Why iron–dithiocarbamates ensure detection of nitric oxide in cells and tissues

The in vivo mechanism of NO trapping by iron-dithiocarbamate complexes is considered. Contrary to common belief, we find that in biological systems the NO radicals are predominantly trapped by ferric iron-dithiocarbamates. Therefore, the trapping leads to ferric mononitrosyl complexes which are diamagnetic and cannot be directly detected with Electron Paramagnetic Resonance spectroscopy. The ferric mononitrosyl complexes are far easily reduced to ferrous state with L-cysteine, glutathione, ascorbate or dithiocarbamate ligands than their non-nitrosyl counterpart. When trapping NO in oxygenated biological systems, the majority of trapped nitric oxide is found in diamagnetic ferric mononitrosyl iron complexes. Only a minority fraction of NO is trapped in the form of paramagnetic ferrous mononitrosyl iron complexes with dithiocarbamate ligands. Subsequent ex vivo reduction of biological samples sharply increases the total yield of the paramagnetic mononitrosyl iron complexes. Reduction also eliminates the overlapping EPR spectrum from Cu(2+)-dithiocarbamate complexes. This facilitates the quantification of yields from NO trapping.     

Reduction enhances yields of nitric oxide trapping by iron-diethyldithiocarbamate complex in biological systems.

The mechanism of NO trapping by iron-diethylthiocarbamate complexes was investigated in cultured cells and animal and plant tissues. Contrary to common belief, the NO radicals are trapped by iron-diethylthiocarbamates not only in ferrous but in ferric state also in the biosystems. When DETC was excess over endogenous iron ligands like citrate, ferric DETC complexes were directly observed with EPR spectroscopy at g=4.3. This was the case when isolated spinach leaves, endothelial cultured cells were incubated in the medium with 2.5mM DETC or mouse liver was perfused with 100mM DETC solution. After trapping NO, the nitrosylated Fe-DETC adducts are mostly in diamagnetic ferric state, with only a minor fraction having been reduced to paramagnetic ferrous state by endogenous biological reductants. In actual in vivo trapping experiments with mice, the condition of excess DETC was not met. The substantial quantities of iron in animal tissues were bound to ligands other than DETC, in particular citrate. These non-DETC complexes appear as roughly equal mixtures of ferric and ferrous iron. The presence of NO favors the replacement of non-DETC ligands by DETC. In all biological systems considered here, the nitrosylated Fe-DETC adducts appear as mixture of diamagnetic and paramagnetic states. The diamagnetic ferric nitrosyl complexes may be reduced ex vivo to paramagnetic form by exogenous reductants like dithionite. The trapping yields are significantly enhanced upon exogenous reduction, as proven by NO trapping experiments in plants, cell cultures and mice.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

Comparison of the reactivity of nitric oxide and nitroxyl with heme proteins. A chemical discussion of the differential biological effects of these redox related products of NOS

Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO(-)) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO.     

Formation of paramagnetic nitrosyl complexes of non heme iron in animals with participation of nitric oxide from exogenous and endogenous sources

It was found that thiosulfate has a stabilizing effect on exogenous and endogenous dinitrosyl-iron complexes in mice treated with bacterial lipopolysaccharide. It was assumed that thiosulfate protects dinitrosyl-iron complexes from the destructive influence of superoxide and peroxinitrite whose enhanced synthesis, together with the synthesis of nitric oxide, is initiated in mice by the lipopolysaccharide. For the first time, the formation of dinitrosyl-iron complexes was demonstrated, which occurs with the participation of nitric oxide generated enzymatically via the L-arginine-dependent pathway. The injection of exogenous dinitrosyl-iron complexes with thiosulfate, which, together with diethyldithiocarbamate, provide the formation of exogenous mononitrosyl iron-diethyldithiocarbamate complexes, made it possible to use the ABC method, which markedly enhances the efficiency of scavenging of endogenous nitric oxide in mice treated with lipopolysaccharides.     

EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.     

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.     

EPR spin-trapping study of nitric oxide formation during bilateral carotid occlusion in the rat

The formation of nitric oxide (NO) radicals was demonstrated by electron paramagnetic resonance spectroscopy in the rat during varying degrees of brain ischemia. Diethyldithiocarbamate and Fe-citrate were used as in vivo spin-trapping reagents. The signal of NO spin adducts increased in accordance with the degree of ischemic insults. The formation of NO radicals was inhibited by N^G-nitro]l-arginine methyl ester.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and 15N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and ¹⁵N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.     

Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial

The nitrophorins comprise an unusual family of proteins that use ferric (Fe(III)) heme to transport highly reactive nitric oxide (NO) from the salivary gland of a blood sucking bug to the victim, resulting in vasodilation and reduced blood coagulation. We have determined structures of nitrophorin 4 in complexes with H2O, cyanide and nitric oxide. These structures reveal a remarkable feature: the nitrophorins have a broadly open distal pocket in the absence of NO, but upon NO binding, three or more water molecules are expelled and two loops fold into the distal pocket, resulting in the packing of hydrophobic groups around the NO molecule and increased distortion of the heme. In this way, the protein apparently forms a 'hydrophobic trap' for the NO molecule. The structures are very accurate, ranging between 1.6 and 1.4 A resolutions.     

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.     

Reactive complexes in myoglobin and nitric oxide synthase

In this overview some of our crystallographic and spectroscopic studies on reactive complexes in myoglobin and nitric oxide synthase are summarised. Myoglobin and nitric oxide synthase are both haemoproteins with some similar reaction intermediates. For myoglobin we have studied different intermediates generated in the reaction with hydrogen peroxide by X-ray diffraction, single-crystal microspectrophotometry, electron paramagnetic resonance spectroscopy, Mössbauer spectroscopy, resonance Raman spectroscopy and quantum refinement. Several of these myoglobin states are quite susceptible to radiation-induced changes during crystallographic data collection, and we have observed a radiation-induced change of the ferric resting myoglobin to aqua ferrous myoglobin, of myoglobin compound II to a proposed intermediate H, and of myoglobin compound III to peroxy myoglobin. For the myoglobin compound II/ intermediate H we observe a single-bonded Fe^IV-O species, which is probably protonated. The long Fe-O bond seen in the crystal structure can be supported by the observation of a new ¹⁸O-sensitive resonance Raman mode at 687 cm⁻¹. For nitric oxide synthase we detected with cryobiochemical methods in electron paramagnetic resonance spectra the first biopterin radical serving as electron donor to the ferrous-oxy complex, and that biopterin serves as a proton donor as well, in addition we could observe formation of the Fe(NO) complex with a amino-pterin cofactor capable to form a reactive radical.     

Rat liver-mediated metabolism of hydroxyurea to nitric oxide

Hydroxyurea is an approved treatment for sickle cell disease. Oxidation of hydroxyurea results in the formation of nitric oxide (NO), which also has drawn considerable interest as a sickle cell disease therapy. Although patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of crude rat liver homogenate to convert hydroxyurea to nitrite/nitrate, evidence for NO production. Nitrite/nitrate form at therapeutic concentrations of hydroxyurea in a clinically relevant time frame. Electron paramagnetic resonance (EPR) studies show the formation of iron nitrosyl complexes during this incubation and experiments with labeled hydroxyurea show the NO derives from the drug. Gas chromatography-mass spectrometry measurements indicate the hydrolysis of hydroxyurea to hydroxylamine in this system. Incubation of hydroxylamine with crude rat liver homogenate also generates nitrite/nitrate and iron nitrosyl complexes. A line of evidence including inhibitor studies, EPR spectroscopy, and nitrite/nitrate detection identifies catalase as a possible oxidant for the conversion of hydroxyurea to NO. These results reveal the ability of liver tissue to convert hydroxyurea to nitric oxide and provide insight into the metabolism of this drug.     

Effect of Rubia cordifolia, Fagonia cretica linn, and Tinospora cordifolia on free radical generation and lipid peroxidation during oxygen-glucose deprivation in rat hippocampal slices

The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis, and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC), and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. Hippocampal slices were subjected to oxygen-glucose deprivation (OGD) and divided into three groups, control, OGD, and OGD+drug treated. Cytosolic reduced glutathione (GSH), nitric oxide [NO, measured as nitrite (NO2)]. EPR was used to establish the antioxidant effect of RC, FC, and TC with respect to superoxide anion (O*2-), hydroxyl radicals (*OH), nitric oxide (NO) radical, and peroxynitrite anion (ONOO-) generated from pyrogallol, menadione, DETA-NO, and Sin-1, respectively. RT-PCR was performed for the three herbs to assess their effect on the expression of gamma-glutamylcysteine ligase (GCLC), iNOS, and GAPDH gene expression. All the three herbs were effective in elevating the GSH levels and expression of the GCLC. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as revealed by electron paramagnetic resonance spectroscopy, diminishing the expression of iNOS gene. RC, FC, and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression levels and may be effective therapeutic tool against ischemic brain damage.     

Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation.

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.     

Experimental evidence suggesting that nitric oxide diffuses from tissue into blood but not from blood into tissue

The aim of this study was to evaluate in vivo whether nitric oxide (NO) is able to diffuse from blood into tissues and vice versa from tissues into blood. We used an in vivo model of intestinal ischemia (superior mesenteric artery occlusion) selectively increasing NO levels in intestinal tissue and an infusion of L-arginine selectively increasing NO levels in blood. In this model we followed formation of nitrosyl complexes of hemoglobin (Hb-NO) in blood and nitrosyl-diethyldithiocarbamate-iron complexes (DETC--Fe--NO) in ischemic intestine and normoxic tissues by means of electron paramagnetic resonance spectroscopy. NO trapping by DETC--Fe in the tissues resulted in a reduction of Hb--NO levels in blood accompanied by the formation of water-insoluble DETC--Fe-NO complexes in ischemic intestine and normoxic tissues both during ischemia and during reperfusion. Administration of L-arginine increased NO levels in blood but neither in ischemic intestine nor in normoxic tissue. Our data suggest that NO released in blood from endothelial cells does not diffuse into tissue. In contrast, NO formed in tissue diffuses into blood. The latter indicates that NO formed in tissues may exert its biological activities systematically.     

Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species

It has been established that albumin-bound dinitrosyl iron complexes can be destroyed by superoxide radicals generated in a xanthine-xanthine oxidase system. It was shown that peroxynitrite also effectively destroyed albumin-bound dinitrosyl iron complexes. At the same time, hydrogen peroxide and tert-butyl hydroperoxide did not stimulate the destruction of albumin-bound dinitrosyl iron complexes up to concentrations one order higher than the content of NO. The data have been obtained indicating that dinitrosyl iron complexes possess the vasodilatory activity. It has been proposed that peroxynitrite and superoxide radical, by causing the destruction of albumin-bound dinitrosyl iron complexes, affect the physiological properties of nitric oxide.     

Caloric restriction increases free radicals and inducible nitric oxide synthase expression in mice infected with Salmonella Typhimurium

It is well known that CR (caloric restriction) reduces oxidative damage to proteins, lipids and DNA, although the underlying mechanism is unclear. However, information concerning the effect of CR on the host response to infection is sparse. In this study, 6-month-old mice that were fed AL (ad libitum) or with a CR diet were infected with Salmonella serovar Typhimurium. EPR (electron paramagnetic resonance; also known as ESR (electron spin resonance)) was used to identify FRs (free radicals). These results were subsequently correlated with SOD (superoxide dismutase) catalytic activity, iNOS [inducible NOS (nitric oxide synthase) or NOSII] expression and NO (nitric oxide) content. EPR analysis of liver samples demonstrated that there was a higher quantity of FRs and iron-nitrosyl complex in infected mice provided with a CR diet as compared with those on an AL diet, indicating that CR was beneficial by increasing the host response to Salmonella Typhimurium. Furthermore, in infected mice on the CR diet, NOSII expression was higher, NO content was greater and spleen colonization was lower, compared with mice on the AL diet. No changes in SOD activity were detected, indicating that the NO produced participated more in the formation of iron-nitrosyl complexes than peroxynitrite. These results suggest that CR exerts a protective effect against Salmonella Typhimurium infection by increasing NO production.