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.     

Intracellular free iron in liver tissue and liver homogenate: studies with electron paramagnetic resonance on the formation of paramagnetic complexes with desferal and nitric oxide.

Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.     

L-arginine--an endogenous source of nitric oxide in animal tissues in vivo]

According to EPR data, NG-mononitro-L-arginine (MNA) being intraperitoneally injected to inbred albino mice in the dose of 70-700 mg/kg strongly decreases the formation of mononitrosyl iron complexes (MNIC) with the exogenous ligand, diethyldithiocarbamate (DETC) in liver cells. Simultaneous injections of experimental mice with MNA (70 mg/kg) and L-arginine (700 mg/kg) are unaccompanied by the formation of MNIC-DETC complexes. It is concluded that nitric oxide (NO) which is produced in mouse liver in vivo and which provides for the formation of MNIC complexes with DETC is generated by L-arginine via an enzymatic reaction which is competitively inhibited by MNA. Besides, MNA causes reversible inhibition and augmented synthesis of NO formed in mouse liver after the injection of the exogenous lipopolysaccharide of E. coli.     

EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.     

On-line detection of nitric oxide formation in liquid aqueous phase by electron paramagnetic resonance spectroscopy.

A method for the detection of the nitric oxide radical (NO) in oxygen-containing aqueous solution by means of electron paramagnetic resonance spectroscopy (EPR) is described. NO evolving from the spontaneous decomposition of 3-morpholinosydnonimine (SIN-1) was trapped by Fe(2+)-diethyldithiocarbamate (DETC) complex dissolved in yeast cell membranes. The resulting mononitrosyl-Fe(2+)-(DETC)2 complex was stable and exhibited a characteristic EPR signal at g perpendicular = 2.04 and g parallel = 2.02 with an unresolved triplet hyperfine structure at g perpendicular in frozen solution and an isotropic triplet signal at gav = 2.03 at 37 degrees C. The amount of NO trapped was calculated from the amplitude of one of the triplet lines calibrated by means of a dinitrosyl-Fe(2+)-thiosulfate standard. The lower detection limit of NO was 0.5 nmol/(ml x h) due to a low background NO signal. The upper detection limit was about 10 nmol NO/40 mg traps (DETC-loaded yeast cells), because of saturation of traps. The trapping efficiency approached 60% under anaerobic conditions and with low concentrations of SIN-1, but decreased progressively with higher concentrations and in the presence of oxygen. Nitrite (up to 0.1 mM) did not increase the background NO level. The sensitivity was sufficient to follow the rate of NO release from SIN-1 on-line at 37 degrees C in a flat quartz cuvette. The time course of NO release detected by EPR spectrometry correlated with the time course of nitrite accumulation measured by diazotation. In conclusion, this method will permit the on-line detection of NO formation from endogenous and pharmacological sources in oxygen-containing aqueous media.     

EPR study of ferric native prostaglandin H synthase and its ferrous NO derivative

Purified prostaglandin H synthase (EC 1.14.99.1) apoprotein, a polypeptide of 72 kDA, was titrated with hemin and EPR spectra of high-spin ferric heme were observed at liquid-helium temperature. With up to one hemin per polypeptide, a signal at g = 6.6 and 5.4, rhombicity 7.5%, evolved owing to specifically bound, catalytic active heme. At higher heme/polypeptide ratios signals at g = 6.3 and 5.9 were observed which were assigned to non-specific heme with no catalytic function. In microsomes from ram seminal vesicles the native enzyme showed the signal at g = 6.7 and 5.2 which could not be increased by the addition of hemin. Cyanide, an inhibitor of the enzyme, reacted at lower concentrations with the specific heme abolishing its signal at g = 6.6 and 5.4. Higher concentrations of cyanide were needed for the disappearance of the signal of non-specific heme. The reduced enzyme reacted with NO and formed two types of NO complexes. A transient complex, with a rhombic signal at gx = 2.07, gz = 2.01 and gy = 1.97, was assigned to a six-coordinate complex. The final, stable complex showed an axial signal at g = 2.12 and g = 2.001 and was assigned to a five-coordinate complex, where the protein ligand was no longer bound to the heme iron. Neither type of signal showed a hyperfine splitting from nitrogen of histidine indicating the absence of a histidine-iron bond in the enzyme. From these results and the similarity of the EPR signal at g = 6.6 and 5.4 to the signal of native catalase (EC 1.11.1.6) we speculated that tyrosinate might be the endogenous ligand of the heme in prostaglandin H synthase.     

The measurement of the amount of iron (III) complexes with desferal in the perfused rat liver by an EPR method

Rat liver was perfused by Hank's solution, containing desferal (deferoxamine). It was shown that in perfusion of the liver spectrum EPR a signal (g = 4.3; H = 63 G) appears. This signal belongs to desferal complexes, containing intracellular Fe/3/. Desferal transfer to the liver tissue and further formation of desferal complexes there takes place within first 5-10 min of liver perfusion by solution, containing 0.5 mM of desferal.     

Characterization of two different five-coordinate soluble guanylate cyclase ferrous-nitrosyl complexes

Soluble guanylate cyclase (sGC), a hemoprotein, is the primary nitric oxide (NO) receptor in higher eukaryotes. The binding of NO to sGC leads to the formation of a five-coordinate ferrous-nitrosyl complex and a several hundred-fold increase in cGMP synthesis. NO activation of sGC is influenced by GTP and the allosteric activators YC-1 and BAY 41-2272. Electron paramagnetic resonance (EPR) spectroscopy shows that the spectrum of the sGC ferrous-nitrosyl complex shifts in the presence of YC-1, BAY 41-2272, or GTP in the presence of excess NO relative to the heme. These molecules shift the EPR signal from one characterized by g 1 = 2.083, g 2 = 2.036, and g 3 = 2.012 to a signal characterized by g 1 = 2.106, g 2 = 2.029, and g 3 = 2.010. The truncated heme domain constructs beta1(1-194) and beta2(1-217) were compared to the full-length enzyme. The EPR spectrum of the beta2(1-217)-NO complex is characterized by g 1 = 2.106, g 2 = 2.025, and g 3 = 2.010, indicating the protein is a good model for the sGC-NO complex in the presence of the activators, while the spectrum of the beta1(1-194)-NO complex resembles the EPR spectrum of sGC in the absence of the activators. Low-temperature resonance Raman spectra of the beta1(1-194)-NO and beta2(1-217)-NO complexes show that the Fe-NO stretching vibration of the beta2(1-217)-NO complex (535 cm (-1)) is significantly different from that of the beta1(1-194)-NO complex (527 cm (-1)). This shows that sGC can adopt different five-coordinate ferrous nitrosyl conformations and suggests that the Fe-NO conformation characterized by this unique EPR signal and Fe-NO stretching vibration represents a highly active sGC state.     

Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes

Certain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria. This may be due to the formation of nitric oxide and iron-thiol-nitrosyl [( Fe-S-NO]) complexes. The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated. A mixture of nitrite (0.72 mM) with iron (1.44 mM) and cysteine (2.16 mM) was found to be extremely inhibitory when autoclaved and diluted into the culture medium. This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 microM-nitrite. If added to the initial culture medium, it prevented growth at 5 microM-nitrite. The mixture was more inhibitory, on the basis of the nitrite concentration used, than the 'Perigo factor', obtained by autoclaving nitrite in growth medium. [Fe-S-NO] compounds of known chemical structure were tested to determine if they were responsible for this effect. Total inhibition of cell growth was observed with the tetranuclear clusters [Fe4S3(NO)7] (Roussin's black salt), [Fe4S4(NO)4] or [Fe4Se3(NO)7], added at concentrations equivalent to 10 microM-nitrite, or with [Fe2(SMe)2(NO)4] (methyl ester of Roussin's red salt), equivalent to 200 microM-nitrite. The rate of hydrogen production in growing cell cultures was inhibited by [Fe4S3(NO)7] at levels equivalent to 2.5 microM-nitrite. EPR spectra of the inhibited cells showed features with g-values of 2.03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1.65. There was no correlation between growth inhibition and the g = 2.03 signal, though there was a better correlation between inhibition and the g = 1.65 signal. The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase. EPR spectra and enzyme assays showed that these proteins were not destroyed by [Fe4S3(NO)7], [Fe4S4(NO)4], [Fe2(SMe)2(NO)4], [Fe(SPh)2(NO)2], or M2 (an autoclaved mixture of 66 mM-cysteine, 3.6 mM-FeSO4 and 0.72 mM-NaNO2) at concentrations higher than those that caused total inhibition of cell growth. Inhibition of cells by [Fe-S-NO] compounds is unlikely to be due to interaction with the preformed enzymes. The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed.     

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics. The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes. These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.     

In vivo distribution and behavior of paramagnetic dinitrosyl dithiolato iron complex in the abdomen of mouse

It has been shown that a dinitrosyl dithiolato iron complex is formed under physiological conditions and that it functions as an NO transporter. In the present study, a diglutathionyl dinitrosyl iron complex [DNIC-(GS)₂] was injected into mice and its abdominal distribution and behavior were examined by using electron paramagnetic resonance (EPR) spectroscopy. The X-band EPR signal intensity of the blood, liver, kidney, and spleen decreased with time but signals from the liver and kidney were readily detectable even 24h after the injection. The time courses of signal intensity were quite similar when the agent was administered via intravenous and subcutaneous injection routes, suggesting that DNIC-(GS)₂ can penetrate readily and rapidly through the membranes. Real-time detection of DNIC-(GS)₂ in the upper abdomen of the living mice was performed by employing an in vivo EPR spectroscopy. These results suggest that DNIC-(GS)₂, an endogenous NO carrier, has an excellent membrane permeability and has a relatively high affinity for the liver and kidney.     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

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.     

Single-crystal electron paramagnetic resonance studies of photolyzed oxy- and nitric oxide-cobalt myoglobins

Low-temperature photodissociation of oxygen from oxy-cobalt myoglobin was studied by single-crystal electron paramagnetic resonance (EPR) spectroscopy at 5 K. The photolyzed oxy-cobalt myoglobin exhibited an EPR spectrum consisting of two nonequivalent sets (species I and II) of the principal values and eigenvectors of the g tensors: g1I = 3.55, g2I = 3.47, and g3I = 2.26 for species I, and g1II = 2.04, g2II = 1.93, and g3II = 1.86 for species II, which resembled neither the deoxy nor the oxy form. Possible models of the photodissociated state of oxy-cobalt myoglobin are proposed by comparison with cobalt porphyrin complexes. The photolyzed product of nitric oxide-cobalt myoglobin exhibited new EPR signals at g = 4.3 and a very broad signal at around g = 2. The principal g values have been determined from the single-crystal EPR measurements: g1 = 4.39, g2 = 4.27, and g3 = 4.00. Analysis of another EPR signal around g = 2 was difficult due to its broadness. Magnetic interactions were observed. An isotropic EPR signal at g = 4.3 suggested a weakly spin-coupled system between cobaltous spin (S = 1/2 or 3/2) and nitric oxide spin (S = 1/2).     

X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine)

Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of (.)NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)(2) were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of (.)NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of (.)NO, but also the redox properties of liver and lung might determine the formation of complexes of (.)NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for (.)NO-metry at room temperature using Fe(DTCS)(2) as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of (.)NO production in a whole organ, such as murine liver.     

EPR study of the oxygen evolving complex in His-tagged photosystem II from the cyanobacterium Synechococcus elongatus

The Mn(4)-cluster and the cytochrome c(550) in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S(0)-state (spin = 1/2) and the S(2)-state (spin = 1/2 and IR-induced spin = 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S(3)-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the S(3)-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at g = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn(4)-cluster in a fraction of centers, while the g = 5.20 and g = 1.51 signals are tentatively attributed to a high-spin state of the Mn(4)-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn(4)-cluster. Biochemical modifications (Sr(2+)/Ca(2+) substitution, acetate and NH(3) treatments) were also investigated. In Sr(2+)-reconstituted PSII, in addition to the expected modified S(2) multiline signal, a signal at g = 5.2 was present instead of the g approximately 4 signal seen in plant PSII. In NH(3)-treated samples, in addition to the expected modified S(2)-multiline signal, a g approximately 4 signal was detected in a small proportion of the reaction centers. This is of note since g approximately 4 spectra arising from the Mn(4)-cluster in the S(2) state have not yet been published in cyanobacterial PSII. The detection of modified S(3)-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH(3)-treated PSII indicate that NH(3) is still bound in the S(3)-state. The acetate-treated PSII behaves essentially as in plant PSII. A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc(550) was perpendicular to the plane of the membrane.     

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.     

Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.     

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.