Lowmolecularweight Dnic With Thiolcontaining Ligands

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The first description of EPR spectra of low-molecular DNIC with various anionic ligands (phosphate, pyrophosphate, arsenate, molybdate, carbonate, maleate, mercaptane, cysteine,

Fig. 2. 2.03 signals from the livers of mice maintained on drinking water with nitrite + 57Fe-citrate complex (curve a) or nitrite + 56Fe-citrate complex (curve b). EPR signals of DNIC with cysteine, containing 57Fe (curves c,f) or 56Fe (curves d,g). Recordings were made at 77 K (curves a-d) or ambient temperature (curves f,g). (From Ref. [15].)

Fig. 2. 2.03 signals from the livers of mice maintained on drinking water with nitrite + 57Fe-citrate complex (curve a) or nitrite + 56Fe-citrate complex (curve b). EPR signals of DNIC with cysteine, containing 57Fe (curves c,f) or 56Fe (curves d,g). Recordings were made at 77 K (curves a-d) or ambient temperature (curves f,g). (From Ref. [15].)

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Fig. 3. 2.03 signals from rabbit liver (A) or yeast cells (B). Recordings were made at ambient temperature. A narrow signal superimposed on the 2.03 signal (B) is due to a low-molecular DNIC. (C) The change of the shape of the EPR signal of DNIC with cysteine at temperature increasing from -196 to 0°C. (From Ref. [13].)

Fig. 3. 2.03 signals from rabbit liver (A) or yeast cells (B). Recordings were made at ambient temperature. A narrow signal superimposed on the 2.03 signal (B) is due to a low-molecular DNIC. (C) The change of the shape of the EPR signal of DNIC with cysteine at temperature increasing from -196 to 0°C. (From Ref. [13].)

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Field (Gs)

Fig. 4. (A) EPR spectrum of DNIC with OH- (pH 11) containing 14NO (a) or 15NO (b) [36]. (B,C) EPR spectra of DNIC with OH- (pH 12) (a,a~), DNIC with H2O (pH 7) (b,b~), DNIC with phosphate (c,c*) or DNIC with cysteine (d,d~) [6]. (D) EPR spectrum of DNIC with OH- containing 15NO and 57Fe (pH 11) [36]. Recordings were made at ambient temperature (A,B,D) or 77 K (C). (With permission.)

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Field (Gs)

Fig. 4. (A) EPR spectrum of DNIC with OH- (pH 11) containing 14NO (a) or 15NO (b) [36]. (B,C) EPR spectra of DNIC with OH- (pH 12) (a,a~), DNIC with H2O (pH 7) (b,b~), DNIC with phosphate (c,c*) or DNIC with cysteine (d,d~) [6]. (D) EPR spectrum of DNIC with OH- containing 15NO and 57Fe (pH 11) [36]. Recordings were made at ambient temperature (A,B,D) or 77 K (C). (With permission.)

thiourea, penicillamine, hydroxyl and other) was given by McDonald et al. in the 1960s. EPR spectra of the complexes in aqueous solutions were recorded at ambient temperature [36]. In dilute solution at X-band frequencies, these complexes have a characteristic EPR spectrum showing an isotropic singlet at g = 2.02-2.04 with resolved HFS from the nuclear magnetic moment of nitrogen atoms in NO ligands. Additional HFS may arise from protons or phosphorus atoms in anionic ligands (Fig. 4). The analysis of EPR characteristics of the complexes indicated that the paramagnetic complexes consist of one iron atom, two NO groups and two anionic ligands. EPR spectra show an additional strong doublet hyperfine (HF) coupling of 1.25 mT from the iron nucleus if the 57Fe isotope is used. This magnitude shows that the unpaired electrons from the nitrosyl ligands are predominantly localized on iron atom (Fig. 4). So, d7 electron configuration and low spin state S = 1/2 were proposed to explain the spectroscopic properties of the complexes [36]. Infrared studies of the intramolecular vibrations suggest that the nitrosyl ligands have donated an electron to the iron and resemble NO+ rather than NO [37]. This nitrosonium character is further corroborated by the small HFS coupling to the nitrogen nucleus in the nitrosyl ligands in DNIC (ca 0.15 mT) [36]. Thus, the electronic structure of DNIC is better represented by the formula {(L-)2Fe+(NO+)2}+. The geometrical structure of DNIC is still a matter of debate and depends on the aggregation state. As will be discussed below, X-ray diffraction has shown tetrahedral surrounding of the iron in crystalline state, but in dilute solution EPR spectroscopy favors square planar configuration.

In frozen solution at X-band, the EPR spectra of DNIC with low-molecular-weight anionic ligands have the shape of a spin S = 1/2 center with either axial or rhombic symmetry. Axial symmetry is mainly characteristic of DNIC with thiol-containing ligands, and the complexes have g-factors with the values of g± = 2.045 or 2.04, g|| = 2.014 (Fig. 5). The spectral shape shows that the coupling tensor has gz < gx = gy, i.e. oblate axial symmetry rather than prolate. At higher frequencies the Zeeman anisotropy is better resolved, and will be discussed g = 2.045 2.04 2.014

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Fig. 5. EPR spectra from frozen solutions of DNIC with cysteine containing 14NO (curve a) or 15NO (curve b) and DNIC with thiosulphate (curve c). Recordings were made at 77 K. (From Ref. [40].)

at the end of this chapter. Lower rhombic symmetry is mainly characteristic of DNIC with various non-thiol-containing ligands (phosphate, halogens, citrate, ascorbate, histidine, etc.), the three elements of the g-tensor spanning the range 2.014-2.05. Nevertheless, some non-thiolate complexes (for example, DNIC with hydroxyl) show axial symmetry with g-factors of gx = 2.014, g|| = 2.05, gav = 2.026 [6,7]. The representative EPR spectra of DNIC with non-thiol-containing ligands are presented in Fig. 4.

DNICs with low-molecular-weight thiol-containing ligands can be divided into two groups in accordance with the shape of central part of the EPR spectrum and the value of gx, g||and gav = (g||+ 2gx)/3. The first group comprises the EPR signals from DNIC with cysteine, glutathione, N-acetylcysteine, homocysteine, N-acetylpenicillamine or dithiothreitol (gx = 2.04, g|| = 2.014 and gav = 2.031). The EPR signals from DNIC with thiosulfate and mercaptotriazole (gx = 2.045, g||= 2.014 and gav = 2.035) constitute the second group [40]. The representative EPR spectra from both groups are presented in Fig. 5. The spectrum of frozen solution of DNIC with cysteine including 15NO ligands is also shown in Fig. 5.

In dilute solution at room temperature, fast tumbling motions averages the axial anisotropy of g-factor resulting in the registration of narrow isotropic EPR signals with giso values at the range 2.029-2.031 for all the mentioned DNIC with thiol-containing ligands. The signals show resolved HFS with 5 or 13 components. The 5 component HFS has 1:2:2:2:1 intensity and is characteristic of DNIC with thiosulfate, N-acetylpenicillamine or mercaptotriazole (Fig. 6, curve c). It is caused by HF interaction with the nitrogen nuclear moments (I = 1) of both NO ligands. At room temperature, the DNIC with other thiol-containing ligands shows resolved 13-component HFS. This multiplicity results from HF interaction with the two nitrogen nuclei of the nitrosyl ligands plus four protons (I = 1 /2) from two methylene groups close to the sulfur atom (Fig. 6, curve a). The HF interaction with the nitrogen may be modified by substitution with the 15N isotope (I = 1/2): With 15NO ligands, the spectrum shows 9 resolved components (Fig. 6, curve b). The description of the HFS formation in the signals is shown on the right side of Fig. 6.

Quite often, the EPR spectra show resolved HFS from four protons from two separate methylene groups. Examples are DNIC with cysteine, glutathione, N-acetylcysteine, homo-cysteine or dithiothreitol ligands. This property demonstrates that the complexes contain two thiol-containing ligands. Therefore, these DNICs are cationic and can be presented as {(RS-)2Fe+(NO+)2}+ as proposed in Ref. [36]. This formula was also found for DNIC with mercaptotriazole (from X-ray analysis described in Ref. [41]). Since DNIC with thiosulfate or N-acetylpenicillamine have similar EPR spectra in solution. Although not yet proven, it is plausible that they have the same formula as well.

It is noteworthy that the values of giso at room temperature differ slightly from the average gav = (g||+ 2gx)/3 as obtained in frozen state. Evidently, it is due to the influence of the solvent on the electronic structure of the complexes in the solution at ambient temperature. As proposed in Refs. [38,39] besides two thiolate and two nitrosyl ligands the DNICs may also contain one or two solvent molecules (usually water). Changing the solvent to dimethyl-formamide, dimethylsulfoxide (DMSO), tetramethylurea or hexamethanol affects the EPR spectra significantly. In particular, it shows a marked decrease from axial to rhombic symmetry of the paramagnetic center (Fig. 7) [42]. Cysteine is highly soluble in these polar organic solvents and the DNICs undergo slow precipitation (on a timescale of several minutes) but the EPR spectra could be observed for a few minutes.

Hyperfine Structure
Fig. 6. EPR spectra from solutions of DNIC with cysteine containing NO (curve a) or NO (curve b) and DNIC (14NO) with thiosulphate (curve c). Recordings were made at ambient temperature. On the right -decoding of hyperfine structure of the signals. (From Ref. [40].)

Usually, only a fraction of the available iron is included in paramagnetic DNIC with thiol-containing ligands: Often a substantial quantity of iron is included in the form of dimeric DNIC, which is favored at acid conditions and lower thiol:iron ratios [43,44]. Dimeric DNIC is diamagnetic due to antiferromagnetic coupling between the DNIC entities. Therefore, at a given iron concentration, the yield of paramagnetic monomeric DNIC is usually decreasing with pH due to dimerization. At neutral pH, only DNIC with cysteine or thiosulfate remain fully monomeric and include all iron. With other ligands, higher pH is usually required. Full incorporation of all available iron in monomeric DNIC is usually achieved at pH ~ 10. The dimerization has a strong effect on the color of the solutions. The monomeric DNIC complexes have a dark green color due to d-d transitions in the wavelength region 600-850 nm (Fig. 8). A much stronger absorption band is observed at 392 nm (£392 = 3580 (Mcm)-1) [45,46] (Fig. 8). Upon decreasing the pH from 10 to 6, the intensity of the EPR signals from these complexes drops reversibly 5-10 times and the color of the solutions turned

Fig. 7. Change of EPR signal shape of DNIC with cysteine (aqueous solution) (curve a) after mixing with dimethylformamide (1:100) (curves b,c), dimethylsulfoxide (1:4) (curve d), tetramethylurea (1:10) (curve e) or hexamethapol (1:10) (curve f) [42]. EPR spectra of DNIC with bovine serum albumin with 56Fe (curves g,h) or 57Fe (curve i) [54]. Recordings were made at 77 K (curves a-g,i) or ambient temperature (curve h).

Fig. 7. Change of EPR signal shape of DNIC with cysteine (aqueous solution) (curve a) after mixing with dimethylformamide (1:100) (curves b,c), dimethylsulfoxide (1:4) (curve d), tetramethylurea (1:10) (curve e) or hexamethapol (1:10) (curve f) [42]. EPR spectra of DNIC with bovine serum albumin with 56Fe (curves g,h) or 57Fe (curve i) [54]. Recordings were made at 77 K (curves a-g,i) or ambient temperature (curve h).

to yellow-orange. The color is characteristic of the solutions of diamagnetic dimeric DNICs with thiol-containing ligands.

The ratio between iron and thiol decides whether the DNIC complexes appear as monomers or dimers. Except for thiosulfate, dimeric DNIC are synthesized at the stoichiometric ratio of iron:thiol of 1:2 (DNIC 1:2) [43,44,47,48]. The dimeric form does not give any EPR signal and has two optical absorption bands at 310 and 360 nm [47,48] (Fig. 8). Dimeric DNIC with cysteine is quantitatively transformed into paramagnetic monomeric DNIC by the addition of excess cysteine (cysteine:iron ratios exceeding 20:1).

In frozen state, the EPR spectra of DNIC do not show resolved HF interactions, except after isotopic substitution of iron with 57Fe (Fig. 2). Still, simulation of the frozen spectrum with only axial Zeeman coupling does not lead to good fits of the EPR lineshape. In particular, the simulated intensity at ^remains smaller than that near the central feature, in conflict with the experimental lineshape [49] (Fig. 9). For the EPR signals from frozen solutions of DNIC with thiol-containing ligands, the amplitude at g| | is considerably higher than that of central component. This phenomenon is attributed to unresolved anisotropic HFS from the nitrogens of the nitrosyl ligands with splitting at g± much higher than for parallel alignment of the complexes with respect to the magnetic field.

McDonald et al. also described in their article [36] the mononitrosyl iron complexes (MNICs) formed from ferrous iron, NO and dithiols. At room temperature, the complexes give rise to triplet HFS at g = 2.027-2.041 with component separations of 1.55 mT (Fig. 10). The EPR spectra are similar to those from MNIC with the derivatives of thiocarbonic acids, dithiocarbamates, xanthogenates, etc. [50-52]. Ferrous iron and cysteine were proved to be capable of generating MNIC also in aqueous solutions treated with nitric oxide at the amount lower than that of iron and cysteine [10,53-55]. The MNIC with cysteine is usually detected

Fig. 8. Top panel: UV-Visible spectra of 0.8 mM 5-nitrosocysteine in 100 mM HEPES buffer, pH 7.4 (curve b) and 0.15 mM monomeric DNIC-cysteine complex (curve a and curve shown in inset) [46]. Bottom panel: Optical spectra of 0.2 mM dimeric DNIC-cysteine complex in 15 mM HEPES buffer, pH 7.4 (curve a). Curve b is the spectrum of monomeric DNIC after 30 min in air. (From Ref. [47].)

at the first step of DNIC-cysteine formation when gaseous NO begins to penetrate the ferrous-cysteine solutions. Further exposure to NO results in MNIC-cysteine being transformed to DNIC-cysteine [10,53]. When recorded at ambient temperature, the MNIC-cysteine gives clearly resolved triplet HFS at g = 2.04 (Fig. 10). Frozen solutions show a comparatively wide asymmetrical singlet EPR signal at gav = 2.04 (gx = 2.055, g|| = 2.01) and a slightly resolved triplet HFS from the nitrogen nucleus (I = 1) at g||(Fig. 10) [53].

Interestingly, the ferrous-ethylxanthogenate complex can form both MNIC and DNIC when exposed to NO in dimethylformamide or DMSO solutions [56]. The DNIC complexes dominate if some nitrogen dioxide is added together with gaseous NO. In frozen solution, DNIC-ethylxanthogenate has an anisotropic EPR spectrum at gav = 2.035 (Fig. 11). In solution at room temperature, the isotropic EPR signal at g = 2.03 shows resolved quintet HFS structure (Fig. 11). Isotopic labeling with 57Fe (I = 1/2) of DNIC-ethylxanthogenate gives signal broadening at 77 K in frozen solution. In solution at room temperature, the spectrum shows additional doublet HFS splitting of the signal from 57Fe (Fig. 11).

So far we discussed the DNIC complexes with d7 configuration, the {Fe(NO)2}7 structure in Enemark-Feltham notation [57], but the DNICs may undergo redox transformations to

Fig. 9. Calculated shape of the EPR signals from polycrystalline samples with axial symmetry of g-factor at various values of S = \H± — H\\\/AHsp where AHsp is the width of a spin-packet [49]. AHsp = T2

where y, Hi, T\ and T2 are electron gyromagnetic ratio, amplitude of magnetic component of microwave field, times of spin-lattice and of spin-spin relaxations, respectively.

Fig. 9. Calculated shape of the EPR signals from polycrystalline samples with axial symmetry of g-factor at various values of S = \H± — H\\\/AHsp where AHsp is the width of a spin-packet [49]. AHsp = T2

where y, Hi, T\ and T2 are electron gyromagnetic ratio, amplitude of magnetic component of microwave field, times of spin-lattice and of spin-spin relaxations, respectively.

other electronic states. DNIC with cysteine or glutathione ligands may be reduced to complexes with {Fe(NO)2}8 (diamagnetic) and {Fe(NO)2}9 (paramagnetic S = 1/2) centers. In frozen solution, the latter show EPR spectra with axial symmetry and g± = 2.01 and g|l = 1.97. In dilute solution, a single line without HFS appears at g = 2.0 (Fig. 12). Isotopic substitution of iron with 57Fe causes broadening of the EPR spectrum and demonstrates that the unpaired electron of the {Fe(NO)2}9 state is localized on the iron atom (Fig. 12) [58-61].

The redox state of DNIC is clearly reflected in the optical absorption as well. The addition of dithionite to a dark-green solution of monomeric DNIC with {Fe(NO)2 }7 changes the color to bright green and new absorption bands at 460 and 660 nm appear [60]. The redox reaction is reversible: Upon readmission to oxygen, the spectroscopic properties of the solution return to those of the {Fe(NO)2}7 complex.

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