materials have been prepared and shaped as cathode of half cells using the proton-conducting electrolyte BaCe_0.9Y_0.1O_3 − δ (BCY10): two perovskite compounds, Ba_0.5Sr_0.5Co_0.8Fe_0.2O_3 − δ (BSCF) and La_0.6Sr_0.4Fe_0.8Co_0.2O_3 − δ (LSFC), and the praseodymium nickelate Pr₂NiO_4 + δ (PRN) having the K₂NiF₄-type structure. The electrochemical properties of these compounds have been studied under zero current conditions (two-electrode cell) and under polarization (three-electrode cell). Their measured area-specific resistances were about 1–2 Ω cm² at 600 °C. Under direct current polarization, it appears that the three compounds show almost similar values of current densities at 625 °C; however, at lower temperatures, BSCF appears to be the most efficient cathode material.     

A novel Et<Subscript>4</Subscript>NBF<Subscript>4</Subscript> and LiPF<Subscript>6</Subscript> blend salts electrolyte for supercapacitor battery

Electrolytes of 1 M blend salts (LiPF₆ and tetraethylammonium tetrafluoroborate, Et₄NBF₄) have been investigated in supercapacitor battery system with composite LiMn₂O₄ and activated carbon (AC) cathode, and Li₄Ti₅O₁₂ anode. The results obtained with the blend salts electrolytes are compared with those obtained with cells build using standard 1 M LiPF₆ dissolved in ethylene carbonate + dimethyl carbonate + ethyl (methyl) carbonate (EC + DMC + EMC, 1:1:1 wt.%) as electrolyte. It is found that the blend salts electrolyte performs better on both electrochemical and galvanostatic cycling stability, especially cycled at 4 C rate. When the concentration of LiPF₆ is 0.2 M and Et₄NBF₄ is 0.8 M, the capacity retention of the battery is 96.23% at 4 C rate after 5,000 cycles, much higher than that of the battery with standard 1 M LiPF₆ electrolyte, which is only 62.35%. These results demonstrate that the blend salts electrolyte can improve the galvanostatic cycling stability of the supercapacity battery. Electrolyte of 0.2 M LiPF₆ + 0.8 M Et₄NBF₄ in EC + DMC + EMC (1:1:1 wt.%) is a promising electrolyte for (LiMn₂O₄ + AC)/Li₄Ti₅O₁₂.     

Oxygen reduction mechanism and performance of Y1Ba2Cu3O7−d as a cathode material in a high-temperature solid-oxide fuel cell

The high-Tc Y₁Ba₂Cu₃O_7−δ superconductor with oxygen ion vacancies was employed as the cathode for a high-temperature solid-oxide fuel cell (SOFC). The cathodic current-overpotential characteristics were studied in the temperature range from 500 to 800 °C and the oxygen pressure range from 10⁻⁴ to 0.21 atm. The delocalization of the triple-phase boundary and the oxygen reduction mechanism were identified. The delocalized triple-phase boundary of Y₁Ba₂Cu₃O_7−δ improves the cathodic polarization in SOFCs. By using a mathematical simulation and a particular experimental design, the oxygen adsorption step in the oxygen reduction process was demonstrated to be rate limiting. A layer of strong oxygen-adsorption catalyst such as Pt or Ag coated on the Y₁Ba₂Cu₃O_7−δ electrode was found to be able to largely enhance the activity of oxygen reduction by improving the ability of oxygen to be adsorbed on the electrode surface. Received: 28 October 1997 / Accepted: 16 January 1998     

Preparation of NOx modified PMMA-EGDM composite membrane for the recovery of chromium (VI)

A non-interpenetrating cross-linked poly (methyl methacrylate-ethylene glycol dimethacrylate) copolymer ultrafiltration membrane on a microporous ceramic support has been prepared from the monomers in two stages. The polymer membranes thus obtained have been nitrated using NO_x (a mixture of NO and NO₂) by the gas phase reaction at 80 °C. Separation experiments on the chromium (VI) salt solution have been carried out using unmodified (giving 68% rejection per pass) and nitrated membranes (giving as much as 67% rejection per pass). For nitrated membranes, the water flux and the solute flux increased with time of nitration about hundred folds because of the increase in the hydrophilicity as well as the pore size.     

FTIR studies of butane, toluene and nitric oxide adsorption on Ag exchanged NaMordenite

In this work, we studied the adsorption of butane, toluene and nitric oxide on NaMordenite exchanged with different amounts of silver. The reactions that occurred when the adsorbed hydrocarbons interacted with NO and the effect of water adsorption were also addressed. Different silver species were formed after ion exchange and they were detected by TPR analysis. Highly dispersed Ag₂O particles were reduced at temperatures lower than 300 °C whereas Ag⁺ exchanged ions showed two TPR peaks, which can be ascribed to species exchanged at different mordenite sites. The TPD experiments after adsorption of NO at 25 °C showed that the only desorbed species was NO₂ which was formed by the total reduction of Ag₂O particles. When the adsorbed butane was exposed to NO (1000 ppm), isocyanate species were formed on Ag⁺ ionic sites as well as Ag⁺–(NOx)–CO species. Toluene adsorption was stronger than butane since adsorbed toluene molecules were held even at 400 °C. The characteristic bands of the aromatic ring C=C bond was observed as well as that of methyl groups interacting with Ag⁺ and Na⁺ ions. However, the appearance of carboxylic groups at temperatures above 300 °C in inert flow indicated the partial oxidation of toluene due to Ag₂O species present in the samples. After contacting adsorbed toluene with NO, different FTIR bands correspond to organic nitro-compounds, isocyanate, cyanide and isocyanide species adsorbed on Ag⁺ ions, were detected. The presence of water inhibited the formation of NO₂ species and the hydrocarbon adsorption on Na⁺ sites but did not affect the toluene-Ag⁺ interaction.     

Self-consistent reaction field calculation of solvent reorganization energy in electron transfer: a dipole-reaction field interaction model

Based on the continuum dielectric model, this work has established the relationship between the solvent reorganization energy of electron transfer (ET) and the equilibrium solvation free energy. The dipole-reaction field interaction model has been proposed to describe the electrostatic solute-solvent interaction. The self-consistent reaction field (SCRF) approach has been applied to the calculation of the solvent reorganization energy in self-exchange reactions. A series of redox couples, O₂/O⁻ ₂, NO/NO⁺, O₃/O⁻ ₃, N₃/N⁻ ₃, NO₂/NO⁺ ₂, CO₂/CO⁻ ₂, SO₂/SO⁻ ₂, and ClO₂/ClO⁻ ₂, as well as (CH₂)₂C-(-CH₂-) _n -C(CH₂)₂ (n=1 ∼ 3) model systems have been investigated using ab initio calculation. For these ET systems, solvent reorganization energies have been estimated. Comparisons between our single-sphere approximation and the Marcus two-sphere model have also been made. For the inner reorganization energies of inorganic redox couples, errors are found not larger than 15% when comparing our SCRF results with those obtained from the experimental estimation. While for the (CH₂)₂C–(–CH₂–) _n –C(CH₂)₂ (n=1 ∼ 3) systems, the results reveal that the solvent reorganization energy strongly depends on the bridge length due to the variation of the dipole moment of the ionic solute, and that solvent reorganization energies for different systems lead to slightly different two-sphere radii. Received: 19 April 2000 / Accepted: 6 July 2000 / Published online: 27 September 2000     

Enzyme biosensor based on the immobilization of HRP on SiO<Subscript>2</Subscript>/BSA/Au composite nanoparticles

In this work, an enzyme biosensor based on the immobilization of horseradish peroxidase (HRP) on SiO₂/BSA/Au/thionine/nafion-modified gold electrode was fabricated successfully. Firstly, nafion was dropped on the surface of the gold electrode to form a nafion film followed by chemisorption of thionine (Thi) as an electron mediator via the ion-exchange interaction between the Thi and nafion. Subsequently, the SiO₂/BSA/Au composite nanoparticles were assembled onto Thi film through the covalent bounding with the amino groups of Thi. Finally, HRP was immobilized on the SiO₂/BSA/Au composite nanoparticles due to the covalent conjugation to construct an enzyme biosensor. The surface topographies of the SiO₂/BSA/Au composite nanoparticles were investigated by using scanning electronic microscopy. The stepwise self-assemble procedure of the biosensor was further characterized by means of cyclic voltammetry and chronoamperometry. The enzyme biosensor showed high sensitivity, good stability and selectivity, a wide linear response to hydrogen peroxide (H₂O₂) in the range of 8.0 × 10^-6 ∼ 3.72 × 10^-3 mol/L, with a detection limit of 2.0 × 10^-6 mol/L. The Michaelies-Menten constant K_M^app K_M^{app} value was estimated to be 2.3 mM.     

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated‐tyrosine rings in single‐tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non‐destructive method. Thus, we have resorted to ¹H‐¹⁵N heteronuclear single quantum coherence(HSQC) spectra to identify the ³J(N? H) correlation between a ¹⁵N‐tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the ¹H‐¹³C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.     

Electrocatalytic activity and stability of Ti/IrO2 + MnO2 anode in 0.5 M NaCl solution

Ti/IrO₂(x) + MnO₂(1-x) anodes have been fabricated by thermal decomposition of a mixed H₂IrCl₆ and Mn(NO₃)₂ hydrosolvent. Cyclic voltammetry (CV) and polarization curve have been utilized to investigate the electrochemical behavior and electrocatalytic activity of Ti/IrO₂(x) + MnO₂(1-x) anodes in 0.5 M NaCl solution (pH = 2). Ti/IrO₂+MnO₂ anode with 70 mol% IrO₂ content has the maximum value of q*, indicating that Ti/IrO₂(0.7) + MnO₂(0.3) anode has the most excellent electrocatalytic activity for the synchronal evolution of Cl₂ and O₂ in dilute NaCl solution. Tafel lines displayed two distinct linear regions with one of the slope close to 62 mV dec⁻¹ in the low potential region and the other close to 295 mV dec⁻¹ in the high potential region. Electrochemical impedance spectroscopic is employed to investigate the impedance behavior of Ti/IrO₂(x) + MnO₂(1-x) anodes in 0.5 M NaCl solution. It is observed that as the R _ct, R _s and R _f values for Ti/IrO₂(0.7) + MnO₂(0.3) anode become smaller, electrocatalytic activity of Ti/IrO₂(0.7) + MnO₂(0.3) anode becomes better than that of other Ti/IrO₂ + MnO₂ anodes with different compositions. Ti/IrO₂(0.7) + MnO₂(0.3) anode fabricated at 400 °C has been observed to possess the highest service life of 225 h, whereas the accelerated life test is carried out under the anodic current of 2 A cm⁻² at the temperature of 50 °C in 0.5 M NaCl solution (pH = 2).     

FT-IR study of the nature and stability of NOx surface species on ZrO2, VOx/ZrO2 and MoOx/ZrO2 catalysts

The adsorption of NO, NO/O₂ mixtures and NO₂ on pure ZrO₂ and on two series of catalysts supported on ZrO₂, one containing vanadia and the other molybdena (ZV and ZMo, respectively), has been investigated. The V and Mo surface contents of the latter were ≤3 atoms nm⁻² and ≤5 atoms nm⁻², respectively. All samples had been previously submitted to a standard oxidation treatment. On all samples, only extremely minor amounts of NO_x surface species are formed by NO interaction at room temperature (RT). NO_x surface species are formed in greater amounts on pure ZrO₂ when NO and O₂ are coadsorbed at RT; they are mainly nitrites, small amounts of nitrates, and small amounts of (O₂NO−H)^δ− species; when ZrO₂ is warmed to 623 K in the NO/O₂ mixture, nitrites decrease, nitrates and (O₂NO−H)^δ− species increase. The same NO_x species as on the ZrO₂ surface free from V (or Mo) are formed on ZV (or ZMo) samples with surface V (or Mo) density <1.5 atoms nm⁻²; however, they occur in decreased amount with increasing V (or Mo) coverage. On ZV samples with a surface V density of 1.5–3 atoms nm⁻² (or ZMo samples with a surface Mo density of 1.5–5 atoms nm⁻²) when NO and O₂ are coadsorbed at RT, there is formation of small amounts of nitrites, nitrates (both on ZrO₂ surface free from V (or Mo) and at the edges of V- or Mo-polyoxoanions) and NO₂ ^δ+ species, associated with V⁵⁺ (or Mo⁶⁺) of very strong Lewis acidity; when samples are warmed up 623 K in the NO/O₂ mixture, nitrites disappear, nitrates increase, NO₂ ^δ+ species remain constant or slightly decrease. When NO₂ is allowed into contact at RT with oxidized samples, surface situations almost identical to those obtained for each sample warmed to 623 K in NO/O₂ mixture is reached. The NO_x surface species stable at 623 K, the temperature at which catalysts show the best performance in the selective catalytic reduction (SCR) of NO by NH₃, are nitrates, both on ZrO₂ and on polyvanadates or polymolybdates at high nuclearity. On the contrary, nitrites and NO₂ ^δ+ species are unstable at 623 K.