1 INTRODUCTION
1.2 Biochemistry of protein nitration
The nitration of tyrosine residue appears to represent a prominent in vivo pathway of protein oxidative modification occurring in many different pathological conditions such as atherosclerosis [12, 13], asthma and lung diseases [14-16], neurodegenerative disease [17-19], chronic hepatitis and cirrhosis [20, 21], diabetes [22] and other disorders. Only a selective number of proteins are modified by nitration in vivo, and this selectivity may be caused by a combination of several factors such as (1), the proteins are in close proximity to the site of generation of nitrating agents; (2), the chemical selectivity of the nitrating reagent; (3), the relative abundance of the target proteins; (4), the accelerated turnover of some of the nitrated proteins; (5), the proteins contain tyrosine residues in a specific primary sequence or in a specific environment and (6), the “repair” of nitrated proteins by a putative enzyme called
“denitrase” [23, 24].
The knowledge regarding nitration of proteins is to the most part derived from in vitro experiments with well known proteins or with free tyrosine, and the physiological relevance of these findings remains to be defined. The major in vivo mechanisms for protein nitration are summarized in Figure 1 and described in the following [25].
Figure 1: Different pathways for protein tyrosine nitration. Peroxynitrite (ONOO–) and heme peroxidase-dependent protein nitration are the most likely mechanisms. Other mechanisms, whose physiological relevance remains to be understood, include protein
Protein
Protein
(NO2)n ONOO- NO2-/H2O2
+
heme peroxidase Cu/Zn-SOD
Catalase Hemaglobin
Myoglobin Protein
Protein
(NO2)n ONOO- NO2-/H2O2
+
heme peroxidase Cu/Zn-SOD
Catalase Hemaglobin
Myoglobin
Shortly after the discovery of the free radical, nitric oxide (•NO) as a cellular messenger, its reaction with superoxide (O2•–) to form peroxynitrite was proposed in order to explain the toxicity linked to their excess formation [26, 27]. Peroxynitrite (PN) can react with a wide range of different biological molecules including lipids [28], DNA [29-31], proteins [32-34] and lead to changes in structure and function. The level of ONOO- was found to be increased in several disorders such: acute lung injury [35], cystic fibrosis [36], asthma [37], neurodegenerative disease [38, 39], atherosclerosis [40], and diabetes [41].
Figure 2A shows the typical morphology of lung tissue as a control and Figure 2B the immunohystochemisty (using a polyclonal anti 3-NT antibody) indicate the severity of lesions and the formation of 3-nitrotyrosine as a maker of peroxynitrite formation in patients suffering of sever lung infection [42].
Figure 2: Immunoreactivity to nitrotyrosine (NT) in lung tissue (A): control group; (B): peroxynitrite induced nitration and implicit damage in the lung cell. [42]
The main proposed pathway of peroxynitrite (PN) formation is the reaction between nitric oxide and superoxide; the reaction being normally controlled by the action of two enzymes, nitric oxide synthase (NOS) and superoxide dismutase (SOD) (s.
Figure 3).
(A) (B)
Figure 3: Formation of peroxynitrite from nitric oxide and superoxide radicals. PN anion it self is unreactive for tyrosine, but protonation to the conjugate acid or Lewis adduct formation with carbon dioxide generates biological nitrating reagent –nitrogen dioxide radical.
NO• in the cell is synthesised by three isoformes of NO-synthases (NOS), which belong to the P450- protein family. They use L-arginine as substrate and release NO• and L-citruline, via formation of an intermediate, L-hydroxy arginine, according to the equation (1.1)
2L-Arginine + 4O2 + 3NADPH + 3 H+ 2L-Citruline + 2 •NO + 4H2O + 3 NADP+ (1.1)
At pH 7, superoxide (O2• –) is a short lived radical with a rather low reactivity. Its short life time is due to its fast self-dismutation in aqueous solutions (s. eq. 1.2). By the reaction with metals and other reactive species superoxide can generate hydroxyl radicals, which may damage nearly all existing biomolecules. In vivo there are two enzymatic systems which keep the O2• – concentration low, the Mn-SOD (only in mitochondria) and Cu, Zn-SOD (in cytosol) releasing hydrogen peroxide as a major decomposition product (s. eq.1.3).
2O2• – + 2H+ O2 + H2O2 k = 7.3x105 M-1 s-1 (1.2)
• – + SOD
Arginine
Citrulline NOS
ONOO ONOOH HO•---•NO2
Peroxynitrous „Caged-radicals“
acid
•O2–
Superoxide
Peroxynitrite
O2 Mitochondria
P450oxidases SOD H2O2
NO•
Nitric oxide Arginine
Citrulline NOS
ONOO ONOOH HO•---•NO2
Peroxynitrous „Caged-radicals“
acid
•O2–
Superoxide
Peroxynitrite
O2 Mitochondria
P450oxidases SOD H2O2
NO•
Nitric oxide
Disproportionation of O2• – occurs with k = 108 M-1 s-1 for the Mn-SOD catalyzed reaction and with k = 2x109 M-1 s-1 under catalysis of Cu, Zn-SOD. Hydrogen peroxide is scavenged in the cell to oxygen and water by catalase. As previously mentioned, PN can be formed in vivo by the nearly diffusion controlled reaction of NO• and O2• –:
NO• + O2• – ONOO-
k = 6.7x109 M-1 s-1 (1.4)
The velocity of the nitric oxide and superoxide reaction (1.4) is a factor of 2-10 faster compared to the velocities of the reactions of Mn- and Cu, Zn-SOD with superoxide.
For the suppression of PN formation in vivo the NO• concentration is essential. If the nitric oxide concentration gets too high, SOD’s cannot complete with it for the superoxide anion and formation of peroxynitrite will be favoured [43].
It is well accepted that peroxynitrite (ONOO-) is stable only in alkaline solution. The unusual stability of ONOO- is due to its folding into the cis-conformation, which can not directly isomerise to the much more stable form, nitrate. After protonation, ONOO- can isomerise to a trans-conformation or trans-peroxynitrous acid (ONOOH).
Figure 4 shows a compilation of the protonation, conformation equilibrium and pathways for the decomposition of PN, as well as isomerisation and pKa-values [44, 45]. Trans-peroxynitrous acid (ONOOH) is a strong oxidant and decays rapidly to hydroxyl radical and nitrogen dioxide as a pair of caged radicals [46]. These two radicals undergo electron transfer to form nitronium ion (NO2+) and hydroxide (HO-) or may escape the solvent cage as free radicals. Trans-ONOOHis toxic by oxidative mechanisms which result in oxidation of sulphydryls, lipid peroxidation, and nitration of amino acid residues.
Figure 4: Cis-trans isomerisation of peroxynitrite anion (ONOO-) and decomposition of trans- peroxynitrite conjugate acid (trans-ONOOH) in to hydroxyl radical HO• and nitric dioxide radicalNO2• two highly toxic radicals.
Nitric oxide is neutral and hydrophobic, capable of traversing membranes, while superoxide is anionic at neutral pH (pKa = 4.8), so that PN formation occurs predominantly close to the sites of superoxide formation [47]. In turn, the half-time of peroxynitrite, of ca. 1s, seems to be sufficient to traverse membrane by passive diffusion as its conjugate acid (ONOOH, pKa = 6.8) or in the anionic form.
Peroxynitrite is more reactive than its precursors nitric oxide and superoxide. First, PN reacts directly with certain amino acid residues such as cysteine and methionine [48]. Second, prosthetic groups, and particularly transition metal centers, are likely to react with peroxynitrite [49, 50]. Third, secondary radicals derived from PN (hydroxyl, carbonate and nitrogen dioxide radicals) can also react with protein residues such as tyrosine, phenylalanine, tryptophan and histidine [7, 51].
O
For in vitro studies, a solution of peroxynitrite can be prepared by treating acidified hydrogen peroxide with a solution of sodium nitrite, followed by addition of sodium hydroxide. Its concentration is indicated by absorbance at 320 nm (pH 12, λ302 = 1670 M-1 cm-1) [52]. Preformed PN may be directly used as a nitrating agent for peptides or proteins in 50 mM phosphate solution or even for enzymes in presence of chelating agents (Diethylene-triaminepentaacetic acid, DTPA or Ethylene-dinitrilo-tetraacetic acid, EDTA) that sequesters metal ions so they cannot combine with other compounds. Appreciable amounts of 3-nitro-tyrosine may be produced by peroxynitrite formed in situ by a continuous generation of NO• and O2• – using different donors systems such as: PAPA NONOate1 and xanthine oxidase with pterin as a substrate [53] or directly using SIN-1 (3-morpholinosydnonimine, which is a co-donor of •NO and O2•−) [54].
Another possible mechanism for tyrosine nitration is the oxidation of NO2
by peroxidases (horseradish peroxidase, myeloperoxidase or eosinophil peroxidase) in the presence of hydrogen peroxide, leading to •NO2 as a nitrating species. This pathway needs higher concentrations of NO2-
and H2O2 that can be achieved under inflammatory conditions.
Tyrosine nitration is a covalent protein modification resulting from the addition of a nitro- (NO2) group onto one of the two equivalent carbons CE1 and CE2 in the ortho position relative to the hydroxyl group of tyrosine residue and is believed to depend on the simultaneous availability of tyrosyl (Tyr•) and nitrogen dioxide (•NO2) radicals (s. Figure 5) [55, 56]. The rate-limiting step in tyrosine nitration is its oxidation to Tyr• (Step A in Figure 5), which may proceed more slowly than the rate at which •NO2
reacts with the Tyr• (k = 3×109 M-1s-1, Step B in Figure 5) [57].
Figure 5: Proposed reaction mechanism of 3-nitrotyrosine formation by radicals derived from peroxynitrite. The reaction is initiated by one-electron oxidation of tyrosine to the tyrosyl radical which reacts with nitrogen dioxide radical forming a 3-nitro-tyrosine residue.
A selective targeting of peroxynitite to specific tyrosine residues (site-specific nitration) has been suggested for some proteins, including glutamine synthetase [58], prostacyclin synthase [59], Mn-superoxide dismutase [60], lysozyme, ribonuclease A [61] and tyrosine hydroxylase [62]. One important factor governing nitration seems to be the localization of tyrosine in hydrophobic domains, since the level of nitration of a hydrophobic tyrosine probe located in a lipid bilayer has been reported to be higher than that measured for tyrosine in an aqueous solution [63]. Furthermore, the pH in the external bulk and inside the protein showed to be crucial for nitration, since the oxidative chemistry of peroxynitrite and the nature of radicals formed from its decay are strictly linked to pH [46]. Moreover, the presence of neighbouring negative charges to the tyrosine residue [64], the location of the tyrosine residue in a loop structure and absence of proximal cysteine or methionine residues [61] may increase the yield of nitration. Thus, the specificity of peroxynitrite-dependent tyrosine nitration seems to depend on the secondary and tertiary structure of proteins and the local
OH
1.3 Analytical methods for identification and structural characterization