Dinitrosyl iron complexes with thiol-containing ligands as a base for developing drugs with diverse therapeutic activities: Physicochemical and biological substantiation

Dinitrosyl iron complexes (DNICs) with thiol-containing ligands occur in animal tissues as paramagnetic (EPR-active) mononuclear and diamagnetic (EPR-silent) binuclear species with the presence of nitric oxide. They provide stabilization and storage (within protein-bound DNICs) of nitric oxide, as well as its transport (within low-molecular-weight DNICs) to biological targets to serve as donors of not only nitric oxide itself but also of the nitrosonium ion (NO⁺). The latter function determines the ability of DNICs to S-nitrosylate various thiol-containing proteins. In this way, the complexes participate in a wide range of physiological and biochemical processes. With respect to the high and diverse level of biological activity and dose dependence of DNICs, they mimic the endogenous nitric oxide system. Taken together with the broad occurrence of DNICs (predominantly in the diamagnetic form) in animal tissues, this fact points to their role as an “operating form” of nitric oxide. It is thought that drugs designed on the basis of DNICs can substantially improve the efficiency of modern medical practice.     

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.     

The inhibitory effect of dinitrosyl iron complexes (NO donors) on myeloperoxidase activity

The effect of synthetic analogues of dinitrosyl mononuclear iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the activity of myeloperoxidase (MPO) was studied, and their efficiency was evaluated. It was shown that the enzyme MPO is the molecular target of DNICs. It was found that six DNICs inhibited the activity of MPO and one compound potentiated it. The evaluation of their efficiency showed that two DNICs effectively inhibited the activity of MPO by 50% at IC₅₀ = 2 × 10^–4 M and IC₅₀ = 5 × 10^–7 M.     

Effect of Dinitrosyl Iron Complexes (NO Donors) on the Metabolic Processes in Human Fibroblasts

The results of the study of the effect of mononuclear dinitrosyl iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the cell viability and metabolism of human lung fibroblasts are presented, and the efficiency of their action is evaluated. It was shown that cationic DNICs increased the cell viability of fibroblasts and demonstrated the cytoprotective properties. Fluorescent analysis revealed that the DNICs compounds decrease the mitochondrial membrane potential but do not have a significant effect on the level of glutathione and reactive oxygen species in fibroblasts. It is assumed that the DNICs have the therapeutic potential for treating cardiovascular diseases.     

Dinitrosyl iron complexes--structure and biological functions

Formation of dinitrosyl iron complexes (DNICs), which can be described by general formula Fe(NO)2(L)2, where L is carbonyl-, nitrosyl- or imino- complexing ligand, was observed in many kinds of living organisms, in a wide spectrum of physiological conditions associated with inflammation, ischemia/reperfusion and cancer. Accumulation of DNICs coincides with intensified production of nitric oxide in macrophages, neurons, endothelial cells, Langerhans' cells and hepatocytes. Low-molecular thiol-containing DNICs (DNIC-(RS)2) show vasodilatory action and they are proposed to play a role of nitric oxide transducers and stabilizers. DNICs have been shown to modulate redox potential of the cell via inhibition of glutathione-dependent enzymes, such as glutathione reductase, S-transferase and peroxidase. Although there is a convincing experimental evidence for their NO and NO+ donating function, the nature of DNICs formed in biological systems, their stability and biological role is still a matter of discussion.     

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.     

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.     

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.     

Protein-bound dinitrosyl-iron complexes appearing in blood of rabbit added with a low-molecular dinitrosyl-iron complex: EPR studies.

The formation of protein-bound dinitrosyl-iron complexes (DNIC) in blood plasma and packed red cell fraction has been demonstrated by the EPR method in the experiments on rabbits which were i/v injected with the low-molecular DNIC with thiosulphate. This formation was ensured by transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNIC onto serum albumin or hemoglobin molecules. Protein-bound DNICs appeared immediately after low-molecular DNIC injection followed with gradually decreasing their amounts. The complexes could be detected by EPR technique during more than two days. The addition of water-soluble NO scavenger, the iron complex with N-methyl-d-glucamine dithiocarbamate (MGD) resulted in decomposition of a part of protein-bound DNICs and in effective excretion of secondary products (mainly mononitrosyl-iron complexes with MGD) from the blood flow.     

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.     

Reactivity pathways for nitric oxide and nitrosonium with iron complexes in biologically relevant sulfur coordination spheres

The interaction of nitric oxide (NO) with iron-sulfur cluster proteins results in the formation of dinitrosyl iron complexes (DNICs) coordinated by cysteine residues from the peptide backbone or with low molecular weight sulfur-containing molecules like glutathione. Such DNICs are among the modes available in biology to store, transport, and deliver NO to its relevant targets. In order to elucidate the fundamental chemistry underlying the formation of DNICs and to characterize possible intermediates in the process, we have investigated the interaction of NO (g) and NO(+) with iron-sulfur complexes having the formula [Fe(SR)(4)](2-), where R=(t)Bu, Ph, or benzyl, chosen to mimic sulfur-rich iron sites in biology. The reaction of NO (g) with [Fe(S(t)Bu)(4)](2-) or [Fe(SBz)(4)](2-) cleanly affords the mononitrosyl complexes (MNICs), [Fe(S(t)Bu)(3)(NO)](-) (1) and [Fe(SBz)(3)(NO)](-) (3), respectively, by ligand displacement. Mononitrosyl species of this kind were previously unknown. These complexes further react with NO (g) to generate the corresponding DNICs, [Fe(SPh)(2)(NO)(2)](-) (4) and [Fe(SBz)(2)(NO)(2)](-) (5), with concomitant reductive elimination of the coordinated thiolate donors. Reaction of [Fe(SR)(4)](2-) complexes with NO(+) proceeds by a different pathway to yield the corresponding dinitrosyl S-bridged Roussin red ester complexes, [Fe(2)(mu-S(t)Bu)(2)(NO)(4)] (2), [Fe(2)(mu-SPh)(2)(NO)(4)] (7) and [Fe(2)(mu-SBz)(2)(NO)(4)] (8). The NO/NO(+) reactivity of an Fe(II) complex with a mixed nitrogen/sulfur coordination sphere was also investigated. The DNIC and red ester species, [Fe(S-o-NH(2)C(6)H(4))(2)(NO)(2)](-) (6) and [Fe(2)(mu-S-o-NH(2)C(6)H(4))(2)(NO)(4)] (9), were generated. The structures of 8 and 9 were verified by X-ray crystallography. The MNIC complex 1 can efficiently deliver NO to iron-porphyrin complexes like [Fe(TPP)Cl], a reaction that is aided by light. Removal of the coordinated NO ligand of 1 by photolysis and addition of elemental sulfur generates higher nuclearity Fe/S clusters.     

Sources of divalent sulfur allow recovery of the Fnr [4Fe-4S]<Superscript>2+</Superscript> center in <Emphasis Type="Italic">Escherichia coli</Emphasis> incubated with nitric oxide donors

Dinitrosyl iron complexes (DNICs) with various thiol ligands, the known donors of nitric oxide, markedly inhibited aidB gene expression in E. coli cells by destroying the [4Fe-4S]²⁺ center of its regulator protein Fnr. Therewith, the cells accumulated DNICs in the protein-bound form, identified by the EPR signal with g _⊥ = 2.04 and g _‖ = 2.014. Subsequent addition of sulfur sources L-cysteine or N-acetylcysteine, DTT as well as Na₂S to the DNIC-treated cells significantly restored the reporter gene expression. Simultaneously, the above-specified EPR signal was partly or completely replaced with a narrower signal (g _⊥ = 2.032, g _‖ = 2.02) identical to that of DNICs with persulfide (R-S-S⁻) ligands, which result from interaction of S²⁻ with thiols; inorganic sulfide proved to be the most efficient agent. These data corroborate the central role of S²⁻ in recovery of the protein [4Fe-4S] center disrupted by the NO donors.     

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.     

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.     

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.     

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.     

Dinitrosyl-iron complexes with thiol-containing ligands: spatial and electronic structures.

Parameters of the EPR signals of monomeric dinitrosyl-iron complexes with 1H-1,2,4-triazole-3-thiol (DNIC-MT), obtained by treating MT+ferrous iron in DMSO solution with gaseous NO, have been compared with those of the crystalline monomeric DNIC-MT with tetrahedral structure. Dissolved DNIC-MT were characterized by the isotropic EPR signal centered at g=2.03 with half-width of 0.7 mT and quintet hyperfine structure when recorded at ambient temperature or the anisotropic EPR signal with g( perpendicular)=2.045, g( parallel)=2.014 from frozen solution at 77 kappa, Cyrillic. DNIC-MT in crystalline state showed the structure-less symmetrical singlet EPR signal centered at g=2.03 and half-width of 1.7 mT at both room and liquid nitrogen temperature. The Lorentz shape of this signal indicates the strong exchange interaction between these complexes in the DNIC-MT crystal. Being dissolved in DMSO the crystalline sample of DNIC-MT demonstrated the EPR signal typical for DNIC-MT, obtained by treating MT+ferrous iron in DMSO solution with gaseous NO. Low spin (S=1/2) d(9) electron configuration of DNIC-MT with tetrahedral structure (formula [(MT-S(.))(2)Fe(-1)(NO(+))(2)](+)) was suggested to be responsible for the signal of DNIC-MT in crystalline state. Dissolving of the crystals of DNIC-MT may result in the change of their spatial and electronic structure, namely, tetrahedral structure of the complexes characterized by low spin d(9) electronic configuration transforms into a plane-square structure with d(7) electronic configuration and low spin S=1/2 state (formula [(MT- S(-))(2)Fe(+)(NO(+))(2)](+)). The latter was suggested to be characteristic of other DNICs with various thiol-containing ligands in the solutions. The proposed mechanism of these DNICs formation from ferrous iron, thiol and NO shows that the process could be accompanied by the ionization of NO molecules to NO(+) and NO(-) ions in the complexes. Detailed analysis of the shape of the EPR signals of these complexes provided additional information about the exchange interaction typical for DNIC-MT in crystals.     

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.     

Dinitrosyl iron complexes [E5Fe(NO)2] (E = S, Se): A precursor of Roussin’s black salt [Fe4E3(NO)7]

[PPN][Se₅Fe(NO)₂] (1) and [K-18-crown-6-ether][S₅Fe(NO)₂] (2′) were synthesized and characterized by IR, UV-Vis, EPR spectroscopy, magnetic susceptibility, and X-ray structure. [PPN][Se₅Fe(NO)₂] easily undergoes ligand exchange with S₈ and (RS)₂ (R = C₇H₄SN (5), o-C₆H₄NHCOCH₃ (6), C₄H₃S (7)) to form [PPN][S₅Fe(NO)₂] and [PPN][(SR)₂Fe(NO)₂]. The reaction displays that [E₅Fe(NO)₂]⁻ (E = Se (3), S (4)) facilely converts to [Fe₄E₃(NO)₇]⁻ by adding acid HBF₄ or oxidant [Cp₂Fe][BF₄] in THF, respectively. Obviously, complexes 1 and 2′ serve as the precursors of the Roussin’s black salts 3 and 4. The electronic structure of {Fe(NO)₂}⁹ core of [Se₅Fe(NO)₂]⁻ is best described as a dynamic resonance hybrid of {Fe⁺¹(NO)₂}⁹ and {Fe⁻¹(NO⁺)₂}⁹ modulated by the coordinated ligands. The findings, EPR signal of g = 2.064 for 1 at 298 K, implicate that the low-molecular-weight DNICs and protein-bound DNICs may not exist with selenocysteine residues of proteins as ligands, since the existence of protein-bound DNICs and low-molecular-weight DNICs in vitro has been characterized with a characteristic EPR signal at g = 2.03. In addition, complex 2′ treated human erythroleukemia K562 cancer cells exposed to UV-A light greatly decreased the percentage survival of the cell cultures.