The Balancing of Peroxynitrite Detoxification between Ferric Heme-Proteins and CO₂: The Case of Zebrafish Nitrobindin

Abstract

Nitrobindins (Nbs) are all-β-barrel heme proteins and are present in prokaryotes and eukaryotes. Although their function(s) is still obscure, Nbs trap NO and inactivate peroxynitrite. Here, the kinetics of peroxynitrite scavenging by ferric Danio rerio Nb (Dr-Nb(III)) in the absence and presence of CO₂ is reported. The Dr-Nb(III)-catalyzed scavenging of peroxynitrite is facilitated by a low pH, indicating that the heme protein interacts preferentially with peroxynitrous acid, leading to the formation of nitrate (~91%) and nitrite (~9%). The physiological levels of CO₂ dramatically facilitate the spontaneous decay of peroxynitrite, overwhelming the scavenging activity of Dr-Nb(III). The effect of Dr-Nb(III) on the peroxynitrite-induced nitration of L-tyrosine was also investigated. Dr-Nb(III) inhibits the peroxynitrite-mediated nitration of free L-tyrosine, while, in the presence of CO₂, Dr-Nb(III) does not impair nitro-L-tyrosine formation. The comparative analysis of the present results with data reported in the literature indicates that, to act as efficient peroxynitrite scavengers in vivo, i.e., in the presence of physiological levels of CO₂, the ferric heme protein concentration must be higher than 10⁻⁴ M. Thus, only the circulating ferric hemoglobin levels appear to be high enough to efficiently compete with CO₂/HCO₃⁻ in peroxynitrite inactivation. The present results are of the utmost importance for tissues, like the eye retina in fish, where blood circulation is critical for adaptation to diving conditions.

1. Introduction

Nitrosative stress plays a key role in the etiology of human diseases, such as atherosclerosis, inflammation, cancer, and neurological diseases, being particularly relevant in the onset of retinopathies and glaucoma [1,2,3]. In fact, reactive nitrogen species (RNS) can cause protein, DNA, and lipid nitration, impairing their functions [4,5,6,7,8,9,10,11]. One of the most potent biological nitrosative agents is peroxynitrite (ONOO⁻), which is produced when nitric oxide (^●NO) and superoxide (^●O₂⁻) are combined at extremely rapid rates [4,5,9,10,11,12,13]. ONOO⁻ diffuses through membrane anion channels as well as the conjugated peroxynitrous acid ONOOH (pK_a = 6.9) [13]. ONOO⁻ is relatively stable, while ONOOH decays rapidly, with an apparent half-life of 1-2 s at physiological pH, yielding ~70% nitrate (NO₃⁻) and H⁺, and ~30% nitrogen dioxide (^●NO₂) and hydroxyl (^●OH) radicals via the homolysis of the O–O bond. The secondary reactions of ^●NO₂ and O^●− lead to NO₂⁻ and O₂ [4,9,10,11,12,13]. ONOO⁻ reacts with biomolecules mainly by a direct reaction or immediately after ONOOH is homolyzed to ^●NO₂ and ^●OH [4,9,10,11,12,13].

At the end of the last century, carbon dioxide (CO₂) was reported to react with ONOO⁻ [14,15]. Since the CO₂ concentration is relatively high in vivo (~1.2 × 10⁻³ M at physiological pH in equilibrium with ~2.4 × 10⁻² M HCO₃⁻ with a pK_a ≈ 6.3 at 25.0 °C), most of the ONOO⁻ rapidly reacts with the CO₂/HCO₃⁻ species (depending on pH), leading mostly to the formation of 1-carboxylato-2-nitrosodioxidane adduct (ONOOCO₂⁻), which displays an apparent half-life of 0.5–3 ms. This compound further decays (via homolysis of the O–O bond), yielding the reactive species trioxocarbonate^●− (CO₃^●−) and ^●NO₂ (3% to 35%), which then proceed towards CO₂ and NO₃⁻ (or by direct yielding of NO₃⁻ and CO₂ (65% to 90%) [9,11,12,13,14,15,16,17,18,19,20,21]). As a whole, the complexity of the peroxynitrite inactivation mechanism can be sketched, as is shown in Scheme 1 [13,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41], where Dr-Nb(III) represents the contribution of heme proteins.

Moreover, the reaction of ONOO⁻ with CO₂/HCO₃⁻/CO₃²⁻ redirects the ONOO⁻/ONOOH-based oxidation of aromatic and aliphatic amino acid residues, facilitating the nitration of tyrosine and tryptophan and limiting the oxidation of methionine and cysteine [5,9,10,11,12,13,14,15,20,21,42,43,44,45]. Remarkably, ^●NO₂ and CO₃^●− radicals are much stronger oxidant species than their precursors ^●NO, O₂^●−, and ONOO⁻/ONOOH [9,10,11,12,13,20,21,46].

Tyrosine nitration through the peroxynitrite pathway (see Scheme 1) is operative in most fish, including zebrafish (Danio rerio), under stressed conditions [47,48]. Further, the effect of the enrichment of CO₂ in the atmosphere brings about the raising of CO₂ levels also in the oceans [49]. Although the increased levels of CO₂ create only limited behavioral effects, they may significantly affect the body response to external insults. In addition, the increased concentration of CO₂ in the blood is of particular relevance in fish, for which retinal circulation is of crucial importance due to the high hydrostatic pressure of the environment where they live [50,51].

Here, the kinetics of ONOO⁻/ONOOH (hereafter peroxynitrite) inactivation by all-β-barrel ferric Danio rerio nitrobindin (Dr-Nb(III)), in the absence and presence of CO₂, are reported and analyzed in parallel with those of all-α-helical globins. The present results open a question on the competition between the protecting activity of heme proteins (which inactivate the damaging reactions of peroxynitrite on L-tyrosine nitration) and the non-protecting action of CO₂/HCO₃⁻, which indeed depends on the levels of ferric heme-proteins. Thus, the levels (and the scavenging activity) of most heme proteins might be too low to act as peroxynitrite scavengers in vivo; only circulating ferric hemoglobin (Hb(III)) levels appear to be high enough to efficiently compete with CO₂/HCO₃⁻/CO₃^2- in peroxynitrite inactivation.

2. Materials and Methods

2.1. Materials

Dr-Nb(III) was cloned, expressed, and purified as already reported [52]. The Dr-Nb(III) concentration was determined spectrophotometrically by measuring the absorbance at 407 nm (ε = 1.57 × 10⁵ M⁻¹ cm⁻¹) [52]. Apo-Dr-Nb was prepared, as already reported [53]. Peroxynitrite was obtained from Cayman Chemical (Ann Arbor, MI, USA). The concentration of peroxynitrite was determined spectrophotometrically at 302 nm (ε = 1.705 × 10³ M⁻¹ cm⁻¹) [12]. L-tyrosine (obtained from Merck KGaA, Darmstadt, Germany) was dissolved in 5.0 × 10⁻² M Bis-Tris propane buffer, at pH 7.0 and 22.0 °C; the final L-tyrosine concentration was 1.0 × 10⁻⁴ M [25,30,36,39]. All the other chemicals were purchased from Merck KGaA (Darmstadt, Germany). All chemicals were of analytical grade and were used without further purification.

2.2. Methods

In the absence and presence of CO₂, peroxynitrite isomerization by Dr-Nb(III) and apo-Dr-Nb was investigated at pH 5.8, 7.0, and 8.5 (5.0 × 10⁻² M Bis-Tris propane buffer) and 22.0 °C, under anaerobic conditions. In the presence of CO₂, peroxynitrite isomerization by Dr-Nb(III) and apo-Dr-Nb was investigated by adding NaHCO₃ (final concentration, 5.0 × 10⁻¹ M) to the Dr-Nb(III) and apo-Dr-Nb solutions; this NaHCO₃ concentration corresponds to different concentrations of CO₂, HCO₃⁻, and CO₃^2- depending on pH. After the addition of NaHCO₃, the Dr-Nb(III) and apo-Dr-Nb solutions were allowed to equilibrate for at least 5 min; then, the pH was readjusted to the desired pH if needed [25,27,28,30,36,39].

Peroxynitrite isomerization by Dr-Nb(III) and apo-Dr-Nb was investigated via rapid mixing of the Dr-Nb(III) and apo-Dr-Nb solutions (final concentration ranging between 5.0 × 10⁻⁶ M and 3.5 × 10⁻⁵ M) with the peroxynitrite solution (final concentration ranging between 2.5 × 10⁻⁵ M and 2.0 × 10⁻⁴ M); no gaseous phase was present. Peroxynitrite isomerization was monitored at 302 nm [9,12,25,26,27,30,36,39] by the SFM-20/MOS-200 rapid-mixing stopped-flow apparatus (BioLogic Science Instruments, Claix, France); the light path of the observation chamber was 10 mm and the dead-time was 1.3 ms.

Values of the pseudo-first-order rate constant for peroxynitrite isomerization by Dr-Nb(III) and apo-Dr-Nb (i.e., k_obs) were determined according to Equation (1) [13,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54,55]:

[peroxynitrite]_t = [peroxynitrite]_i × e ^{− kobs × t}

(1)

Values of the second-order rate constant for peroxynitrite isomerization by Dr-Nb(III) (i.e., k_on), and of the first-order rate constant for the spontaneous decay of peroxynitrite (i.e., k₀), were obtained from the dependence of k_obs on the Dr-Nb(III) concentration, according to Equation (2) [13,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54,55]:

k_obs = k_on × [Dr-Nb(III)] + k₀

(2)

The reaction of peroxynitrite with L-tyrosine was investigated at pH 7.0 and 22.0 °C, as reported elsewhere. The relative nitro-L-tyrosine yield (%) corresponds to: (yield with added Dr-Nb(III) or apo-Dr)/(yield with no Dr-Nb(III) or apo-Dr-Nb) × 100 [13,25,30,34,35,36,39,55,56].

The percentage of NO₃⁻ and NO₂⁻ obtained from peroxynitrite isomerization was determined spectrophotometrically at 543 nm by using the Griess reagent and vanadium(III) chloride (VCl₃) to catalyze the conversion of NO₃⁻ to NO₂⁻, according to literature [25,30,36,39,56].

Kinetic data were analyzed using the GraphPad Prism program, version 5.03 (GraphPad Software, San Diego, CA, USA). The results are given as the mean values of at least four experiments plus and minus the standard deviation. Data were analyzed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) and were expressed as mean values ± standard deviation (SD). Statistical analysis was performed using either the Student’s t-test or the one-way ANOVA comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

3. Results and Discussion

In the absence and presence of CO₂, the absorbance at 302 nm decreases upon mixing the Dr-Nb(III), apo-Dr-Nb, and peroxynitrite solutions over the whole pH range explored. According to the literature [13,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,54,55], this reflects peroxynitrite isomerization. No absorbance spectroscopic changes were observed in the Soret region.

Under all the experimental conditions, the time course of peroxynitrite isomerization in the absence and presence of Dr-Nb(III), apo-Dr-Nb, and CO₂ was fitted to a single-exponential decay for more than 93% of its course, according to Equation (1) (Figure 1, Figure 2 and Figure 3, panels A and B). The values of the pseudo-first-order rate constant for peroxynitrite isomerization catalyzed by Dr-Nb(III) (i.e., k_obs) increase with the heme protein concentration (Figure 1, Figure 2 and Figure 3, panels C and D). Previous experiments have shown that, once bound to a heme protein (likely as Fe(III)-ONOOH), peroxynitrite isomerizes to NO₃⁻ at a rate of ~70 s⁻¹ [13]. Therefore, since the values of k_obs range between 1 × 10⁻¹ s⁻¹ and 8 s⁻¹ (Figure 1, Figure 2 and Figure 3, panel C), they indicate that, under our conditions, the formation of the Dr-Nb(III)-OONOH species represents the rate-limiting step of the catalytic process. Notably, the Dr-Nb(III)-OONOH conversion to Dr-Nb(III) and NO₃⁻ is faster by at least 10-fold than the formation of the Dr-Nb(III)-OONOH complex.

The analysis of the data shown in Figure 1, Figure 2 and Figure 3 (panels C and D), according to Equation (2), allowed us to determine the values of k_on and k₀, corresponding to the slope and the y-intercept of the linear plots, respectively (

Table 1. Effect of pH and CO₂ on the values of k_on and k₀ for Dr-Nb(III)-induced isomerization of peroxynitrite at 22.0 °C ^a.

pH	− CO2		+ CO2
	kon (M−1 s−1)	k0 (s−1)	kon (M−1 s−1)	k0 (s−1)
5.8	(2.2 ± 0.3) × 105	(8.1 ± 0.8) × 10−1	(2.8 ± 0.3) × 105	4.1 ± 0.4
7.0	(4.7 ± 0.5) × 104	(3.1 ± 0.3) × 10−1	n.d.	(1.5 ± 0.2) × 101
8.5	(1.1 ± 0.1) × 104	(4.9 ± 0.5) × 10−2	n.d.	(4.8 ± 0.5) × 101

^a The CO₂ concentration was 1.2 × 10⁻³ M. 5.0 × 10⁻² M Bis-Tris propane buffer. n.d., not determined.

). Moreover, the values of k₀ were measured in the absence of Dr-Nb(III) via the rapid mixing of the peroxynitrite solution with the appropriate Bis-Tris propane buffer solution (Figure 1, Figure 2 and Figure 3, panels E and F). Both in the absence and presence of CO₂, the values of k₀ obtained by the different methods match well with each other (Table 1) and agree well with those previously reported [12,13,25,27,28,30,33,34,36,39,54] (see

Table 2. Effect of pH and CO₂ on values of k_on and k₀ for heme-protein-induced isomerization of peroxynitrite.

Heme-Protein	− CO2		+ CO2
	kon (M−1 s−1)	k0 (s−1)	kon (M−1 s−1)	k0 (s−1)
Ma-Pgb(III) a	3.8 × 104	2.8 × 10−1	n.d.	n.d.
Mt-trHbN(III) b	6.2 × 104	2.7 × 10−1	n.d.	n.d.
Ph-trHbO(III) c	2.9 × 104	2.8 × 10−1	n.d.	n.d.
Cj-trHbP(III) d	9.6 × 105	3.0 × 10−1	8.8 × 105	2.1 × 101
Efc-Mb(III) e	2.9 × 104	3.5 × 10−1	7.7 × 104	1.7 × 101
Pc-Mb(III) f	1.6 × 104	n.d.	n.d.	n.d.
Hs-Hb(III) e	1.2 × 104	3.0 × 10−1	3.9 × 104	1.7 × 101
Mt-Nb(III) g	6.9 × 104	2.6 × 10−1	n.d.	n.d.
At-Nb(III) g	3.7 × 104	3.0 × 10−1	n.d.	n.d.
Dr-Nb(III) h	4.7 × 104	3.1 × 10−1	n.d.	1.5 × 101
Hs-Nb(III) i	3.4 × 104	2.6 × 10−1	n.d.	n.d.

^a pH 7.4 and 20.0 °C. From [55]. ^b pH 7.0 and 20.0 °C. From [34]. ^c pH 7.0 and 20.0 °C. From [35]. ^d pH 7.3 and 25.0 °C. From [36]. ^e pH 7.0 and 20.0 °C. From [25]. ^f pH 7.5 and 20.0 °C. From [27]. ^g pH 7.2 and 25.0 °C. From [40]. ^h pH 7.0 and 22.0 °C. Present study. ⁱ pH 7.1 and 25.0 °C. From [39]. n.d., not determined.

).

As shown in Table 1, the values of k_on for the interaction between Dr-Nb(III) and peroxynitrite were unaffected by CO₂/HCO₃⁻/CO₃²⁻, suggesting that CO₂/HCO₃⁻/CO₃²⁻ does not alter the binding properties of Dr-Nb(III). The values of k_on for peroxynitrite isomerization via all-α-helical globins and all-β-barrel nitrobindins ranged between 1.2 × 10⁴ M⁻¹ s⁻¹ for Hs-Hb(III) [25] and 6.9 × 10⁴ M⁻¹ s⁻¹ for Mt-Nb(III) [40] (Table 2), suggesting that the very different structural organization [40,57,58,59,60,61] is not at the root of the different rate of peroxynitrite isomerization. However, the coordination of the heme-Fe(III) atom, the in- or out-of-plane position of the metal with respect to the pyrrole nitrogen atoms of the porphyrin, the ligand accessibility of the heme-Fe(III) atom, and its Lewis acidity may tune the kinetics of the related peroxynitrite decomposition [36].

To outline the role of the metal center, the values of k_obs have been determined as a function of (i) apo-Dr-Nb concentration (at a fixed peroxynitrite concentration) and (ii) peroxynitrite concentration (at fixed Dr-Nb(III) concentration). Apo-Dr-Nb does not induce the isomerization of peroxynitrite; in fact, the values of k_obs and k₀ match each other in the presence of apo-Dr-Nb (Figure 1, Figure 2 and Figure 3, panels E–H), as reported for apo-Efc-Mb, apo-Hs-Hb, and apo-Hs-Nb [25,39]. As shown in Figure 4, the values of k_obs slightly decrease with an increasing peroxynitrite concentration, reflecting either the slow isomerization process of peroxynitrite-peroxynitrous acid dimers or their slow dissociation preceding the Dr-Nb(III)-catalyzed isomerization of peroxynitrite [30,36].

As shown in Figure 1, Figure 2 and Figure 3 (panel D) and in Figure 4 (panel B) it turns out that, in the presence of 5.0 × 10⁻¹ M CO₂/HCO₃⁻/CO₃²⁻, the role of Dr-Nb(III) in characterizing the rate constant of peroxynitrite isomerization becomes progressively less relevant as the pH is raised since values of k₀ for peroxynitrite isomerization obtained in the presence of CO₂/HCO₃⁻/CO₃²⁻ are faster by about two orders of magnitude than those obtained in its absence (Table 1). This indicates that CO₂/HCO₃⁻/CO₃²⁻ dramatically speeds up the decay of peroxynitrite through direct interaction, without interfering with peroxynitrite binding to Dr-Nb(III) [9,12,13,25,27,28,30,32,33,34,36].

To clarify the differential roles of various ferric heme proteins and CO₂/HCO₃⁻/CO₃²⁻ levels in peroxynitrite detoxification, the overall effect of pH and CO₂/HCO₃⁻/CO₃²⁻ on the observed rate of peroxynitrite isomerization was investigated. Thus, the protonation equilibria of OONO⁻/HOONO and CO₂/HCO₃⁻/CO₃²⁻ were considered. The effect of CO₂ on peroxynitrite isomerization in the absence of heme proteins (i.e., k_obs) can be described by Equation (3):

$$\begin{array}{c}{k}_{\mathrm{obs}}=\left(\frac{K_{\mathrm{P}}}{\left[{\mathrm{H}}^{+}\right]+K_{\mathrm{P}}}\right)\times \left\{k_0+\left[\mathrm{L}\right]\times \left(\frac{k_{\mathrm{c}1}\times {\left[{\mathrm{H}}^{+}\right]}^2+k_{\mathrm{c}2}\times K_{\mathrm{c}1}\times \left[{\mathrm{H}}^{+}\right]+k_{\mathrm{c}3}\times K_{\mathrm{c}1}\times K_{\mathrm{c}2}}{{\left[{\mathrm{H}}^{+}\right]}^2+K_{\mathrm{c}1}\times \left[{\mathrm{H}}^{+}\right]+K_{\mathrm{c}1}\times K_{\mathrm{c}1}}\right)\right\}+\\ +\left(\frac{\left[{\mathrm{H}}^{+}\right]}{\left[{\mathrm{H}}^{+}\right]+K_{\mathrm{p}}}\right)\times \left\{k_0^{\mathrm{H}}+\left[\mathrm{L}\right]\times \left(\frac{k_{\mathrm{c}1}^{\mathrm{H}}\times {\left[{\mathrm{H}}^{+}\right]}^2+k_{\mathrm{c}2}^{\mathrm{H}}\times K_{\mathrm{c}1}\times \left[{\mathrm{H}}^{+}\right]+k_{\mathrm{c}3}^H\times K_{\mathrm{c}1}\times K_{\mathrm{c}2}}{{\left[{\mathrm{H}}^{+}\right]}^2+K_{\mathrm{c}1}\times \left[{\mathrm{H}}^{+}\right]+K_{\mathrm{c}1}\times K_{\mathrm{c}2}}\right)\right\}\end{array}$$

(3)

where k₀ and k₀^H are the intrinsic degradation rates of OONO⁻ and HOONO, respectively, [H⁺] (= 10^−pH) is the proton concentration, K_P (= ([OONO⁻] × [H⁺])/[HOONO]) is the protonation constant of peroxynitrite, [L] is the reactant concentration (in our case [HCO₃⁻] = 5.0 × 10⁻¹ M), K_c1 ([H₂O]) (= ([HCO₃⁻] × [H⁺])/[CO₂] = 10^−6.34) is the protonation equilibrium constant between HCO₃⁻ and CO₂, K_c2 (= ([CO₃²⁻] × [H⁺])/[HCO₃⁻] = 10^−10.25) is the protonation equilibrium constant between CO₃²⁻ and HCO₃⁻; k_c1 and k_c1^H are the second-order rate constants for the reaction of CO₂ with OONO⁻ and HOONO, respectively, k_c2 and k_c2^H are the second-order rate constants for the reaction with HCO₃⁻ of OONO⁻ and HOONO, respectively, and k_c3 and k_c3^H are the second-order rate constants for the reaction with CO₃²⁻ of OONO⁻ and HOONO, respectively.

The pH-dependence of peroxynitrite degradation in the absence of CO₂/HCO₃⁻/CO₃²⁻ (i.e., [L] = 0, see Equation (3)), allowed us to determine the values of k₀ (= 3.0 × 10⁻² s⁻¹), k₀^H (= 8.3 × 10⁻¹ s⁻¹) and K_P (= 10^−6.8) (Figure 5, panel A), showing that HOONO decays to NO₃⁻ faster than OONO⁻, with a pK_a ≈ 6.8 [13,36]. On the other hand, as already outlined before [9,13,22,27,28,30,36,39], when CO₂/HCO₃⁻/CO₃²⁻ is present (i.e., [L] = 5.0 × 10⁻¹ M), the rate of peroxynitrite degradation becomes much faster and increases with a rising pH (see Table 1 and [36]). Therefore, employing Equation (3) and knowing the values of K_c1 and K_c2 (see above), the pH dependence of peroxynitrite degradation (Figure 5, panel B) gives information on the different values of k_ci and k_ci^H (with i = 1, 2, 3). The inspection of

Table 3. Values of the parameters fitting data with Equation (3).

Parameter	Value
k0 (s−1)	(3.0 ± 0.5) × 10−2
k0H (s−1)	(8.3 ± 1.0) × 10−1
KP (M)	(1.6 ± 0.3) × 10−7
Kc1 (M)	(4.6 ± 0.9) × 10−7
Kc2 (M)	(5.6 ± 1.2) × 10−11
kc2 (M−1s−1)	(6.2 ± 1.3) × 101
kc3 (M−1s−1)	(1.7 ± 0.4) × 103
kc1H (M−1s−1)	8.0 ± 2.1

allows for the following considerations:

(i) k_c1, k_c2^H, and k_c3^H are not playing any role, likely because either one or both reactants are too scarcely populated over the pH range investigated for the pseudo-first-order rate constant to have a detectable value, which can then be considered as ≈0 s⁻¹;

(ii) At pH ≤ 6.2, the peroxynitrite degradation rate is mostly characterized by the reaction between HOONO and CO₂, corresponding to k_c1^H = 8.0 M⁻¹ s⁻¹);

(iii) For 6.2 < pH < 8.2, the peroxynitrite degradation rate is mostly characterized by the reaction between OONO⁻ and HCO₃⁻, corresponding to k_c2 = 6.2 × 10¹ M⁻¹ s⁻¹;

(iv) For pH ≥ 8.2, the peroxynitrite degradation rate is mostly characterized by the reaction between OONO⁻ and CO₃²⁻, corresponding to k_c3 = 1.8 × 10³ M⁻¹ s⁻¹.

Obviously, the value of k_obs and its pH dependence depend on the CO₂/HCO₃⁻/CO₃²⁻ levels (i.e., [L]), envisaging that the relative levels of the ferric heme protein and CO₂/HCO₃⁻/CO₃²⁻ are crucial in defining the respective role for peroxynitrite detoxification. Of note, data reported in Figure 1, Figure 2 and Figure 3 (panel D) refer to [CO₂/HCO₃⁻/CO₃²⁻] = 5.0 × 10⁻¹ M, while in the bloodstream, the physiological levels of CO₂/HCO₃⁻/CO₃²⁻ (~3.0 × 10⁻² M) are about 10-20 fold lower. Therefore, under these conditions, k_obs decreases significantly even in the presence of CO₂/HCO₃⁻/CO₃²⁻, as indicated by Equation (3) (Figure 5, panel C). The pH-dependent mechanism of peroxynitrite degradation can be described by Scheme 2, which reports the different pathways for peroxynitrite degradation.

According to Equation (3), the effects of the heme protein and CO₂/HCO₃⁻/CO₃²⁻ levels were simulated (Figure 5, panel C), with the values of the rates at [CO₂/HCO₃⁻/CO₃²⁻] = 3.0 × 10⁻² M and [Dr-Nb(III)] = 3.0 × 10⁻⁵ M (see Figure 1, Figure 2 and Figure 3, panels C). At acidic pH values, the role of Dr-Nb(III) (= 3.0 × 10⁻⁵ M) is prevalent for peroxynitrite isomerization, while at a neutral pH the role of Dr-Nb(III) (= 3.0 × 10⁻⁵ M) is equivalent to that of the CO₂/HCO₃⁻/CO₃²⁻ system (Figure 5C). For alkaline pH values, Dr-Nb(III) levels higher than 3.0 × 10⁻⁵ M would be required to play a relevant role in peroxynitrite detoxification (see Figure 5, panel C).

The relative importance of the heme proteins and CO₂/HCO₃⁻/CO₃²⁻ catalyzing the peroxynitrite isomerization is crucial since, according to the literature [9,12,25,30,36,62,63], ferric heme proteins prevent L-tyrosine nitration, which, instead, occurs either in the presence of apo-Dr-Nb and/or of CO₂/HCO₃⁻/CO₃²⁻ (Figure 6) (see also Scheme 1). In fact, the relative yield of NO₃⁻ and NO₂⁻, obtained from peroxynitrite isomerization catalyzed by Dr-Nb(III), ranged between 89 and 92%, and between 7 and 12%, respectively. However, in the absence of Dr-Nb(III) and/or in the presence of apo-Dr-Nb and/or CO₂/HCO₃⁻/CO₃²⁻, the values of the relative yield of NO₃⁻ and NO₂⁻ ranged between 69 and 74%, and between 7 and 12%, respectively (

Table 4. Percentage of NO₃⁻ and NO₂⁻ obtained from peroxynitrite isomerization at pH 7.0 and at 22.0 °C ^a.

Dr-Nb(III)	Apo-Dr-Nb	CO2	NO3−	NO2−
(M)	(M)	(M)	(%)	(%)
0.0	0.0	0.0	73 ± 8	28 ± 3
0.0	0.0	1.2 × 10−3	69 ± 7	30 ± 3
0.0	3.5 × 10−5	0.0	71 ± 8	29 ± 3
0.0	3.5 × 10−5	1.2 × 10−3	74 ± 7	25 ± 2
3.5 × 10−5	0.0	0.0	89 ± 9	12 ± 2
3.5 × 10−5	0.0	1.2 × 10−3	93 ± 8	7 ± 1
3.5 × 10−5	3.5 × 10−5	0.0	91 ± 9	10 ± 1
3.5 × 10−5	3.5 × 10−5	1.2 × 10−3	92 ± 9	8 ± 1

^a The peroxynitrite concentration was 2.0 × 10⁻⁴ M; 5.0 × 10⁻² M Bis-Tris propane buffer.

). The great relevance of these observations is confirmed by the fact that, under stressed conditions, zebrafish triggers a defense mechanism through nitrosative stress and peroxynitrite production for which its degradation may be, on one side, enhanced by the elevated levels of CO₂ [48,49], but, on the other side, inhibited by the consequent lowering of the pH level. In any event, the correlation between the peroxynitrite degradation by ferric heme proteins and CO₂ levels appears to be relevant within in vivo models of zebrafish, and it may have important consequences for the O₂ supply to poorly oxygenated tissues, such as the retina [50,51].

4. Conclusions

Ferric heme proteins and the CO₂/HCO₃⁻/CO₃²⁻ system are the major players in peroxynitrite detoxification, for which efficiency depends on both the concentration of these two actors and the pH level. CO₃²⁻ is much more effective at peroxynitrite inactivation, as compared to CO₂, with HCO₃⁻ displaying an intermediate activity. Of note, the CO₃²⁻ levels were much lower than those of the other two components in the system under physiological conditions. Although ferric heme proteins are intrinsically much more effective than the CO₂/HCO₃⁻/CO₃²⁻ system, their levels are usually significantly lower than CO₂/HCO₃⁻/CO₃²⁻. Since over the physiological pH range the rate of peroxynitrite detoxification by CO₂/HCO₃⁻/CO₃²⁻ (i.e., ~3.0 × 10⁻² M) is ~1.5 s⁻¹, values of k_obs (= k_on × [heme-Fe(III)]), for peroxynitrite scavenging by ferric heme-proteins must be larger than 2 s⁻¹. Overall, only the level of the circulating Hs-Hb(III) (~2.0 × 10⁻⁴ M; corresponding to about 2–3% of the total Hs-Hb in red cells) appears to be sufficient to play a relevant role in the detoxification by peroxynitrite under physiological conditions.

Furthermore, it must be remarked that acidification, which often occurs in the bloodstream of fish when they dive to depth [64,65,66] and can even be magnified by increased levels of CO₂ [49], decreases the effect of the CO₂/HCO₃⁻/CO₃²⁻ system, rendering the role of ferric heme proteins even more crucial. This is especially important in the ocular system and in the protection of the retina against oxidative stress linked to peroxynitrite [3,50]. Therefore, under specific environmental conditions (typical of diving fish), Dr-Nb(III) indeed may play a relevant physiological role in peroxynitrite scavenging from poorly oxygenated tissues, such as the retina.