Nitration

Overview

Nitration is a post-translational modification of mostly tyrosine residues that is caused by one-electron oxidation. First, a tyrosine radical is formed by one-electron oxidation followed by a reaction with nitrogen dioxide resulting in 3-nitrotyrosine.

pKaNCLossGainDeltamassHAAUV-SpecPattern
Acidic
7.25
NoHNO2Av: 44.9976
M: 44.9851
YYes*
Physicochemical properties of nitration that are stored in the modification database of Prot pi (NC: Native charge; H: Relative hydrophobicity; AA: Modified amino acid; Pattern: Regex for sequence-motif recognition).

*For the calculation of the molar absorption coefficients the UV/Vis spectrum of 3-nitrotyrosine was taken.

In-depth mechanism

In contrast to many protein modifications, nitration is a naturally occuring chemical and not anenzymatic process. Through nitration, a nitro group is covalently attached to the carbon in 3-position of the aromatic ring of tyrosine residues forming 3-nitrotyrosine​1–6​. Recent studies suggest that the mechanism of tyrosine nitration is mediated by free radical reactions. In a first step, a tyrosine radical is formed by one-electron oxidation. Common oxidants for this reactionare hydroxyl, nitrogen dioxide, carbonate, peroxyl and alkoxyl radicals as well as oxo-metal compounds and compounds I and II of hemoperoxidases​2,3​. The tyrosine radical can then react with a nitrogen dioxide radical forming 3-nitrotyrosine​1–5​. Although this reaction is favored, the tyrosine radical can undergo different reactions with other radicals such as hydroxyl, superoxideor nitric oxide radicals forming 3,4-dihydroxyphenylalanine respectively tyrosine hydroperoxide or 3-nitrosotyrosine. In addition, the tyrosine radical can react with another tyrosine radical forming 3,3’-dityrosine. It is important to note that these modifications can be reversed by reductans such as ascorbate or glutathione or by intramolecular electron transfer reactions with other amino acids such as cysteine​2,3​. It is assumed that the denitration is mediated by enzymes​3​. The abbreviated version of tyrosine nitration is shown in figure 1a. A more detailed mechanism together with an overview of reactions with other radicals has been compiled by Radi and is shown in the figure 1b​3​.

Mechanism of tyrosine nitration. a) Abbreviated mechanism of tyrosine nitration. First, the carbon in 3-position of the aromatic ring of a tyrosine residue is oxidized by one of various one-electron oxidants resulting in a tyrosine radical. The tyrosine radical then reacts with nitrogen dioxide resulting in 3-nitrotyrosine. b) Detailed mechanism of tyrosine nitration which also gives an overview of other possible reactions of tyrosine radicals​3​.

Tyrosine nitration increases the molecular mass of the modified protein by 45 Da and strongly affects the pKa value of the phenol. Through the neighbouring group participation of the nitro group the pKa is lowered from its original value of 10.0 – 10.5 to 7.0 – 7.5​1–6​. This means that
approximately 50 % of the 3-nitrotyrosines are deprotonated while tyrosines are almost completely protonated at physiological pH. In addition, the nitro group changes the absorption behaviour of the protein in the UV range. The influence of the nitro group on the absorbance was determined by the difference of the UV spectra of tyrosine and 3-nitrotyrosine. The absorbance at 275 nm of 3-nitrotyrosine is approximately four times higher than of a non modified tyrosine residue. Moreover, similar to S-nitrosylation, 3-nitrotyrosine also absorbs light in the range between 320 – 450 nm​7​. Furthermore, the nitro group is a bulky and hydrophobic substituent which may cause local steric restrictions. This can trigger conformational changes and interfere with tyrosine phosphorylation​1,3​. Tyrosine nitration is a highly selective process. Generally only 1 – 5 out of 10’000 tyrosine residues are nitrated. However, there are some proteins where tyrosine nitration is more common​2​.

References

  1. 1.
    Abello N, Kerstjens HAM, Postma DS, Bischoff R. Protein Tyrosine Nitration: Selectivity, Physicochemical and Biological Consequences, Denitration, and Proteomics Methods for the Identification of Tyrosine-Nitrated Proteins. Journal of Proteome Research. 2009;8:3222–3238. doi:10.1021/pr900039c
  2. 2.
    Bartesaghi S, Radi R. Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration. Redox biology. 2018;14:618–625. doi:10.1016/j.redox.2017.09.009
  3. 3.
    Radi R. Protein tyrosine nitration: biochemical mechanisms and structural basis of functional effects. Accounts of chemical research. 2013;46:550–559. doi:10.1021/ar300234c
  4. 4.
    Corpas FJ, Chaki M, Leterrier M, Barroso JB. Protein tyrosine nitration. Plant Signaling & Behavior. 2009;4:920–923. doi:10.4161/psb.4.10.9466
  5. 5.
    Turko IV, Murad F. Protein Nitration in Cardiovascular Diseases. Pharmacological Reviews. 2002;54:619 LP – 634. doi:10.1124/pr.54.4.619
  6. 6.
    Zhan X, Wang X, Desiderio DM. Mass spectrometry analysis of nitrotyrosine-containing proteins. Mass spectrometry reviews. 2015;34:423–448. doi:10.1002/mas.21413
  7. 7.
    Crow JP, Beckman JS. Quantitation of Protein Tyrosine, 3-Nitrotyrosine, and 3-Aminotyrosine Utilizing HPLC and Intrinsic Ultrviolet Absorbance. Methods. 1995;7:116–120. doi:https://doi.org/10.1006/meth.1995.1017

Mass spectrum simulator in Prot pi version 2.2

A new mass spectrum simulator comes with the current version 2.2 of Prot pi. This simulator brings some weighty benefits such as to define the ion adduct, charge state and resolution. An awesome feature is the possibility to integrate multiple compounds in one mass spectrum. This gives amongst others the opportunity to simulate multiple charge state of a compound. In […]

A mass spectrometry imaging software: Quantinetix™

ImaBiotech, founded in 2009 by a team of experts, offers new molecular imaging (called MALDI imaging) services and products to life science industries. This new technology is capable of visualizing and quantifying biological compounds that play role in many pathologies like Cancer, Alzheimer or Parkinson diseases. It is also an interesting tool to follow new […]

Fast ultra-high resolution mass spectrum simulation with Prot pi version 2.1

Version 2.1 of the Protein Tool comes with a completely new software architecture. Even ultra-high resolution (up to 1’000’000) mass spectra of large proteins can now be simulated within sub-seconds. To get the mass spectrum, Prot pi calculates the accurate isotopic distribution using isotope-frequencies and mass defects of the chemical elements. Also the overall calculation time […]

Peptide Tool Released with Prot pi 2.0

We just released the version 2.0 of Prot pi which introduces the Peptide Tool, a brand new software architecture and many improvements for higher performance and stability. Prot pi | Peptide Tool brings a lot of useful features to analyse peptide sequences. Major improvements New Peptide Tool New software architecture Modification data base Minor improvements Several graphical […]

MS-based Glycan Analysis of the Glycoprotein Ricin

1  Background N-glycosylations affect important properties such as the specificity, efficacy, immunogenicity and stability of proteins. A portfolio of methods for glycan analysis was developed, using the murine monoclonal antibody 1RK1, an IgG1κ, to enable N-glycosylations of proteins to be analysed (Fig. 1). The methods developed were subsequently applied to the plant glycoprotein ricin.

Enhanced processing power for faster calculations

The new version 1.2.0.27 of Prot pi is released on 15 March 2015. In this version several computationally expensive procedures were optimized for more efficiency. The overall calculation time is now shortened by up to a factor of five. Major improvements Enhanced processing power and performance optimized algorithm enables faster calculations Optimized scroll behavior for mobile […]

Prediction of potential modification sites in proteins

Some sequence motifs in proteins tend to be modified. Thus, for example an N-terminal glutamine or glutamic acid residue can spontaneously lead to a cyclization. This reaction results in an N-terminal pyroglutamic acid with elimination of ammonia or water, respectively. Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions can spontaneously occur, if the subsequent amino acid […]

Molecular mass and isoelectric point calculator for glycosylated proteins

Glycosylations should not be neglected for the correct calculation of the molecular mass, the isoelectric point and the mass-specific UV absorption coefficient. Therefore Prot pi provides a tool to draw glycans as a posttranslational modification of proteins. This short guide deals with how to add two complex-type N-Glycosylation G1 with a sialic acid (N-acetylneuraminic acid) […]