Re-examination of the formation of dinitrosyl–iron complexes during reaction of S-nitrosothiols with Fe(II)

doi:10.1016/S0020-1693(01)00402-9

Inorganica Chimica Acta

Volume 318, Issues 1–2, 18 June 2001, Pages 1-7

https://doi.org/10.1016/S0020-1693(01)00402-9 Get rights and content

Abstract

The reaction of S-nitrosothiol compounds with ferrous ions in solution has been investigated and the generated dinitrosyl–iron complexes have been characterized. During the reaction of S-nitrosocysteamine with Fe(II) in water solution in the presence of a twofold excess (with respect to iron) of cysteamine hydrochloride (CSH), an EPR-silent dinuclear iron complex (complex A of formula [Fe₂(RS)₂(NO)₄]) was formed as the major species and was characterized by FAB MS⁺, UV–Vis, NMR and IR spectroscopies. In the presence of a large excess of CSH (CSH/Fe(II)=20:1), a green paramagnetic mononuclear complex (complex B of formula [Fe(RS)₂(NO)₂]⁻) was formed. From EPR and UV–Vis data, and also on the basis of the few crystallographic structures known for similar complexes, complex B is proposed to display a distorted tetrahedral geometry (C_2v), approaching a trigonal bipyramid with a missing ligand, with the unpaired electron mainly localized on the d_z² orbital of the iron characterized by a d⁹ electronic configuration.

Introduction

Nitric oxide (NO) is involved in a large number of physiological processes including neurotransmission [1], immune system regulation [2], [3], smooth muscle relaxation [4], [5], [6] and platelet inhibition [7], [8]. The high reactivity of NO with regard to molecular oxygen [9], superoxide anion [10], and heme [11] as well as nonheme iron [12] and the ready availability of these reactants in the plasma and cellular environment suggest that NO is stabilized in vivo by incorporation into a carrier molecule that prolongs its lifetime and preserves its biological activity. In these regards, S-nitrosothiols (RSNOs) [14], [13] and dinitrosyl–iron complexes (DNICs) [15], have been claimed to subserve the function of transport and storage of NO [16]. RSNOs formed either by low molecular weight thiols or thiol-containing proteins have been detected in vivo [17], [18] and were shown to elicit physiological and biochemical responses similar to those elicited by NO [19], [20], [21]. The DNICs are formed during the reaction of NO with Fe(II) in the presence of low molecular weight thiols, aminoacids, peptides or proteins (through their cysteine and histidine residues). Such complexes were first observed in biological systems about 30 years ago by their characteristic EPR signal [22], [23], [24], but their physiological significance is still a matter of investigation. They have been proposed as endothelium derived relaxing factor (EDRF) candidates [25] and shown to exhibit antiplatelet [26], vasorelaxant [27] and blood pressure lowering activity [28]. Whereas the DNICs bound to protein thiols occur only as paramagnetic forms, DNICs with low molecular weight thiols are known to exist in two forms, paramagnetic and diamagnetic, which are interconvertible [29].

Metal ions have been shown to promote the decomposition of RSNOs [30], [31], [32], [33]. Recently we showed that the iron chelators greatly stabilized RSNOs in solution, suggesting that iron ions, as well, were able to react with RSNOs [31]. In this work, we report the results of a study on the solution chemistry of reaction mixtures of S-nitrosocysteamine (CSNO) and Fe(II) in water and show that the DNICs, characterized by a variety of spectroscopic methods, are formed spontaneously, in agreement with the preliminary reports [34], [35]. This opens the possibility that NO can be transferred from thiols to iron also in biological systems, thus defining RSNOs and DNICs as the two major interconvertible stable biological forms of NO.

Section snippets

Materials and methods

Cysteamine hydrochloride and HEPES were purchased from Sigma; FeSO₄·7H₂O (99.999%) and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) were purchased from Aldrich, tert-butyl nitrite (90%) (tBuONO) was purchased from Fluka. 1-Amino-2-methylpropane-2-thiol has been synthesized according to Ref. [31]. Milli-Q ultrapure water has been used in all experiments.

Oxyhemoglobin (HbO₂) was prepared from red blood cells according to the procedure described in Ref. [36]. Deoxyhemoglobin (deoxyHb) was

Results

The reaction of CSNO with a ferrous salt in water at pH 7.8 was studied and it was found that, under these conditions, two different complexes were obtained, depending on the excess of CSH present in the reaction mixture. At low excess (twofold with respect to iron) a yellow diamagnetic complex (A) was obtained, whereas at large excess (at least twentyfold with respect to Fe), a green paramagnetic complex (B) was the only product of the reaction.

Discussion

These iron complex species have been previously reported by Vanin et al. [35] but the products of the reaction were not fully characterized. Here, the use of a variety of spectroscopic methods allowed us to identify the complex species in solution coming out from the reaction (UV–Vis as well as mass spectra and high filed EPR spectra are shown for the first time).

One of the complexes, complex B, is a S=1/2 paramagnetic species, as shown by EPR spectroscopy. The EPR signal at room temperature is

Conclusions

We have shown that during reaction of RSNOs with ferrous ions, NO can be transferred quantitatively from the sulfur atom to iron. Depending on the excess of free thiol present in solution, either a paramagnetic or a diamagnetic complex is formed and the two species are interconvertible (Fig. 8). The diamagnetic species is a dinuclear iron-dinitrosyl species in which each iron atom is coordinated to two NO groups and the two iron atoms are bridged by the sulfur atoms of two cysteamine molecules.

Acknowledgements

This work was supported by C.N.R., Progetto Finalizzato Biotecnologie. We thank Dr. C. Toia for performing high-field EPR spectra.

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¹: *Corresponding author. Tel.: +39-095-738 5093; fax: +39-095-58 0138; [email protected]

²: *Corresponding author. Tel.: +33-4-76-88 9103; fax: +33-4-76-88 9124; [email protected]

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Re-examination of the formation of dinitrosyl–iron complexes during reaction of S-nitrosothiols with Fe(II)

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Conclusions

Acknowledgements

FEBS Lett.

Cell

Biochem. Biophys. Acta

Biochem. Biophys. Acta

FEBS Lett.

FEBS Lett.

Arch. Biochem. Biophys.

Chem. Phys. Lett.

Polyhedron

Coord. Chem. Rev.

Annu. Rev. Physiol.

Science

FASEB J.

Angew. Chem., Int. Ed. Engl.

Angew. Chem., Int. Ed. Engl.

Angew. Chem., Int. Ed. Engl.

Proc. Natl. Acad. Sci. USA

Proc. Natl. Acad. Sci. USA

Chem. Res. Toxicol.

Am. J. Physiol.

FASEB J.

Anal. Biochem.

Biochemistry (Moscow)

Proc. Natl. Acad. Sci. USA

Proc. Natl. Acad. Sci. USA

Nature