Dinitrosyl iron complexes with thiol-containing ligands in plant tissues

The formation of dinitrosyl iron complexes with thiol-containing ligands in plant tissues (parsley and apple leaves) in the presence of nitric monoxide was demonstrated using electron paramagnetic resonance. In two types of tissues dinitrosyl iron complexes are predominantly represented by the binuclear diamagnetic form. This diamagnetic form can be transformed in EPR-detectable mononitrosyl iron complexes with diethyldithiocarbamate due to the ability of diethyldithiocarbamate to accept the iron-mononitrosyl groups from iron-dinitrosyl fragments of binuclear complexes. A similar transformation was observed under the effect of diethyldithiocarbamate on a mononuclear paramagnetic form of dinitrosyl iron complexes. The significant amount of binuclear dinitrosyl iron complexes found in plant tissues suggests that these complexes can be considered as a “working form” of nitric monoxide, which is recognized now as a universal regulator of metabolic processes in plants as well as in other organisms.     

EPR Characterization of Dinitrosyl Iron Complexes with Thiol-Containing Ligands as an Approach to Their Identification in Biological Objects: An Overview

The overview demonstrates how the use of only one physico-chemical approach, viz., the electron paramagnetic resonance method, allowed detection and identification of dinitrosyl iron complexes with thiol-containing ligands in various animal and bacterial cells. These complexes are formed in biological objects in the paramagnetic (electron paramagnetic resonance-active) mononuclear and diamagnetic (electron paramagnetic resonance-silent) binuclear forms and control the activity of nitrogen monoxide, one of the most universal regulators of metabolic processes in the organism. The analysis of electronic and spatial structures of dinitrosyl iron complex sheds additional light on the mechanism whereby dinitrosyl iron complex with thiol-containing ligands function in human and animal cells as donors of nitrogen monoxide and its ionized form, viz., nitrosonium ions (NO⁺).     

Estimation of nitric oxide level in vivo by microdialysis with water-soluble iron-N-methyl-d-dithiocarbamate complexes as NO traps: A novel approach to nitric oxide spin trapping in animal tissues

It was found that microdialysis, i.e., passage of aqueous solutions of iron-N-methyl-d-glucamine dithiocarbamate complexes through dialysis fibers implanted into heart, kidney and liver tissues of narcotized rats, was accompanied by effective binding of the complexes to nitric oxide from interstitial fluid. The walls of dialysis fibers used in this study were permeable for compounds with molecular weight not exceeding 5 kDa. The dialyzate samples collected every 20 min and containing diamagnetic nitrosyl Fe³⁺-MGD adducts were reduced to the paramagnetic state with sodium dithionite; their concentration was measured by the EPR method. The basic level of the adducts, which represented mononitrosyl iron complexes with MGD (MNIC–MGD), in the dialyzate samples of all tested organs were similar (1 μМ). Treatment of animals with the water-soluble nitroglycerine analog Isoket or a low-molecular dinitrosyl iron thiosulfate complex as a NO donor increased the concentration of MNIC–MGD with going out into a plateau. The novel approach allows determination of nitric oxide levels in tissue interstitial fluid from concentration of MNIC–MGD formed during microdialysis.     

The Free-Radical Nature of Nitric Oxide Molecules as a Determinant of their Conversion to Nitrosonium Cations in Living Systems

Biophysics - This paper presents new results that confirm our previous inference that the binuclear form of biologically active dinitrosyl iron complexes (B-DNICs) with thiol-containing ligands...     

Antiviral Activity of Nitrosonium Cations against SARS-CoV-2 on a Syrian Hamster Model

Biophysics - The antiviral action of binuclear dinitrosyl iron complexes with glutathione along with diethyldithiocarbamate against the SARS-CoV-2 virus has been demonstrated on a Syrian hamster...     

Autowave mode of the functioning of the nitric oxide + free iron + thiols system as a basis for control of the biological action of nitric oxide and its endogenous compounds

The NO + Fe²⁺ + thiols system in an aqueous solution has been found earlier to exhibit temporal oscillatory changes in the concentration of paramagnetic dinitrosyl iron complexes with thiol-containing ligands and S-nitrosothiols, as well as in the concentration of free iron (not included in the complexes). It is proposed that autowaves can appear in this system characterized by periodic changes in the concentrations of its components in time and space. Such changes may form a basis for the control of the physiological effects of nitric oxide, dinitrosyl iron complexes, and S-nitrosothiols as agents affecting various cellular and tissue targets.     

Iron nitrosyl complexes as models for biological nitric oxide transfer reagents

Owing to the indiscriminate reactivity of the free NO radical, intricate control mechanisms are required for storage, transport and transfer of NO to its various biological targets. Among the proposed storage components are protein-bound thionitrosyls (R_protein–SNO) and protein-bound dinitrosyl iron complexes. Current knowledge suggests the latter are derived from iron–sulfur cluster degradation in the presence of excess NO. Mobilization of protein-bound NO could involve NO or Fe(NO)₂ unit transfer to small serum molecules such as glutathione, free cysteine, or iron-porphyrins. The study reported is of a reaction model which addresses the key steps in NO transfer from a prototypal iron dinitrosyl complex. While the N,N′-bis(2-mercaptoethyl)-N,N′-diazacyclooctane (bme-daco) ligand typically binds in square-planar N₂S₂ coordination, it also serves as a bidentate dithiolate donor for tetrahedral structures in the preparation of the (H⁺bme-daco)Fe(NO)₂ derivative (Chiang et al., J. Am. Chem. Soc. 126:10867–10874, 2004). The removal of one NO produces the mononitrosyl complex, (bme-daco)Fe(NO), and simplifies studies of NO release mechanisms. We have used heme-type model complexes, Fe or Co porphyrins as NO acceptors, yielding (porphyrin)M(NO), where M is Fe or Co, and monitored reactions by ν(NO) Fourier transform IR spectroscopy. Reaction products were verified by electrospray ionization mass spectrometry. Rudimentary mechanistic studies suggest a role for HNO in the NO release from the dinitrosyl; the mononitrosyl benefits as well from acid catalysis. Other NO uptake complexes such as [(N₂S₂)Fe]₂ [N₂S₂ is bme-daco or N,N’-bis(2-mercapto-2-methylpropyl)-daco] are shown to form Fe(NO) mononitrosyls with stability and spectroscopic signatures similar to those of the porphyrins.     

The antitumor activity of the S-nitrosoglutathione and dinitrosyl iron complex with glutathione: Comparative studies

The antitumor activity of the binuclear form of dinitrosyl iron complexes with glutathione against Lewis lung carcinoma was found earlier with intraperitoneal administration of the complexes. This activity was also observed when this preparation was injected subcutaneously. The complex inhibited the tumor growth by 43% upon subcutaneous injection at a daily dose of 100 µM/kg (as calculated per one iron atom in the binuclear dinitrosyl iron complex) for 10 or 15 days. The effect was observed during the first 2 weeks after tumor transplantation. After this, the tumors began to grow at a rate that was equal to or even higher than that for the control animals. The mean survival time for the treated mice exceeded the control values by 30%. Binuclear dinitrosyl iron complexes were also effective against Ca-755 adenocarcinoma with intraperitoneal administration. In this case, however, the mean survival time for the treated animals only increased by 7%. It was also shown that S-nitrosoglutathione inhibited the growth of Lewis lung carcinoma and Ca-755 adenocarcinoma by 70 and 90%, respectively. However, in contrast to binuclear dinitrosyl iron complexes, the antitumor effect of S-nitrosoglutathione decreased with an increase in the daily dose of the compound from 200 to 400 µM/kg. The initial antitumor effect of binuclear dinitrosyl iron complexes and S-nitrosoglutathione is suggested to be due to NO that is released from both compounds. The subsequent suppression of the effect is caused by the activation of antinitrosative and antioxidant defense systems in tumors.     

Nitrite protonation as a necessary stage in the generation of nitric oxide from nitrite in biological systems

The yields of nitric oxide from 1 mM and 10 mM sodium dithionite in 5 or 150 mM solutions of HEPES buffer (pH 7.4) differed by a factor of 200. Dithionite acted as both a strong reducing agent and an agent responsible for local acidification of the solutions without significant changes in pH. The concentration of nitric oxide was estimated by electron paramagnetic resonance (EPR) by monitoring its incorporation into water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which resulted in the formation of EPR-detectable mononitrosyl complexes of iron. Ten seconds after dithionite addition, the concentration of mononitrosyl iron complexes reached 2 μM, whereas it did not become greater than 0.01 μM in 5 mM HEPES buffer. It has been suggested that this difference results from a longer lifetime of a localized decrease in pH in a weaker buffer solution. This time could be long enough for the protonation of some nitrite molecules. Nitrous acid thus formed decomposed to nitric oxide. A difference in nitric oxide formation from nitrite in weak and strong buffer solutions was also observed in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, in the weak buffer the nitric oxide yield was only three-four times greater than in the strong buffer. An increase in the nitric oxide yield from nitrite was observed in solutions containing both proteins. A significant amount of nitric oxide from nitrite was formed in mouse liver preparation subjected to freezing and thawing procedure followed by slurrying in 150 mM HEPES buffer (pH 7.4) and dithionite addition (10 mM). We suggest that the presence of zones with lowered pH values in cells and tissues may be responsible for the predominance of the acidic mechanism of nitric oxide formation from nitrite. The contribution of nitric oxide formation from nitrite catalyzed by heme-containing proteins as nitrite reductases may be minor under these conditions.     

Autowave distribution of nitric oxide and its endogenous derivatives in biosystems strongly enhances their biological effects: A working hypothesis

It is hypothesized that in cells producing nitric oxide (NO), NO and its endogenous derivatives (low-molecular S-nitrosothiols and dinitrosyl iron complexes (DNIC) with thiol-containing ligands) can move in the intracellular space not only by diffusion but also in an autowave mode. This hypothesis is based on the previously obtained data on autowave distribution of DNIC with glutathione following application of a drop of a solution of Fe²⁺ + glutathione onto the surface of a thin layer of a S-nitrosoglutathione solution. The appearance of autowaves is conditioned by a self-regulating self-sustained system arising in the process. This system consists of self-convertible DNIC and S-nitrosothiols as well as free ferrous iron ions, thiols and NO and can function in the autowave regime for several seconds with subsequent passage to a steady state maintained by chemical equilibrium between DNIC and their constituent components (free Fe²⁺ ions, thiols, S-nitrosothiols and NO). Possible advantages of autowave distribution of NO and its endogenous derivatives in the intracellular space over free diffusion, which might entail higher efficiency of their biological action, are discussed.     

Effects of the donor of nitric oxide,dinitrosyl iron complex with glutathione,on blood circulation in healthy animals

We have found that the hypotensive effect of the nitric oxide donor dinitrosyl iron complex with glutathione was caused by a decrease in general peripheral resistance in healthy rats. This effect did not impair microcirculation and was accompanied by an increase in the myocardial contractile activity. Under the hypotension condition induced by dinitrosyl iron complex with glutathione, we did not find any changes in the oxygen or carbon dioxide tensions in the blood as compared to the control or any change in the acidic-basic blood state. Thus, the possible inhibitory influence of this complex on some enzymes and proteins in the animal body was not accompanied by effects on the heart, vessels, or blood. The dinitrosyl iron complex with glutathione induced a decrease in the arterial pressure only. We hypothesize that a new type of drugs for the treatment of cardiovascular diseases can be developed on the basis of such complexes and complexes with other thiol-containing ligands.     

Detection of basal NO production in rat tissues using iron–dithiocarbamate complexes

We probe endogenous NO production in WKY rats by trapping NO with iron–dithiocarbamate complexes. The aim was to detect non-stimulated NO production in small organs like kidneys of juvenile rats. The yields of mononitrosyl Fe–dithiocarbamate complexes are small and difficult to quantify in the presence of strong contaminating signals from Cu²⁺–DETC complexes. We evaluate four methods to improve the detection of mononitrosyl Fe–dithiocarbamate adducts: progressive microwave saturation, tissue perfusion, spectral subtraction, and finally, reduction of the tissue with sodium dithionite. While the first three were only moderately useful, reduction was very helpful for quantification of the mononitrosyl Fe–dithiocarbamate yield. The increase in sensitivity allows the detection of non-stimulated NO release in small organs of juvenile rats.     

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 Antitumor Properties of Dinitrosyl Iron Complexes with Thiol-Containing Ligands and S-Nitrosoglutathione in Experiments

Biophysics - Abstract—The antitumor activity of S-nitrosoglutathione, bi- and mononuclear dinitrosyl iron complexes with different thiol-containing ligands such as glutathione,...     

Autowave mode of functioning of the system nitric oxide + free iron + thiols may ensure the regulation of biological action of nitric oxide and its endogenic compounds

It has been shown earlier that, in a system NO + Fe2+ + thiols in aqueous solution, an oscillatory mode of changes with time in the concentration of paramagnetic dinitrosyl iron complexes with thiol-containing legends and S-nitrosothiols formed in this system and in the concentration of free iron (not included into dinitrosyl iron complexes) can be realized. It is assumed that, in this system, autowaves can arise, which ensure periodic changes with time and space in the concentration of the system constituents. These changes may underlie the regulation of the physiologic effect of nitric oxide, dinitrosyl iron complexes, and S-nitrosothiols as agents affecting various intracellular and tissue targets.     

Dinitrosyl iron complexes with thiol ligands promote skin wound healing in animals

Dimeric dinitrosyl iron complexes (DNIC) with cysteine or glutathione as NO donors accelerated the healing of experimental skin wound in rats, as demonstrated by histological and histochemical examination. After two injections of an aqueous DNIC solution into the wound (total 5 μmol) on days 1 and 2 after surgery, the granulocyte volume in wound tissue on day 4 was 3–4 times greater than in the control. Higher DNIC doses provoked inflammation in the wound. Similar experiments with another NO donor S-nitrosoglutathione in equivalent amounts (10 μmol) adversely affected the wound. Addition of 2.5 μmol glutathione DNIC for 40 min produced EPR-detectable protein-bound DNIC (2.5 nmol) in wound tissue. Under the same conditions, 5 μmol S-nitrosoglutathione produced less than 10 pmol of protein-bound DNIC; an EPR-active nitrosyl hemoglobin complex was mainly formed (1.5–2.0 nmol) in this case. The beneficial effect of DNIC on the wound was suggested to be due to the delivery of NO to its targets without pronounced formation of cytotoxic peroxynitrite in wound tissue. In contrast, peroxynitrite could form upon administration of rapidly decomposed S-nitrosoglutathione, thereby aggravating the wound condition.     

Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.     

Dinitrosyl iron complexes with glutathione in rat myocardial tissue during regional ischemia and postischemic reperfusion

Injection of dinitrosyl iron complexes with glutathione at the onset of 40-min regional myocardial ischemia in rat was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After introducing the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that in postischemic reperfusion, the rate of decrease in the content of these complexes in the ischemic area increases, which reflects effective scavenging of short-lived reactive oxygen species by the dinitrosyl iron complexes.     

Effect of exogenous donors of nitric oxide and inhibitors of its enzymatic synthesis on experimental ischemic thrombosis in conjunctive veins of the rabbit eyes

The beneficial action of dinitrosyl iron complex with glutathione on conjunctive veins of eyes in rabbits with experimental thrombosis of conjunctive veins has been demonstrated. Aqueous solutions of dinitrosyl iron complexes were added subconjunctively at doses of 5.4-8.1 micromole per eye. The average duration of thrombosis by the action of dinitrosyl iron complex decreased from 6.4 days in control animals to 2 days. The addition of dinitrosyl iron complex resulted in blood flow recovery in occlusive vessels and prevented ischemia and necrosis of tissues. The enhancement of hemorrhagic activity induced by dinitrosyl iron complexes was abrogated with combined addition of the nonselective NO synthase inhibitor N-nitro-L-arginine. In contrast, S-nitrosoglutathione affected adversely the veins: the duration of thrombosis in experimental thrombosis of conjunctive veins increased to 7 days. Intensive hemorhage developed in the conjunctive. The formation of protein-bound dinitrosyl iron complexes was observed by the EPR method in eye tissues after the subconjunctive or parabulbar addition of dinitrosyl iron complex with glutathione. This was not the case when the complex was injected intravenously. It was shown that dinitrosyl iron complex with glutathione induces the blockade of pellet aggregation or strengthens the fibrinolytic activity of plasma of patients with eye vessel pathology. The beneficial action of dinitrosyl iron complexes on conjunctive veins was proposed to be due to the capacity of dinitrosyl iron complexes to donate NO primarily to its biological targets. The release of free NO molecules in large amounts is not characteristic for dinitrosyl iron complexes. This process is characteristic of S-nitrosoglutathione, which sharply increases the probability of the accumulation of peroxynitrite, which produces a toxic effect on cells and tissues.     

Prospects of using magnetic nanoparticles to potentiate the anticarcinogenic action of dinitrosyl iron complexes with thiol ligands

Low-molecular dinitrosyl iron complexes with thiol-containing ligands (cysteine or glutathione) have recently been shown to be capable of modulating apoptosis. Being in the intact state, the complexes prevent apoptosis, and when decomposed, they initiate it. The possibility is considered of delivering the complexes to tumor tissues by ferromagnetic nanoparticles driven by an external magnetic field.