G

Fig. 3. ESR spectra of selected DNIC. Spectra were recorded in 0.1 M phosphate buffer saturated with N2 (20°C). A stock solution of 0.1 M phosphate buffer (pH 7.20) saturated with NO gas (~1.8 mM) was prepared by rst deoxygenating the medium with N2 for 20 min and then gassing with NO gas that was passed through a gas trap containing KOH. NO was produced by dropping sulfuric acid to sodium nitrite. Spectra 1 Fe(NH4)2(SO4)2 (0.2 mM) in the presence of cysteine (1 mM), DTT (5 mM), or N-methyl-d-glucamine dithio-carbamate (MGD) 0.5 mM. 2 Fe(NH4)2(SO4)2, cysteine, and NO (0.2 mM). 3 Fe(NH4)2(SO4)2, MGD, and NO in the absence (dashed lines) and the presence (solid lines) of cysteine. 4 Fe(NH4)2(SO4)2, DTT, and NO in the absence (a) and the presence (b) of cysteine. With dashed line is presented spectrum 2.

Fig. 3. ESR spectra of selected DNIC. Spectra were recorded in 0.1 M phosphate buffer saturated with N2 (20°C). A stock solution of 0.1 M phosphate buffer (pH 7.20) saturated with NO gas (~1.8 mM) was prepared by rst deoxygenating the medium with N2 for 20 min and then gassing with NO gas that was passed through a gas trap containing KOH. NO was produced by dropping sulfuric acid to sodium nitrite. Spectra 1 Fe(NH4)2(SO4)2 (0.2 mM) in the presence of cysteine (1 mM), DTT (5 mM), or N-methyl-d-glucamine dithio-carbamate (MGD) 0.5 mM. 2 Fe(NH4)2(SO4)2, cysteine, and NO (0.2 mM). 3 Fe(NH4)2(SO4)2, MGD, and NO in the absence (dashed lines) and the presence (solid lines) of cysteine. 4 Fe(NH4)2(SO4)2, DTT, and NO in the absence (a) and the presence (b) of cysteine. With dashed line is presented spectrum 2.

Thiolato-nitrosyl-iron complexes are formed spontaneously in solutions of a thiol, Fe2+, and NO (Fig. 3). In the case of cysteine (Fig. 3, spectrum 2), the unstable bis(cysteinato) dinitrosyliron^2+) complex is formed, which is then converted to cystinatodinitrosyl-iron^2+) [59]. Similarly, compounds with vicinal thiol functions form iron-nitrosyl complexes [60,61], even in the presence of equimolar concentrations of cysteine (Fig. 3, spectra 3 and 4, respectively) or GSH (data not shown).

In iron-nitrosyl complexes, the charge on the nitrogen atom is presumed to be a major determinant in the reactivity of the ligated NO. The principal bonding scheme of Fe2+ NO complexes has been described by Enemark and Fetham [71]. These complexes were classi-ed as [FeNO] [72], and it has been proposed that they show bent Fe NO units with radical character on the nitrosyl ligand. However, this description leaves room for a large variation of the electronic structure mediated by metal-ligand covalency. A charge transfer between NO and Fe may lead to the formation of either NO+ or NO- as a ligand, where NO+ is expected to interact with thiols to form RSNOs; in contrast, NO- oxidizes thiols to disul des [17,49,73]. The charge transfer in transition metal-nitrosyl complexes are often classi ed on the basis of their NO stretching frequencies in the infra red spectrum as coordination compounds containing either the NO+(vno+ = 1500 2000 cm-1) or the NO- ligand (vno- = 1080 1500 cm-1) [74]. However, concerns have been raised that this classi -cation may be misleading, as a series of iron-nitrosyl complexes with stretching frequencies between 1080 and 1500 cm-1 have been shown to contain hyponitrite, nitrito, or nitro function, rather than NO ([62] and the references therein). If the assumption that stretching frequencies of 1600 1640 cm-1 are associated with double bonds, then the presence of NO- as a ligand in many iron-nitrosyl complexes cannot be ruled out [75,76]. In support of the latter hypothesis, Pearsall and Bonner have reported that Fe2+ reduces NO to nitroxyl (HNO) and forms iron-dinitrosyl complexes bearing both NO+ and NO- as cis positioned ligands [77]. Granozzi et al. have provided quantum mechanical calculations indicating that in the bis-cyclopentadienyl-bis(^-nitrosyl)iron2+ complex, a considerable amount of charge is withdrawn from the cyclopentadienyl ion; a small part of this charge is retained by the metal ion while the remaining charge is channeled through the metal to the bridging NO ligand [78]. Hence, iron-nitrosyl complexes may either trans-S-nitrosate cellular thiols via transfer of NO+ [17,79] or cause oxidation of SH functions via the intermediate formation of S-derived hydroxylamines [73]. An alternative mechanism is suggested by the studies of Kijima et al. [70], whereby the oxidation of thiols by Fe2+ and NO could be envisaged without the intermediate formation of S-nitrosothiols (Scheme 2). This mechanism is similar to the oxidation of the bis(cysteinato)Fe2+ complex by O2, except that NO plays the role of an oxidant (2 ^ 3); in turn, Fe3+ could be reduced by one of the ligated SH functions, which would set the stage for an intramolecular disul de ring closure (3 ^ 4). In the presence of oxygen, 5 readily undergoes oxidation to 6 with release of superoxide anion radical [32,33].

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