Characterization of the Site‐Specific Acid‐Base Equilibria of 3‐Nitrotyrosine

The complete macro‐ and microequilibrium analyses of 3‐nitrotyrosine, a biomarker of oxidative stress damage, are presented for the first time. The protonation macroconstants were determined by 1H‐NMR‐pH titration, while microconstants were elucidated by a combination of deductive and NMR methods, in which properties of the methyl ester derivative as an auxiliary compound were also studied. Combination of the NMR‐pH characterization of the title and auxiliary compounds and the pair‐interactivity parameters of 3‐iodotyrosine provided the sufficient system to evaluate all the microconstants. NMR‐pH profiles, macroscopic and microscopic protonation schemes, and species‐specific distribution diagrams are included. The phenolate basicity of 3‐nitrotyrosine is 500 times below that of tyrosine, and it is even lower than that of 3‐iodotyrosine. This phenomenon can be explained by the stronger electron withdrawing and the negative mesomeric effect of the nitro group. Based on our results, 89 % of the phenolic OH groups are deprotonated in 3‐NT molecules at the pH of the blood plasma.


3-Nitrotyrosine (3-NT,
) is the earliest discovered biomarker of nitrosative stress. It is formed through the nitration of tyrosine (Tyr) by peroxynitrite. Peroxynitrite is not a free radical itself but it can become a source of free radicals by an electron accepting process. Being an isomeric form of nitrate, the lifetime of peroxynitrite is a fraction of a second at physiological pH, thus Tyr nitration represents its most important footprint in biological systems. The formation of peroxynitrite in organisms takes place by diffusion-controlled reaction of NO * (nitric oxide) and O 2 *À (anion superoxide), in various, enzyme-associated process, [1] starting mainly from L-arginine. Some in vivo experiments demonstrated its release from nitrones by the action of hydroxyl radical. [2] Peroxynitrite, the deprotonated form of peroxynitrous acid can decompose to * OH and * NO 2 , the latter being the most important nitration agent of Tyr. [3] Tyr nitration takes place in two steps: i) production of tyrosyl radical (Tyr * ) by one-electron oxidation of the phenolic ring and ii) Tyr * and * NO 2 radical-radical coupling reaction with a rate constant of 3 × 10 9 M À 1 s À 1 . Oxidants that can achieve the one-electron oxidation of Tyr are: * OH, The target of electrophilic reagents and/or oxidants in the Tyr structure is the position 3 of the aromatic ring. The nitration of Tyr in proteins in most cases leads to inactivation of the enzymes. Several properties of 3-NT and the proteins, containing it have been described. For example, at low pH, 3-NT is more hydrophobic than Tyr, while at high pH, it is more polar than Tyr. [4,5] Tyr nitration obviously alters the protein structures and concomitantly the physiological effects. These modifications are strongly related to diseases, mainly with degenerative and inflammatory symptoms.
Although the role of 3-NT has been studied in oxidative stress, no previous report was devoted to its microspeciation. Therefore, our aim was to characterize the acid-base properties of 3-NT at the site-specific level. Macroscopic protonation constants (K) are known, based on a previous work which used capillary zone electrophoresis. [6] Nevertheless, we also determined the K values by 1 H-NMR-pH titration method. Macroscopic equilibrium constants of multiprotic molecules give information only on the acid-base properties of the compound as a whole. Site-specific, submolecular basicities can be obtained when microconstants are determined. The site-specific acid-base characterization of 3-NT is therefore of fundamental importance to allow the interpretation of its biological functions at site-specific level.

Results and Discussion
3-NT is a triprotic molecule with phenolate, amino and carboxylate protonation sites. As a triprotic molecule, the total number of microspecies and microconstants are 8 and 12, respectively. The macroscopic-and microscopic protonation schemes are shown in Figure 2.
The relationships between the micro-and macroconstants which has been used for the calculation of microspeciation are as follows:

Equation 3
can be written in six different and equivalent ways based on the Hessian-relationship of protonation constants which means that the sum of protonation constants in logarithmic units is constant between the same start and end-points regardless of the path of protonation.

Determination of Protonation Macroconstants
Evaluation of the protonation constants from 1 H-NMR-pH titration curves was based on the principle that non-exchanging NMR nuclei near the basic site sense different electronic environments upon protonation, and change their chemical shifts accordingly. All carbon-bound protons could be observed.
The protonation macroconstants of 3-NT and its methyl ester derivate were determined by investigating the chemical shift changes of the CH proton and the aromatic proton next to the nitro group in 1 H-NMR-pH titrations ( Figure 3). Since the effect of protonation on the chemical shifts diminishes along with increasing distance from the site of protonation, the aromatic proton signals were much more sensitive for the protonation of the phenolate group, while the chemical shifts of the aliphatic protons changed more upon the protonation of the amino and the carboxylate groups. Some representative NMR spectra at different pH values were depicted in the Supporting Information ( Figure S1).
The protonation constants were determined by nonlinear curve fitting of Equation 19 to the collected data. The values are shown in Table 1.
The macroconstant values determined by capillary electrophoresis earlier and our values are in good agreement.

Complete Microspeciation of 3-Nitrotyrosine
For the determination of all microconstants, the macroconstants of 3-NT and its carboxymethyl deriva-  The data in column marked with * are from the capillary zone electrophoresis-pH studies of Ren et al. [6] Chem. Biodiversity 2019, 16, e1900358 tive, and the pair-interactivity parameters of 3-iodotyrosine from our previous work were used. [7] Protonation of a basic site reduces the basicity of the other basic sites in the molecule. This basicityreducing effect is strong if the sites are in nearby positions, but it gradually fades away along with the increasing distance. The basicity-modifying effect between two moieties can be quantified by the pairinteractivity parameter. For a triprotic molecule, three different pair-interactivity parameters are defined. For example, the interactivity parameter (E) in log units between the amino and the carboxylate sites is: The pair-interactivity parameter is the most invariant and best transferable parameter between molecules having analogous moieties. [8] Due to the similarity between the nitro-and iodo-group, the pairinteractivity parameter of 3-iodotyrosine can be used in the microspeciation of 3-NT as well. The three pairinteractivity parameters were calculated in a previous work, [7] while the standard deviations were based on the Gaussian propagation of uncertainty ( Table 2).
In classical Tyr microspeciation works, [9] UV-pH titrations were successfully used, owing to the overlapping protonations of the phenolate and amino sites, and the selective pH-dependence of the phenolate UV-absorption. For 3-NT, however, this method should not work, due to the negligible overlap between the phenolate and amino protonation regions.
Using the Hessian relationship with the macroconstants of 3-NT and methyl 3-nitrotyrosinate, logk C was calculated as follows: where the protonation macroconstants of 3-NT are in bold, the protonation macroconstants of methyl 3nitrotyrosinate are in normal fonts. After the value of logk C is determined, logk C O , logk C N and logk C O;N can be calculated by using the pair-interactivity parameters and the appropriate form of Equation 4: Applying Equations 1 and 3 for methyl 3-nitrotyrosinate: Furthermore, logk N C;O can be expressed from Equation 4: Then, logk N C;O was introduced into Equation 10: Equations 9 and 12 constitute a system of equations with two unknown parameters (logk O C and logk N C ), resulting in values of 7.06 and 7.41 in log units. Equations 9 and 12, however, do not contain information on the assignments, i. e., which of the above values belong to logk O C and logk N C . The chemical shift-pH profiles (Figure 3), chemical evidences and microconstant values in Table 3 unequivocally indicate that insertion of the electron-withdrawing nitro group in the aromatic ring reduces the basicity of the phenolate to a much greater extent than that of the amino.
Taking also the low diversity of the logk N C values into account, the two parameters could be identified, which also afforded the calculation of the remaining six protonation microconstants using the pair-interactivity parameters and the appropriate form of Equation 4: After all the microscopic protonation constants of 3-NT were determined, some of the values could be verified by the self-consistence of the system. The value of K 1 can be calculated using Equation 1; addi-  Table 4.
The pH-dependent distribution of the eight microspecies (Figure 4) was calculated based on the protonation microconstants in Table 4.
Comparing the microconstants of Tyr, 3-iodotyrosine and 3-NT, it is found that the basicity of the carboxylate and amino sites are similar, unlike the phenolate. This is straightforward to interpret, considering how distant the iodo or nitro group is from the protonation sites. The microconstants describing the phenolate basicity in three compounds are collected  in Table 5 (while all of the microconstants in Tyr, 3iodotyrosine and 3-NT were summarized in the Supporting Information, Table S1).
The substitution of tyrosine with iodine decreases all the respective phenolate microconstants by some 1.4 log units. Changing the iodo group to nitro group results in greater changes and even lower protonation constants. Compared to tyrosine, the decrease is around 2.7 log units. This phenomenon can be explained by the stronger electron withdrawing effect and the negative mesomeric effect of the nitro group. Due to the phenolate microconstants, 89 % of the phenolic OH groups are deprotonated in 3-NT molecules at the pH of the blood plasma (7.40).

Conclusions
Proteins with 3-NT obtain an extra negative site at physiological pH, with several intra-and intermolecular consequences: the 'new' anionic site attracts cationic counterparts, such as arginine guanidinium, lysine, ornithine, terminal ammonium, histidine imidazolium moieties, typically present in the same peptide or protein molecule, causing inevitable conformational modifications. Also, the anionic phenolate is prone to act as an anchor unit to associate with other biomolecules of cationic loci, and to bind metal ions. These changes in conformation, association and composition may all bring about alterations in the biological behavior. [3,4,10,11]

NMR Spectroscopy Measurements
All NMR measurements were carried out on a Varian 600 MHz spectrometer with a dual 5 mm inversedetection gradient probe head at 25°C. The NMR-pH titrations were performed in a mixture of 5 % D 2 O/ 95 % H 2 O (v/v). For titration at 0.15 M ionic strength acidic and basic stock solutions were prepared with concentrations of 0.1 M HCl/NaOH and 0.05 M KCl to ensure 0.15 M ionic strength. The spectra were referenced to internal DSS (sodium 3-(trimethylsilyl)-1propanesulfonate). The sample volume was 600 μl, the titrant and pH indicator concentrations were 5 mM. In the 1 H-NMR experiments, pH values were determined by internal indicator molecules optimized for NMR. [12,13] The concentration of the analytes was 1 mM for the titration. The water resonance was diminished by a double pulse field gradient spin echo sequence (number of transients = 16, number of points = 16384, acquisition time = 3.33 s, relaxation delay = 1.5 s).

Data Analysis
For the analysis of NMR titration curves of proton chemical shifts versus pH, the software Origin Pro 8 (OriginLab Corp., Northampton, MA, USA) was used. In 1 H-NMR-pH titrations the non-linear curve fitting regression analysis option was used with the following function: [8] d obs pH ð Þ ¼ where d L 2À is the chemical shift of the unprotonated ligand (L 2À ), d H i L iÀ 2 values stand for the chemical shifts of successively protonated species of L 2À , n is the maximum number of protons that can bind to the unprotonated ligand, β is the cumulative protonation macroconstant. The standard deviations of logβ values from the regression analyses were used to calculate the Gaussian propagation of uncertainty for the other equilibrium constants.