Long-lasting hypotensive action of stable preparations of dinitrosyl-iron complexes with thiol-containing ligands in conscious normotensive and hypertensive rats

Previously we established the hypotensive action of nitric oxide donors, dinitrosyl-iron complexes (DNIC) with thiol-containing ligands, stored in frozen solution at 77K. In the present study, we tested recently designed water soluble dry powder preparations of DNICs keeping their characteristics in dry air for a long time. The complexes dissolved in PBS were injected intravenously into normotensive Wistar and spontaneously hypertensive SHR rats. The average arterial pressure (AAP) was recorded through preliminary implanted catheter in a carotid artery. The initial hypotensive action of DNIC with cysteine (DNIC-cys) was comparable to action of nitroprusside (SNP) but, in contrast to the latter, lasted for 20-120min depending on a doze. The blood DNIC content as detected by electronic paramagnetic resonance steadily decreased at this time. The hypotensive action of S-nitrosocysteine was similar to SNP while binding of iron in DNIC by batophenantroline-disulphonate prevented its hypotensive effect. These data suggest that long-lasting hypotensive action of DNICs may be caused by stable protein-bound DNICs forming in the process of transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNICs to thiol protein ligands. The relative initial dose-dependent effect of DNIC-cys was similar in Wistar and SHR but secondary AAP reduction was more profound in SHR. A substitution of cysteine in DNIC by thiosulphate resulted in markedly less initial AAP reduction while long-lasting effect was similar and substitution by glutathione smoothed initial AAP decline and stabilized AAP level in the second phase. Prolonged AAP reduction induced by DNIC-cys was considerably shortened in narcotized rats. Thus, dry preparations of DNICs preserve prolonged hypotensive activity.     

Study of dinitrosyl-iron complexes pharmacokinetics and accumulation in depot in rat organs

Protein-bound dinitrosyl-iron complexes (DNIC) in rat whole blood and organs were studied after intravenous injection of this substance with glutathione ligand (DNIC-GH). The effect of DNIC-GH injection on NO level (including NO physiological forms) in hydrophobic areas of rat tissues was also studied in normal physiological blood circulation condition. It has been shown, that after DNIC-GH injection the concentration of protein-bound DNICs in rat whole blood and organs rapidly reached maximum values, and then gradually decreased, that pointed to decomposition of DNIC molecules, coupled with NO release. At the beginning of the experiment the rates of DNIC decay in rat heart and lung were substantially higher, as compared with those in liver and kidney. By spin trappping it has been demonstrated that DNIC-GH, as a source of NO physiological forms (including S-nitrosothiols), in normal physiological blood circulation influence heart more selectively, as compared with the other organs.     

Distribution and pharmacokinetics of dinitrosyl iron complexes in rat organs

Dinitrosyl iron complexes (DNICs) have been traced in rat blood and organs after intravenous infusion of Oxacom. It is shown that the active principle (DNIC with glutathione) is rapidly distributed through the organism and deposited in blood and organs as protein-bound DNICs. The specific levels of DNIC in the main body organs are comparable, whereas its apparent lifetimes relate as blood < heart = lung < liver < kidney. Spin trapping assays indicate that protein-bound DNICs are a major but not the only form of NO deposition; the next largest depot is most probably formed by S-nitrosothiols. The gradual release of NO from such pools ensures the smooth and prolonged hypotensive effect of Oxacom.     

Activation of soluble guanylate cyclase by NO donors--S-nitrosothiols, and dinitrosyl-iron complexes with thiol-containing ligands.

We studied the capability of dimeric forms of dinitrosyl-iron complexes and S-nitrosothiols to activate soluble guanylate cyclase (sGC) from human platelet cytosol. The dinitrosyl-iron complexes had the ligands glutathione (DNIC-GS) or N-acetylcysteine (DNIC-NAC). The S-nitrosothiols were S-nitrosoglutathione (GS-NO) or S-nitrosoacetylcysteine (SNAC). For both glutathione and N-acetylcysteine, the DNIC and S-nitrosothiol forms are equally effective activators of sGC. The activation mechanism is strongly affected by the presence of intrinsic metal ions. Pretreatment with the potent iron chelator, disodium salt of bathophenanthroline disulfonic acid (BPDS), suppressed sGC activation by GS-NO: the concentration of GS-NO producing maximal sGC activation was increased by two orders of magnitude. In contrast, activation by DNIC-GS is strongly enhanced by BPDS. When BPDS was added 10 min after supplementation of DNIC-GS or GS-NO at 4 degrees C, it exerted a similar effect on sGC activation by either NO donor: BPDS only enhanced the sGC stimulation at low concentrations of the NO donors. Our experiments demonstrated that both Fe(2+) and Cu(2+) ions contribute to the decomposition of GS-NO in the presence of ascorbate. The decomposition of GS-NO induced by Fe(2+) ions was accompanied by formation of DNIC. BPDS protected GS-NO against the destructive action of Fe(2+) but not Cu(2+) ions. Additionally, BPDS is a sufficiently strong chelator to remove the iron from DNIC-GS complexes. Based on our data, we propose that S-nitrosothiols activate sGC via a two-step iron-mediated process: In the first step, intrinsic Fe(2+) ions catalyze the formation of DNICs from S-nitrosothiols. In the secondary step, these newly formed DNICs act as the real NO donors responsible for sGC activation.     

Beneficial effect of dinitrosyl iron complexes with thiol ligands on the rat penile cavernous bodies

Dinitrosyl iron complexes (DNIC) with thiol ligands were found to beneficially affect the state of the penile cavernous tissue upon its experimental denervation in rats. Histological and histochemical analysis showed that intracavernous administration of DNIC (twice weekly over six months) almost completely abolished the proliferation of endothelial cells typical of denervated cavernous tissue. On the other hand, this treatment sustained the mitotic activity of smooth myocytes and prevented the appearance of collagenase, a marker of their fibrotic transformation. The DNIC treatment had a pronounced effect on penile erection in neurotomized as well as in intact animals. Introduction of low-molecular DNIC into cavernous tissue was found to cause formation of protein-bound complexes observed by EPR and probably acting as depots of nitric oxide, ensuring steady erection.     

Interaction of reactive oxygen and nitrogen species with albumin- and methemoglobin-bound dinitrosyl-iron complexes.

Destructive effect of superoxide anions O2- derived from KO(2) or xanthine-xanthine oxidase system on dinitrosyl-iron complexes bound with bovine albumin or methemoglobin (DNIC-BSA or DNIC-MetHb) was demonstrated. The sensitivity of DNIC-BSA synthesized by the addition of DNIC with cysteine, thiosulfate or phosphate (DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3, respectively) to destructive action of O2- decreased in row: DNIC-BSA-1>DNIC-BSA-3>DNIC-BSA-2. The estimated rate constant for the reaction between O2- and DNIC-BSA-3 was equal to approximately 10(7)M(-1)s(-1). However, hydrogen peroxide and tert-butyl hydrogenperoxide (t-BOOH) did not induce any noticeable degradation of DNIC-BSA-3 even when used at concentrations exceeding by one order of magnitude those of the complex. As to their action on DNIC-MetHb both hydrogen peroxide and t-BOOH-induced rapid degradation of the complex. Both agents could induce the process due to the effect of alkylperoxyl or protein-derived free radicals formed at the interaction of the agents with ferri-heme groups of MetHb. Peroxynitrite (ONOO(-)) could also initiate protein-bound DNIC degradation more efficiently in the reaction with DNIC-BSA-3. Higher resistance of DNIC-MetHb to peroxynitrite was most probably due to the protective action of heme groups on ONOO(-). However, the analysis allows to suggest that the interaction of protein-bound DNICs with O2- is the only factor responsible for the degradation of the complexes in cells and tissues.     

Nitric oxide-induced bacteriostasis and modification of iron-sulphur proteins in Escherichia coli

The nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids (BCAA) anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (IlvD), an iron-sulphur enzyme essential for the BCAA biosynthesis, is completely inactivated in cells by NO with the concomitant formation of the IlvD-bound dinitrosyl iron complex (DNIC). Fractionation of the cell extracts prepared from the NO-exposed cells reveals that a large number of different protein-bound DNICs are formed by NO. While the IlvD-bound DNIC and other protein-bound DNICs are stable in cells under anaerobic growth conditions, they are efficiently repaired under aerobic growth conditions even without new protein synthesis. Additional studies indicate that L-cysteine may have an important role in repairing the NO-modified iron-sulphur proteins in aerobically growing E. coli cells. The results suggest that cellular deficiency to repair the NO-modified iron-sulphur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions.     

Iron-sulfur proteins are the major source of protein-bound dinitrosyl iron complexes formed in Escherichia coli cells under nitric oxide stress

Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not proteins without iron-sulfur clusters, are modified, forming protein-bound DNICs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of the "chelatable iron pool" in wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of the chelatable iron pool in cells.     

Introduction of dinitrosyl iron complexes with thiol-containing ligands into animal organism by inhalation method

The possibility of water-soluble dinitrosyl iron complexes (DNIC) with thiol-containing ligands introduction into lungs and other tissues of mice by free inhalation of little drops (2–3 microns diameter) of the solutions of these complexes was investigated. Little drops of 2–20 mM solutions of the complexes were obtained by using an inhalation apparatus (compressor nebulizer). A cloud of these little drops was then inhaled by animals in a closed chamber. A maximal amount of protein-bound DNICs formed in mouse lungs was 0.6 micromoles per kilogram of tissue weight. The amount of DNIC in lungs, liver and blood decreased to the undetected level within 2–3 hours after inhalation. No cytotoxic effect of DNIC formed in lungs on Mycobacterium tuberculosis was found in mice infected with these mycobacteria.     

Nitric oxide initiates iron binding to neocuproine.

It was demonstrated that two species of paramagnetic dinitrosyl iron complex (DNIC) with neocuproine form under the following conditions: in addition of neocuproine to a solution of DNIC with phosphate; in gaseous NO treatment of a mixture of Fe(2+) + neocuproine aqueous solutions at pH 6.5-8; and in addition of Fe(2+)--citrate complex + neocuproine to a S-nitrosocysteine (cys-NO) solution. The first form of DNIC with neocuproine is characterized by an EPR signal with g-factor values of 2.087, 2.055, and 2.025, when it is recorded at 77K. At room temperature, the complex displays a symmetric singlet at g = 2.05. The second form of DNIC with neocuproine gives an EPR signal with g-factor values of 2.042, 2.02, and 2.003, which can be recorded at a low temperature only.The revealed complexes are close to DNIC with cysteine in their stability. The ability of neocuproine to bind Fe(2+) in the presence of NO with formation of paramagnetic DNICs warrants critical reevaluation of the statement that neocuproine is only able to bind Cu(+) ions. It was suggested that the observed affinity of neocuproine to iron was due to transition of Fe(2+) in DNIC with neocuproine to Fe(+). In experiments on cys-NO, it was shown that the stabilizing effect of neocuproine on this compound could be due to neocuproine binding to the iron catalyzing decomposition of cys-NO.     

Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.     

The sources of inorganic sulfur in the process of cluster protein Fnr[4Fe-4S]2+ reconstruction in Escherichia coli cells cultivated with NO-donating agents

Dinitrosyl iron complexes (DNICs) with thiol ligands--binuclear and mononuclear--inhibited aidB gene expression in E. coli cells. This process is due to the nitrosylation of the active center in iron-sulfur protein Fnr [4Fe-4S]2+ by low-molecular DNICs. The next step is transformation of the above DNICs into the DNICs with the thiol groups in the apo-form of Fnr protein. These nitrosylated proteins are characterized by the EPR signal with g perpendicular = 2.04 and g parallel 1 = 2,014. An addition of sulfur containing L-Cys or N-A-L-Cys as well as Na2S to the cells lead to the increasing in the aidB gene expression simultaneously with an appearance of the EPR signal with g perpendicular = 2.04 and g parallel = 2.02 as the characteristics of the DNICs with persulfide (R-S-S-) ligands. We suppose that the recovery of the aidB gene activity was due to the accumulation of inorganic sulfur in the cells and reconstruction of the active center in Fnr[4Fe-4S]2+. It appears that the above process is the function of L-cysteine-desulfurase protein which repaired the active center of Fnr[4Fe-4S]2+ protein using the sulfur from L-Cys or N-A-L-Cys after its deacetylation. On the other side the ions of inorganic sulfur being reacted with SH-groups led to the transformation of DNIC with thiol ligands into the persulfides. Na2S was the most potent activator of the aidB gene expression in our experiments.     

The dinitrosyl-iron complexes with cysteine block the development of experimental endometriosis in rats

It has been shown that the administration of 0,5 ml of 5 mM aqueous solution of dinitrosyl-iron complexes (DNIC) with cysteine alleviated the development of experimental endometriosis in rats induced by surgical way: the size of endometriomes decreased 1.85 times when the DNIC was added every day during 10 days. The effect was suggested to be due to cytotoxic action of NO molecules and nitrosonium ions (NO+) released from rapidly decomposed DNIC in animal organism on endometriome tissues.     

Vasorelaxing activity of stable powder preparations of dinitrosyl iron complexes with cysteine or glutathione ligands.

Vasorelaxant activity of new stable powder preparations of dinitrosyl iron complexes (DNIC) with thiol-containing ligands was investigated on rat abdominal aorta rings. The preparations preserve their physicochemical characteristics (EPR and optical absorption) if stored for a long time in dry air (at least half-year). Three preparations of DNIC were tested: diamagnetic dimeric DNIC with glutathione (DNIC-GS 1:2) or cysteine (DNIC-cys 1:2) and paramagnetic monomeric DNIC with cysteine (DNIC-cys 1:20). Being dissolved in physiological solution the preparations induced relaxation of vessel similarly to that by earlier described non-stable DNICs which should be stored in liquid nitrogen. The amplitudes and kinetic characteristics of the relaxation were dependent on the incorporated thiolate ligands. Rapid transient relaxation followed by significant tone recovery to stationary level (plateau) was observed for DNIC-cys 1:2. DNIC-cys 1:20 also induced initial rapid relaxation followed by incomplete tone recovery. DNIC-GS 1:2 induced slow developing and long lasting relaxation. NO scavenger, hydroxocobalamin (2x10(-5)M) eliminated the rapid transitory relaxation induced by DNIC-cys 1:20 and did not influence significantly on the plateau level. SOD increased duration of the DNIC-cys 1:2 and DNIC-cys 1:20 induced relaxation. The addition of 5x10(-5)M DNIC-cys 1:2 or DNIC-cys 1:20 induced long lasting vasorelaxation within 20min and more. However the EPR measurements demonstrated full rapid disappearance (within 1-2min) of both type of DNIC-cys in Krebs medium bubbled with carbogen gas. This was not the case for DNIC-GS 1:2. We suggested that the long lasting vasorelaxation observed during the addition of DNICs-cys was induced by S-nitrosocysteine derived from DNICs-cys and stabilized by EDTA in Krebs medium. The suggestion is in line with the fact that strong ferrous chelator bathophenantroline disulfonate (BPDS) which is capable of rapid degradation of DNICs did not abrogate the vasorelaxtion induced by DNIC addition.     

Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD.

The objective of this study was to elucidate the nitric oxide-forming reactions of the iron-N-methyl-D-glucamine dithiocarbamate (Fe-MGD) complex from the nitrogen-containing compound hydroxyurea. The Fe2+(MGD)2 complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. The reaction of Fe2+(MGD)2 with NO yields the resultant NO-Fe2+(DETC)2 complex, which has a characteristic triplet EPR signal. It is widely believed that only NO reacts with Fe2+(MGD)2 to form the NO-Fe2+(MGD)2 complex. In this report, the mechanism leading to the formation of NO-Fe2+(MGD)2 was investigated using oxygen-uptake studies in conjunction with the EPR spin-trapping technique. We found that the air oxidation of Fe2+(MGD)2 complex results in the formation of the Fe3+(MGD)3 complex, presumably concomitantly with superoxide (O3*-). Dismutation of superoxide forms hydrogen peroxide, which can subsequently reduce Fe3+(MGD)3 back to Fe2+(MGD)2. The addition of NO to the Fe3+(MGD)3 complex resulted in the formation of the NO-Fe2+(MGD)2 complex. Hydroxyurea is not considered to be a spontaneous NO donor, but has to be oxidized in order to form NO. We present data showing that in the presence of oxygen, Fe2+(MGD)2 can oxidize hydroxyurea to yield the stable NO-Fe2+(MGD)2 complex. These results imply that hydroxyurea can be oxidized by reactive oxygen species that are formed from the air oxidation of the Fe2+(MGD)2 complex. Formation of the NO-Fe2+(MGD)2 complex in this case could erroneously be interpreted as spontaneous formation of NO from hydroxyurea. The chemistry of the Fe2+(MGD)2 complexes in aerobic conditions must be taken into account in order to avoid erroneous conclusions. In addition, the use of these complexes may contribute to the overall oxidative stress of the system under investigation.     

Coordination of iron ions in the form of histidinyl dinitrosyl complexes does not prevent their genotoxicity

Formation of dinitrosyl iron complexes (DNICs) was observed in a wide spectrum of pathophysiological conditions associated with overproduction of NO. To gain insight into the possible genotoxic effects of DNIC, we examined the interaction of histidinyl dinitrosyl iron complexes (HIS-DNIC) with DNA by means of circular dichroism. Formation of DNIC was monitored by EPR and FT/IR spectroscopy. Vibrational bands for aquated HIS-DNIC are reported. Dichroism results indicate that HIS-DNIC changes the conformation of the DNA in a dose-dependent manner in 10 mM phosphate buffer (pH 6). Increase of the buffer pH or ionic strength decreased the effect. Comparison of HIS-DNIC DNA interaction with the effect of hydrated Fe²⁺ ion revealed many similarities. The importance of iron ions in HIS-DNIC induced genotoxicity is confirmed by plasmid nicking assay. Treatment of pUC19 plasmid with 1 μM HIS-DNIC did not affect the plasmid supercoiling. Higher concentrations of HIS-DNIC induced single strand breaks. The effect was completely abrogated by addition of deferoxamine, a specific strong iron chelator. Our data reveal that formation of HIS-DNIC does not prevent DNA from iron-induced damage and imply that there is no direct interrelationship between iron–NO coordination and their mutual toxicity modulation.     

Nitric oxide-induced conversion of cellular chelatable iron into macromolecule-bound paramagnetic dinitrosyliron complexes

One of the most important biological reactions of nitric oxide (nitrogen monoxide, *NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g=2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous *NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, *NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from *NO, and SIH added during the *NO treatment "freezes" DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free *NO, because addition of oxyhemoglobin prevents formation from either *NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by *NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free *NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of *NO and its effects on cellular iron homeostasis.     

The mechanisms of S-nitrosothiol decomposition catalyzed by iron.

The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes (DNICs) with thiol- or non-thiol ligands or mononitrosyl iron complex (MNICs) with N-methyl-D-glucamine dithiocarbamate catalyzed by iron(II) ions under anaerobic conditions were studied by monitoring EPR or optical features of the complexes and S-nitrosothiols. The kinetic investigations demonstrated the appearance of short-living paramagnetic mononitrosyl-iron complex with L-cysteine prior to the formation of stable dinitrosyl-iron complex with cysteine in the solution of iron(II)-citrate complex (50-100 microM), S-nitrosocysteine (400 microM), and L-cysteine (20 mM) in 100 mM Hepes buffer (pH 7.4). The addition of deoxyhemoglobin (100 microM) did not influence the process, which points to a direct interaction between S-nitrosocysteine and iron(II) ions to yield DNIC. The reaction of DNIC-cysteine formation is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0 +/- 0.2) x 10(5) M(-2) s(-1) (estimated from EPR results) or (2.0 +/- 0.1) x 10(4) M(-2) s(-1) (estimated by optical method). A similar process of DNIC-cysteine formation was observed in a solution of iron(II)-citrate complex, L-cysteine, and NO-proline (200 microM) as a NO* donor. The appearance of a less stable dinitrosyl-iron complex with phosphate was detected when solutions of iron(II)-citrate containing 100 mM phosphate buffer (pH 7.4) were mixed with S-nitrosocysteine or NO-proline. The rapid formation of DNIC with phosphate was followed by its decay. When the concentration of L-cysteine in solutions was reduced from 20 to 1 mM, the life-time of the DNIC-cysteine diminished notably; this was caused by consumption of L-cysteine in the process of DNIC-cysteine formation from S-nitrosocysteine and iron. Thus, L-cysteine is consumed. Formation of DNIC with glutathione was also observed in a solution of glutathione (20 mM), S-nitrosoglutathione (400 microM), and iron(II) complex (800 microM) in 100 mM Hepes buffer (pH 7.4), but the rate of formation was about 10 times slower than the formation of the DNIC-cysteine. The rate of MNIC-MGD formation from iron(II)-MGD complexes and S-nitrosocysteine was first-order in both reactants. The second-order rate constant for this reaction, estimated from EPR measurements, was 30 +/- 5 M(-1) s(-1). Rate constants of MNIC-MGD formation from iron(II)-MGD and the more stable S-nitrosoglutathione and S-nitroso-D,L-penicillamine were equal to 3.0 +/- 0.3 and 0.3 +/- 0.05 M(-1) s(-1), respectively. Thus, the concerted mechanism of DNIC and MNIC formation from S-nitrosothiols and iron(II) ions can be suggested to be predominant.