The Mechanism Of Rsno Synthesis Catalyzed By Iron

In principle, one could expect that ferric iron catalyzes the synthesis of RS-NO from thiols and NO through the oxidative pathways similar to those shown for Cu2+ ions in Schemes 3 and 4. However, cells and tissues contain mM levels of glutathione. With [GSH]/[GS-SG] ~ 100 firmly on the reduced side, one would expect that endogenous iron be kept dominantly ferrous state. In buffered solutions, the redox state of iron is easily determined but the situation in biological systems is not clear, and the existence of an endogenous pool of ferrous iron in cells and tissues is still a matter of debate. EPR spectroscopy of frozen tissues usually shows a significant quantity of ferric iron complexes in high-spin state (Fe3+ with S = 5/2 is observable with EPR at the characteristic position g ~ 4.3. cf Chapters 2 and 5). But there is consensus that ferrous irons provide at least a large or dominant fraction of the loosely bound iron pool. In recent years, EPR spectroscopy has provided strong experimental support for the catalytic action of ferrous iron in the synthesis of endogenous RS-NO, but the reaction mechanism is very different from that of monovalent Cu+ in that EPR always shows the formation of some quantities of dinitrosyl-iron complexes. Chapter 2 discussed the properties of DNICs with thiol-containing ligands and noted the possibility that the iron atom mediate the disproportionation of the nitrosyl ligands into nitrosonium cation and nitroxyl anion, respectively. Nitrosonium has known capability to S-nitrosate thiols. We propose that the DNIC contribute significantly to the catalytic S-nitrosation of thiols. The disproportion is an important intermediate step in this catalytic cycle and was demonstrated in DNIC with thiol ligands as well as with ligands of the non-thiol class (Chapter 2).

We demonstrated [33,10] that the formation of Cys-NO and GS-NO from thiols and free NO is catalytically accelerated by the presence of ferrous iron. These experiments were carried out in anaerobic aqueous solutions. The first investigation [33] found that exposure of 1-3 mM cysteine or glutathione in HEPES buffer (15 mM, pH 7.0) caused formation of Cys-NO or GS-NO. The S-nitrosothiols were detected by their optical absorption at 340 nm. The yield of RS-NO calculated on a thiol basis reached 30-40%. The formation of RS-NO was enhanced 1.5-2 times by the addition of ferrous salt (to 20 ^M) and completely suppressed by the selective iron chelator o-phenanthroline (250 ^M). These results prove that ferrous iron, whether from spurious contaminations or exogenously added, contributes to the process.

The effects of pH and oxygen were studied in a subsequent investigation [10]. The yields of Cys-NO or GS-NO are higher at low pH (Fig. 15, curve a). The experiments were performed with 50 mM solutions of cysteine or glutathione in 2 ml deoxygenated HEPES buffer (15 mM) in an evacuated Thunberg vial with 100 ml volume. With an NO gas pressure of 125 mm Hg, the quantity of gaseous NO in the head space of the solution was 1.00 ± 0.05 mmol. No exogenous iron was added. Under exclusion of oxygen, only 4-6% of the thiol was converted to nitrosothiol. The conversion increased to 25% when 8 ^mol of O2 was added together with 1 mmol NO in acid and neutral thiol solutions (Fig. 15, curve b). The pH remained stable, showing that quantities of NO2, if formed at all, remained far below the buffering capacity of the HEPES. The conversion ratio was larger if the initial thiol concentration was decreased. Table 1 quotes the conversion ratios for glutathione in HEPES.

In solutions with higher thiol concentration the conversion was incomplete, but could be enhanced by repeated treatment of these solutions with the NO + O2 mixture. The conversion has a clear maximum as a function of the NO dose. At a dose of 8 ^mol O2, the RS-NO yield was highest at NO pressures of 60-150 mm Hg (0.5-1.2 mmol NO in total). At still higher pressures the RS-NO yield decreased, finally approaching zero at a NO pressure of 600-700 mm Hg.

The addition of selective Fe2+ chelator, o-phenanthroline (250 ^M) prior to addition of NO or O2 completely inhibited the formation of RS-NO. The effect is illustrated in Fig. 16. It shows the effect of o-phenanthroline on the optical absorption of a solution with 50 mM glutathione. The formation of GS-NO is completely inhibited by the presence of o-phenanthroline. It shows that NO itself is not a good S-nitrosating agent by itself. Rather, the catalytic action of ferrous iron is needed to achieve the S-nitrosation of glutathione. Further evidence is the enhancement of RS-NO yields by the addition of extra Fe2+ to thiol solutions. GS-NO yields were enhanced by a factor 1.5 if 20-200 ^M Fe2+ was added to the solutions prior to the exposure to the mixture of NO and O2. These remarks apply to pH < 7 (Fig. 15).

Fig. 15. The pH dependence of the yields of Cys-NO (top) and GS-NO (bottom) in 2 ml HEPES after exposure to nitric oxide. The thiol concentration was 50 mM. (Curve a) Low yields after exposure to 1 mmol NO in absence of oxygen and (Curve b) Much higher yields after exposure to a mixture of 1 mmol NO + 8 ^mol O2. The triangles in the lower panel show the yields if 50 ^mol FeSO4 was added prior to the treatment with NO or NO + O2, respectively. (From Ref. [10].)

Fig. 15. The pH dependence of the yields of Cys-NO (top) and GS-NO (bottom) in 2 ml HEPES after exposure to nitric oxide. The thiol concentration was 50 mM. (Curve a) Low yields after exposure to 1 mmol NO in absence of oxygen and (Curve b) Much higher yields after exposure to a mixture of 1 mmol NO + 8 ^mol O2. The triangles in the lower panel show the yields if 50 ^mol FeSO4 was added prior to the treatment with NO or NO + O2, respectively. (From Ref. [10].)

Table 1 Ratios of conversion of glutathione to GS-NO by spurious iron in 2 ml HEPES (15 mM, pH = 8.0) by gaseous NO in presence of oxygen. The NO and O2 doses are 0.65 mmol and 8 ^mol, respectively. The conversion ratios were determined with optical absorption spectroscopy. The reaction proceeded at room temperature for 10 min. The conversion could be inhibited by 250 ^M o-phenanthroline

Conversion of GSH (%) 100 80 60 10

Addition of extra 20-200 ^M Cu2+ did not significantly affect the GS-NO yields. More details of the S-nitrosation of thiols are given in Chapter 9 of this book.

Evidently, if ferrous iron catalyzes the formation of RS-NO, it is unavoidable that a certain quantity of DNIC is formed from spurious iron and free NO molecules. When liganded to iron, the electronic coupling between the nitrosyl ligands induces a certain degree of dismutation,

Nitric Oxide Nitrosation
Fig. 16. UV/Vis spectra from 50 mM glutathione in HEPES (15 mM, pH 7.4). 2 ml of the solution was exposed for 5 min to a gas mixture of NO (1 mmol) and air (8 ^mol O2 administered with air). (Curve a) In absence of phenanthroline and (Curve b) In presence of 250 ^M o-phenanthroline.

and imparts the character of nitrosonium to one of the nitrosyl ligands in the DNICs (the non-equivalence of the two nitrosyl ligands in DNIC was discussed in Chapter 2). Nitrosonium itself is known as a powerful nitrosating agent [7], and may react readily with cysteine to form Cys-NO. It is important to note the stoichiometry of the reactions. In [31], the formation of Cys-NO from Cys-DNIC was shown to be reversible as well as quantitative. It was induced by rapid acidification of the Cys-DNIC solution from pH 7 to 1. Initially, the solution had green color characteristic of Cys-DNIC. This color changed rapidly to pink and a new optical absorption band at 340 nm demonstrated the formation of free Cys-NO. EPR spectroscopy confirmed that the DNIC signal at g = 2.03 had vanished. Within the experimental accuracy of ca 10%, the yield of Cys-NO was equimolar to that of the original Cys-DNIC. The reverse transformation to Cys-DNIC was induced by raising the pH from 1 to 7. The pink color reverted to green and EPR showed that some 50% of the original Cys-DNIC was recovered. We attribute the recovery of Cys-DNIC to the reaction of two Cys-NO molecules with ferrous iron as illustrated above by Scheme 7. The formation of equimolar Cys-NO was attributed to S-nitrosation of one free cysteine molecule by the nitrosonium ligand in Cys-DNIC as shown in Scheme 9.

S-nitrosation of GSH can also be induced by exposing DNIC to citrate, a good chelator for iron [32]. The reaction was demonstrated in the solution of 0.4 mM dimeric Cys-DNIC. As described in Chapter 2, dimeric Cys-DNIC is obtained by bubbling purified NO gas through a solution of ferrous iron and cysteine (Fe: Cys = 1:2). The vial was subsequently evacuated to remove dissolved free NO from the solution. The formation of dimeric DNIC was confirmed by the optical absorbance at 310 and 360 nm (cf Chapter 2). At this stage, nearly all NO is sequestered in the form of dimeric DNIC.

In absence of free NO, the dimeric DNIC will slowly decompose according to the equilibrium in Scheme 5, releasing 2NO and 2NO+ moieties from one dimer. Strong quantitative support for Scheme 5 came from experiments where the decomposition was accelerated by the addition of an iron chelator, in this case citrate. Subsequently, the complexes were incubated with 1.6 M sodium citrate for 90 min. one mM of glutathione was also added to scavenge nitrosonium (NO+) ions released from the DNIC. This scavenging reaction has the end product GS-NO. The optical absorbance at 310 and 360 nm confirmed that citrate completely decomposed the dimeric Cys-DNIC. A new band at 340 nm showed the formation of the Fe3+-citrate complex (£340 = 1800 M-1cm-1), and the optical absorption also confirmed the formation of ca 0.80 ± 0.05 mM S-nitrosothiols. This concentration confirms that at this stage, all available NO+ has been incorporated into the pool of S-nitrosothiols, probably as a mixture of Cys-NO and GS-NO. As before, the NO ligands are believed to be lost as NO gas into the headspace of the reaction vessel.

After subsequent addition of 20 mM cysteine and 0.5 mM Fe2+, the solution took the green color of monomeric DNIC. EPR spectroscopy on a frozen aliquot confirmed the formation of ca 0.40 ± 0.04 mM of monomeric DNIC. Clearly, all available NO in the pool of S-nitrosothiols had been incorporated into the monomeric DNIC. The formation of the DNIC was evidently due to the reaction of cysteine and iron ions with the GS-NO and Cys-NO formed during the decomposition of the initial DNIC 1:2.

The preceding experiments document the reversible S-nitrosation of low-molecular-weight thiols like GSH or cysteine. However, the same reaction pathways also apply to thiol groups in proteins like bovine serum albumin (BSA) or horse hemoglobin (Hb) [10]. Exposure of 1 mM protein-bound DNICs to a combination of NO + O2 causes loss of DNIC and S-nitrosation of the protein [10]. The complexes were obtained by addition of 1 mM DNIC

2.041 A

2.041 A

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