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.     

NO trapping in biological systems with a functionalized zeolite network.

Zeolite-Y powder has been functionalized with ferric iron-diethyldithiocarbamate complexes and applied to trap nitric oxide radicals in liquids and biological systems. The complexes have been assembled in situ in the pores of zeolite-Y and act as traps for nitric oxide radicals. The resulting mononitrosyl-iron complexes form a mixture of diamagnetic ferric and paramagnetic ferrous complexes. The yield of trapped NO may be determined ex situ using electron paramagnetic resonance. The material may be anchored on solid surfaces, mixed into a composite or compressed into small pellets. The material was used to detect endogenous NO in endothelial cell cultures and spinach leaves. The sensitivity of the functionalized zeolite is significantly better than that achieved in conventional trapping of NO with iron-diethyldithiocarbamate complexes.     

Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: implications for NO trapping

Using EPR spectroscopy, we show that the water-soluble mononitrosyl iron complexes with N-methyl-D-glucamine dithiocarbamate (MNIC-MGD) ligands can easily react with superoxide and with peroxynitrite. The reaction with superoxide transforms the paramagnetic MNIC-MGD complex into an EPR silent complex with a reaction rate of 3 x 10(7) (M.s)(-1). Suppletion of ascorbate partially restores the complexes to their original paramagnetic state. We propose that the reaction of MNIC-MGD with either superoxide or peroxynitrite leads to identical EPR silent complexes. Our results have important implications for the technique of NO trapping in biosystems with Fe-dithiocarbamate complexes, where mononitrosyl-iron complexes (hydrophilic as well as hydrophobic) are formed as adducts in the trapping reaction. This principle is illustrated by NO trapping experiments on viable cultured endothelial cells. We find that MNIC-MGD acts as a very potent and water-soluble antioxidant with an efficiency exceeding most SOD mimics. Moreover, by accounting for the EPR silent fraction of iron complexes, the sensitivity of NO trapping can be enhanced considerably. The method was demonstrated for hydrophobic iron-dithiocarbamate complexes in endothelial cell cultures, where sensitivity for NO detection was enhanced by a factor of 5.     

Why does iron abrogate the cytotoxic effect of S-nitrosothiols on human and animal cultured cells?

It was found that dinitrosyl iron complexes (DNIC) with thiol-containing ligands (cysteine or glutathione) of concentrations up to 1 mM produce no cytotoxic effect on cultured cells from human milk gland carcinoma (MCF-7). The cytotoxic action on MCF-7 cells was produced by S-nitrosocysteine: at a concentration of 1 mM, it induced the death of 50% cells. A more stable S-nitrosothiol, S-nitrosoglutathione, did not produce any cytotoxic effect at the same concentration. It is assumed that the negative action of nitrosocysteine is due to its rapid degradation, which results in the accumulation of large amounts of free NO molecules followed by their oxidation by superoxide ions to peroxynitrite, an efficient inhibitor of metabolic processes. These processes seem to be not characteristic of the more stable S-nitrosoglutathione. The cytotoxic effect of nitrosocysteine was completlly abrogated by the addition of 0.2 mM ferrous citrate complex to the medium. When S-nitrosoglutathione NO (0.5 mM) or S-nitrosoglutathione (0.5 mM) + Fe(2+)-citrate (0.2 mM) were added to the medium, protein-bound dinitrosyl iron complexes formed with the involvement of endogenous or exogenous iron were detected in cells. The amount of the complexes in the presence of exogenous iron increased four times, reaching the value of 1.6 nmole/5 x 10(6) cells. Therefore, it was proposed that the blockade of the cytotoxic action of S-nitrosoglutathione by iron complexes is due to Cys-NO transformation of S-nitrosocysteine into dinitrosyl iron complexes. The high stability of these complexes ensures only a gradual accumulation of nitric oxide in cells.     

Endogenous superoxide production and the nitrite/nitrate ratio control the concentration of bioavailable free nitric oxide in leaves

We have quantitatively measured nitric oxide production in the leaves of Arabidopsis thaliana and Vicia faba by adapting ferrous dithiocarbamate spin tapping methods previously used in animal systems. Hydrophobic diethyldithiocarbamate complexes were used to measure NO interacting with membranes, and hydrophilic N-methyl-d-glucamine dithiocarbamate was used to measure NO released into the external solution. Both complexes were able to trap levels of NO, readily detectable by EPR spectroscopy. Basal rates of NO production (in the order of 1 nmol g(-) (1) h(-1)) agreed with previous studies. However, use of methodologies that corrected for the removal of free NO by endogenously produced superoxide resulted in a significant increase in trapped NO (up to 18 nmol g(-) (1) h(-1)). Basal NO production in leaves is therefore much higher than previously thought, but this is masked by significant superoxide production. The effects of nitrite (increased rate) and nitrate (decreased rate) are consistent with a role for nitrate reductase as the source of this basal NO production. However, rates under physiologically achievable nitrite concentrations never approach that reported following pathogen induction of plant nitric-oxide synthase. In Hibiscus rosa sinensis, the addition of exogenous nitrite generated sufficient NO such that EPR could be used to detect its production using endogenous spin traps (forming paramagnetic dinitrosyl iron complexes). Indeed the levels of this nitrosylated iron pool are sufficiently high that they may represent a method of maintaining bioavailable iron levels under conditions of iron starvation, thus explaining the previously observed role of NO in preventing chlorosis under these conditions.     

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.     

Heme ferrous-hydroperoxo complexes: some theoretical considerations

We report density functional calculations on complexes of ferrous hemes with hydroperoxide, where the axial ligand trans to OOH(-) is imidazole, thiolate, or phenoxide. The geometrical parameters and charge distributions within the Fe-O-O-H moiety are identical between the ferrous complexes reported here and their ferric counterparts previously described, even though the latter contain one unpaired electron on iron as opposed to the former, which are diamagnetic. The extra negative charge upon going from a formally ferric state to formally ferrous appears to be distributed essentially on the porphyrin. These findings support recent experimental data showing that the ferrous state of certain hemoproteins can interact with peroxides in a catalytically competent fashion, cleaving the O-O bond heterolytically in a manner reminiscent of the "canonical" ferric-peroxo complexes, and contrary to any expectations based on the Fenton concept commonly invoked in non-heme chemistry.     

Tracing the structure-function relationship of neuroglobin and cytoglobin using resonance Raman and electron paramagnetic resonance spectroscopy

The physiological role of neuroglobin and cytoglobin, two vertebrate globins discovered in the last 5 years, is not yet clearly understood. In this work, we review the structural information on these globins and its implication on the possible protein function, obtained by electron paramagnetic resonance and resonance Raman spectroscopy. All studies reveal a high flexibility in the heme-pocket region of neuroglobin. Together with the observation that the distal ligand of the heme iron is the endogenous E7-histidine in both the ferric and ferrous form of neuroglobin and cytoglobin, the flexibility of the heme environment in neuroglobin will play a crucial role in the globins' ability to bind and stabilize exogenous ligands.     

Redox properties of iron–dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems

While the Fe²⁺–dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe²⁺ complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe³⁺ complexes. Following exposure to NO, diamagnetic NO–Fe³⁺ complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO–Fe³⁺–MGD and lipid soluble NO–Fe²⁺–DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO–Fe²⁺–MGD complexes with yield of up to 50% and the balance is converted to Fe³⁺–MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO–Fe³⁺–MGD into NO–Fe²⁺–MGD (60–90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe²⁺–MGD. With isotope labeling of the NO–Fe³⁺–MGD with ⁵⁷Fe, it was shown that these complexes donate NO to Fe²⁺–MGD. NO–Fe³⁺–MGD complexes were also formed by reversible oxidation of NO–Fe²⁺–MGD in air. The stability of NO–Fe³⁺–MGD and NO–Fe²⁺–MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron–dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.     

Resonance Raman studies of cytochrome P450BM3 and its complexes with exogenous ligands.

Resonance Raman spectra are reported for both the heme domain and holoenzyme of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, palmitate. Comparison of the spectrum of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme, a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, while the binding of substrate induces relatively dramatic changes in the nu(Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is nonexistent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450BM3 and has been suggested to prevent hydroxylation of the terminal, omega, position of long-chain fatty acids.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.     

Gamma-irradiation potentiates L-arginine-dependent nitric oxide formation in mice.

Gamma-irradiation of mongrel mice at a sublethal dose (700 Roentgen) enhanced the formation of nitric oxide (NO) in the liver, intestine, lung, kidney, brain, spleen or heart of the animals. NO formation was determined by the increase in intensity of the EPR signal due to trapping of NO into mononitrosyl iron complexes (MNIC) with exogenous diethyldithiocarbamate (DETC) injected intraperitoneally. The EPR signal of these MNIC-DETC complexes was characterized by g-factor values at g perpendicular values at g perpendicular = 2.035 and g parallel = 2.02 and a triplet hyperfine structure at g perpendicular. The NO synthase inhibitor, NG-nitro-L-arginine, prevented MNIC-DETC complex formation both in liver and intestine, demonstrating the involvement of endogenous NO formed. Thus, gamma-irradiation may enhance endogenous NO biosynthesis in these tissues, presumably by facilitating the entry of Ca2+ ions into the membrane as well as the cytosol of NO-producing cells through irradiation-induced membrane lesions.     

Dinitrosyl Iron Complexes with Thiol-Containing Ligands Exist in Living Organisms Mainly in the Binuclear Form

The levels of the mononitrosyl iron complex with diethyldithiocarbamate that form in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite, or the vasodilating drug isosorbide dinitrate (Isoket®) have been assessed by electron paramagnetic resonance (EPR). The levels of the complex in mice that received binuclear dinitrosyl iron complexes with thiol-containing ligands or S-nitrosoglutathione do not change after the treatment of liver preparations with the strong reducing agent dithionite, in contrast to those formed after nitrite or isosorbide dinitrate administration, whose levels sharply increase after the same treatment. It is inferred that in the latter case an EPR-active mononitrosyl iron complex with diethyldithiocarbamate is produced with the absence or presence of dithionite in the reaction of NO formed from nitrite with Fe²⁺-diethyldithiocarbamate and Fe³⁺-diethyldithiocarbamate complexes, respectively. In the former case, the mononitrosyl iron complex with diethyldithiocarbamate is produced by transition of iron-mononitrosyl fragments from already present iron-dinitrosyl groups of binuclear dinitrosyl complexes, whose content is three to four times higher than the content of the mononuclear form of these complexes in the tissue. The results we obtained indicate that when dinitrosyl iron complexes with thiol-containing ligands, either introduced into the body or produced with the participation of endogenous NO, appear in animal tissues in vivo, these complexes are presented in these tissues mainly in their diamagnetic, EPR-silent binuclear form.

     

Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems

While the Fe(2+)-dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe(2+) complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe(3+) complexes. Following exposure to NO, diamagnetic NO-Fe(3+) complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO-Fe(3+)-MGD and lipid soluble NO-Fe(2+)-DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO-Fe(2+)-MGD complexes with yield of up to 50% and the balance is converted to Fe(3+)-MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO-Fe(3+)-MGD into NO-Fe(2+)-MGD (60-90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe(2+)-MGD. With isotope labeling of the NO-Fe(3+)-MGD with (57)Fe, it was shown that these complexes donate NO to Fe(2+)-MGD. NO-Fe(3+)-MGD complexes were also formed by reversible oxidation of NO-Fe(2+)-MGD in air. The stability of NO-Fe(3+)-MGD and NO-Fe(2+)-MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron-dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.     

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.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

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.