Fig. 3. Solubility in water of nitric oxide under 1 atm NO as a function of temperature. (From Ref. .)
0 20 40 60 80 100
Fig. 3. Solubility in water of nitric oxide under 1 atm NO as a function of temperature. (From Ref. .)
Table 2 Solubility of selected small neutral molecules in pure water under a gas pressure of 1 atm. The solubilities are in millimolar concentration
In pure deoxygenated water and at low concentrations, NO^ is indefinitely stable on a timescale of weeks. In particular, it does not undergo a hydration reaction. However, this stability is only apparent due to slowness of the irreversible disproportionation reaction Eq. (1).
At high concentrations as in compressed gases, the disproportionation reaction (1) is very noticeable and leads to rapid formation of substantial quantities of secondary radical like nitrogen dioxide NO2. The latter radical is a strong one-electron oxidant with a reduction potential of 1.04 V  for the NO2/NO- redox couple. Nitrogen dioxide rapidly decomposes in water. At lower concentrations of nitric oxide, the disproportionation reaction (1) is kinetically inhibited, but NO2 may still be formed by a complicated reaction of NO^ with dioxygen. For biological samples, the formation of this NO2 intermediate is highly significant due to its capacity to generate further species capable of N-nitrosation of amines and S-nitrosation of thiols and organic sulfides. Therefore, a short outline will be given here, with a comprehensive discussion being found in the excellent recent monograph of Williams .
The formation of NO2 from nitric oxide and dioxygen has a small activation energy of AH = 4.6 ± 2.1 kJ/mol. The precise mechanism has not been clarified and two pathways have been proposed. The first is based on the tendency of NO^ to dimerize to dinitrogen dioxide. This species may be oxidized to dimerized nitrogen dioxide.
N2O4 ^ 2NO2
The second proposition involves the formation of an intermediate peroxynitrite radical
ONOONO ^ 2NO2 Both mechanisms have a net balance
Upon completion of reaction (7c), the appearance of nitrosating species like N2O3 and N2O4 is unavoidable. Although not radicals themselves, these species are quite reactive.
This fact is highly relevant for biological systems, where the pool of proteins provides a potential target for subsequent nitrosating reactions of amine and thiol moieties. The equilibrium reactions are
The forward and backward reaction rates of Eq. (8) were reported as 4.5 x 108 (Ms)-1 and 6.9 x 103 s-1, respectively. The forward and backward reaction rates of Eq. (9) were reported an order of magnitude faster with 1.1 x 109 (Ms)-1 and 8.1 x 104 s-1, respectively .
The powerful nitrosating species dinitrogen trioxide N2O3 is easily identified from its weak blue color (£620 ~ 20 Mcm-1). It is a highly polar molecule with a dipole moment of 2.122 D, i.e. exceeding the moment 1.854 D of a water molecule. Experimental evidence supports the usual assumption that dinitrogen trioxide N2O3 is the main nitrosating species in buffered water solutions near physiological pH, in spite of the fact that it may easily react with water molecules [see below, Eq. (12)]. However, the situation is less clear for living systems, as these form a heterogeneous environment with regions of low polarity. In such regions, neutral molecules like NO2 or NO^ tend to accumulate to higher concentration with different equilibrium constants for the equilibria between NO2, NO\ N2O3 and N2O4 . This phenomenon significantly accelerates the autooxidation reaction Eq. (1) . The nitrosation reactions that transform thiol moieties RS—H to nitrosothiols RS—NO are
These nitrosation reactions are fast. For glutathione (GSH), reaction (10b) proceeds with a second order rate of k = 6.6 x 107 (Ms)-1 .
These reactions are distinguished by their primary reaction products being nitrate and nitrite, respectively. In principle, nitrosonium is a nitrosating species also. Its nitrosation reaction with thiols RS—H proceeds as
However, in aqueous environment the concentration of free nitrosonium is negligibly small [cf Eq. (3b) and its discussion].
In aqueous solution, the nitrosation reactions (10a) and (10b) have to compete with an alternative pathway: hydrolysis to nitrite or nitrate according to the reactions
The hydrolysis pathway leaves N2O4 with a lifetime of about 10-3 s in water . Published rates for (12b) vary considerably [42,43], possibly because the reaction is catalyzed by the presence of phosphate. But even in absence of phosphate, reaction (12b) is fast, and the lifetime of N2O3 in water is shorter than ca 1 ms . Therefore, the nitrosation reactions (10a,b) will be significant only at sufficiently high levels of thiol. The critical thiol concentration can be estimated from the nitrosation rate of k = 6.6 x 107 (Ms)-1  for glutathione and the lifetime of 1 ms for N2O3 in water. It means that nitrosation of glutathione dominates hydrolysis if [GSH] > 15 ^M. This condition is easily satisfied in vivo where tissue concentrations of GSH are of the order of 0.5-1 mM (cf Table 1, Chapter 9). Phrased otherwise, at physiological thiol concentrations, the hydrolysis pathway (12a,b) is not significant and thiols act as main target for the nitrosating species N2O4 and N2O3.
It should be kept in mind that N2O3, NO+ and N2O4 are by no means the only compounds with proven capacity of S-nitrosation. Alternative pathways for S-nitrosation have been identified for organic nitrates RONO2 and organic nitrites RONO. More details on these reactions are given in Chapters 9, 10 and 17.
The formation of S-nitrosothiols has significant implications in vivo. The S-nitrosated moieties in tissues reach concentrations upto ca 100 nM. Therefore, these moieties are potentially relevant as an endogenous form of nitric oxide with higher stability and longer lifetime than nitric oxide itself. The S-nitrosothiols are quite stable with a lifetime of several days in aqueous solutions if these solutions are kept cool in the dark and free of trace metal ions. They may release free NO^ radicals by hemolytic cleavage via thermolysis, photolysis or catalytically by trace metal ions in reduced state like Fe2+ or Cu+. The possibility of heterolytic cleavage of the S—N bond under release of free NO+ is still controversial. As will be explained in Chapters 9-11, the homolytic decomposition has high significance for the balance between the various nitrosated species in biological systems.
The balance of the above reactions makes the rate of nitrite formation third order in the constituents d2
The rate of k ~ 5 x 106 M-2s-1 (at 25°C) does not depend on the pH .
The formation of the NO2 intermediate is the rate-limiting step in this oxidation of NO^. Accordingly, it also determines the rate of nitrosation of thiol moieties in biological systems. The low rate guarantees that the uncatalyzed oxidation to nitrite does not significantly affect the lifetime of NO^ in aqueous solutions. Even at full oxygen saturation ([O2] ~ 1 mM), the nanomolar concentrations of NO^ make that this radical would have a lifetime of many hours if kept in aqueous solution. However, in complex biological systems, the presence of a membrane fraction may strongly enhance the observed rate of autooxidation and nitrosating capacity [39,44]. The small dipole moment of NO^ (cf Table 2) allows this radical to concentrate in the hydrophobic phospholipid membranes and reaches a tenfold higher local concentration than in aqueous medium. This concentration effect increases the rate of autooxidation 300-fold . In addition, spurious metal ions or enzymatic reaction centers could act as catalyzers and accelerate the oxidation reaction (7c). The ensuing nitrosation reactions would be accelerated as well. At the moment, it is still unknown whether such nonenzymatic autooxidation reactions are significant for the NO^ lifetime and nitrosation in biological systems.
In addition, enzymatic pathways exist for the oxidation of NOV In biological systems, at least two important irreversible pathways for oxidation of NO^ have been identified: oxy-hemoproteins and superoxide. Hemoglobin (Hb) is a tetrameric heme protein composed of pairs of ferrous a- and P-subunits. The four hemes may be independently nitrosylated and the ferrous forms are paramagnetic. Accordingly, blood and many biological tissues show the prominent EPR absorption as a mixture of a- and P-type heme nitrosylated ferrous state . The spectral shape depends on oxygen concentration and conformation of the Hb (relaxed-R of tense-T). In fully oxygenated blood, the dominant species is a(Fe—O2)2—P(Fe—O2)2 which rapidly oxidizes nitric oxide to nitrate
Equation (14) has a high reaction rate of ~1 x 107 (Ms)-1 and is the dominant reaction pathway for loss of nitric oxide in the normoxic vasculature. At first sight, reaction (14) might look like iron-mediated oxidation of NO^ by dioxygen. However, the rate depends crucially on whether dioxygen or nitrosyl is the first ligand to bind, since nitrosylated Hb itself is remarkably stable against transformation into metHb and nitrate by dioxygen. In oxygenated Tris buffer (50 mM, pH = 7.4) the lifetimes of a(Fe—NO)2—P(Fe—O2)2 and a(Fe—NO)2—P(Fe—NO)2 were 2 h and 41 min, respectively . Interestingly, the partially nitrosylated a(Fe—NO)2—P(Fe—O2)2 retained significant capacity for cooperative binding and transport of oxygen. Myoglobin is an oxygen transporter with a single heme-binding site. Oxymyoglobin also rapidly converts nitric oxide to nitrate with a comparable rate .
An even higher reaction rate applies to the diffusion-controlled reaction with the superoxide radical to peroxynitrite, an isomer of nitrate
Reported reaction rates in water range from 4.3 x 109 to 1.9 x 1010 (Ms)-1 . Per-oxynitrite, though not itself a radical, is a powerful oxidizing agent and has been shown to be highly damaging to intracellular processes. It reacts with many proteins , particularly via the nitration of tyrosine residues and thiols. Peroxynitrite readily acts as a ligand for transition metal ions  and the metal-containing sites of many enzymes, hemoproteins in particular, are prone to modification by peroxynitrite leading to inhibition of catalytic activity (e.g. superoxide dismutase, cytochromes and nitric oxide synthase) or structural degradation (e.g. Zn release from zinc-finger-containing proteins). In water, the peroxynitrite anion is stable (£302 ~ 1704 Mcm-1), but the protonized form (peroxynitrous acid, cis-ONOOH with pKa = 6.8, trans-ONOOH with pKa ~ 8) is unstable, because the trans-isomer is a vibrationally excited state that can rearrange to nitric acid . The rearrangement shortens the lifetime of peroxynitrous acid to less than 1 s.
It was already remarked that NO^ is thermodynamically unstable and prone to dismutation. Recent investigations have shown that transition metal ions may catalyze the autooxidation of NO^ . Dinitrosyl iron complexes (DNIC) with small thiol ligands possess S-nitrosating capacity reminiscent of nitrosonium, and the formation of hydroxylamines was attributed to intermediate nitroxyl NO- anions. It suggests that the iron atom mediates a strong electronic coupling between the nitrosyl ligands and facilitates their dismutation into nitrosonium and nitroxyl. A more detailed discussion is given in Chapter 2.
Finally, a nonnegligible decay channel for NO^ may be its sequestration by ferrous iron as found in heme enzymes like hemoglobin. Although nitric oxide binds to ferrous as well as ferric form, the ferrous binding is so strong as to be considered irreversible on the relevant timescales of minutes to hours. The strong binding of NO^ ligands to ferrous heme forms a very stable paramagnetic mononitrosyl iron complex. It was reported that ca 0.004% of Hb-heme is nitrosylated in healthy human volunteers . Given that blood has a heme concentration of [Hb-heme] = 4 [Hb] ~ 8 mM, the quantity of nitrosylated heme in arterial blood amounts to ca 0.3 ^M. This is a significant quantity indeed. The binding of nitrosyl ligands to iron strongly affects the spectroscopic properties of the complex. The fundamental intramolecular N-O vibration appears at 1878 cm-1 in the gas phase, but is redshifted upon binding to heme iron. The vibration is easily observable by strong IR absorption and moderate intensity of Raman scattering and may be used to discriminate the redox state, degree of coordination and conformation of the protein.
The sequestration by iron allows hemoglobin of the blood to act as a very significant sink for the nitric oxide radicals as produced by the endothelial lining of blood vessels. Numerical simulations indicate that the loss of nitric oxide in the vascular lumen should reduce the NO^ levels in the smooth muscle tissue as well [52,53] to levels below the activation threshold of the guanylate cyclase enzyme. Therefore, it might still be premature to simply equate NO^ with the endothelial relaxation factor (EDRF). We should keep an open mind to the possibility that EDRF be some reaction product of primary NO^ that is more stable against the oxidation in the vascular system and may be either reconverted to truly free NO^ or share certain NOMike properties like vasodilation or inhibition of platelet aggregation. For fulfilling its signaling function, important parameters are the lifetime, diffusion rate and the ability to cross biological membranes. In recent years, a surprisingly large variety of such stabilizing forms of NO^ have been proposed and discussed in the literature. Some of them share only some of the properties of NO^ but lack others. For example, S-nitrosothiols are good inhibitors of platelet aggregation but rather inefficient activators of the guanylate cyclase enzyme. DNIC complexes are excellent vasodilators and inhibitors of platelet aggregation (cf Chapter 3). In the bloodstream of animals, they have much longer lifetime than NO^ itself. However, these complexes also may release some free iron with the risk of toxic effects on tissues or individual cells. Nitrite anions are small and show rapid diffusive transport in water. They are known to cross the membranes of red blood cells, but their entry into other cell types is uncertain. Nitrite fails to prevent platelet aggregation  but can induce relaxation of precontracted vessel rings . The purpose of this book is to give an overview of these alternative forms of NO^ and to compile the diverse and scattered literature in the form of a monograph. Some readers may have noticed the absence of nitrate from the list of topics covered. Although dedicated nitrate reductases exist in certain strains of bacteria, they are not known as a truly mammalian enzyme. Although a modest degree of nitrate reduction is known from symbiotic bacteria in mouth and intestinal tract , this xenobiotic pathway does not seem to contribute significantly to endogenous nitrite or NO. However, certain mammalian enzymes are capable of reducing nitrate under hypoxia . Biological systems are highly complex and often show very unexpected properties. The recent findings about reduction of nitrite anions under hypoxia are a good example of such unexpected results. The progress in the field is very rapid, and the topics discussed should be considered in statu nascendi with many more surprises to come in the future.
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