G 2045 203 2014

Fig. 25. The EPR signals from DNIC-MT dissolved in methanol (a) and crystalline DNIC-MT (b) recorded at 77 K. (c) The shape of the EPR signal obtained by subtracting of signal (a) from signal (b). (From Ref. [40].)

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Fig. 25. The EPR signals from DNIC-MT dissolved in methanol (a) and crystalline DNIC-MT (b) recorded at 77 K. (c) The shape of the EPR signal obtained by subtracting of signal (a) from signal (b). (From Ref. [40].)

the dissolved complex. The change to square-plane structure is reflected in the backtransfer of the unpaired electrons from the iron towards the thiol sulfurs. The sulfurs change their character from thiyl to thiolate, and iron reverts to a d7 configuration and the complex is described by {(RS-)2Fe+(NO+)2}+ structure. Interaction of free thiols with NO+ can result in inclusion of these thiols into DNIC, thus changing the total charge of the complex to negative value, according to the formula {(RS-)2Fe+(NO+.. .RS-)2}- [46,88].

It is hardly possible to keep tetrahedral structure for dissolved DNIC with thiol-containing ligands with d7 electron configuration. In this case, the complexes should be characterized with high spin state (S = 3/2, Scheme 10B). Such state is characteristic of various MNICs. These complexes give the EPR signal with three ^-factor values (4.0, 3.95

and 2) [113-119]. Such type of signal has not been observed for DNIC with thiol-containing ligands [113].

OBSERVATIONS OF DNICs IN CELLS AND TISSUES

As aforementioned, the elucidation of the nature of paramagnetic centers giving 2.03 signal in cells and tissues (respectively called 2.03 centers) was unraveled when it was found that the EPR lineshape of the biological samples resembles spectra from DNIC with low-molecular-weight thiols in frozen solutions [6,7]. Although the coincidence was noted, the formation of dinitrosyl complexes in biological systems seems at first to be improbable because the biological role of NO was not known at the time [3]. The formation of such DNICs was subsequently proven by the appearance of the 2.03 signal in baker yeast cells that were cultured in a growth medium containing CaNO3 (Reader's medium) [9,12]. Independently, the group of Commoner in USA confirmed that the elimination of nitrate from Reader's medium prevented the appearance of the g = 2.03 signal in anaerobically cultivated yeast cells [10]. Evidently, nitric oxide ensuring DNIC formation in yeast cells appeared in these cells due to the accumulation of nitrite. The latter was generated from nitrate which was reduced to nitrite by respective nitrate reductase. Further work demonstrated the positive correlation between the amount of the 2.03 centers in liver tissue from rats fed on a diet containing different carcinogens and the amount of nitrate anions in the ingested drinking water [10,12]. Moreover, the formation of these centers was observed in various organs of mice which were kept on a diet with a high content of KNO3 without any addition of carcinogens (Fig. 26) [12].

Since nitrate reductase has not been found in mammals the reduction of nitrates to nitrites seemed to be caused by intestinal denitrifying bacterial microflora. Subsequent experiments on the addition of nitrite to animal diet demonstrated the formation of the 2.03 centers in animal tissues in higher amounts than that on the addition of nitrate [11,120-126] (Fig. 26). Similar result was obtained when isolated animal tissues were treated with gaseous nitric oxide [8]. These results point unequivocally to the fact that 2.03 centers are the DNICs with thiol-containing ligands.

The remaining constituents of the DNICs from yeast cells, thiols and iron, were identified by the effect of mercuric salts and chelators for ferrous iron, respectively. Exposure to competing alternative ligands caused their transformation into other types of iron-nitrosyl complexes [3]. Interesting effects were observed upon addition of xanthogenate derivatives, i.e. bidentate ligands with two binding sulfurs similar to those found in dithiocarbamate ligands. Addition of xanthogenates caused the formation of paramagnetic mononitrosyl complexes (MNICs) at the expense of DNIC. At 77 K, the EPR spectrum showed resolved triplet HFS (Fig. 27). The transformation to MNIC occurred for DNICs in biological samples, but similarly for DNIC formed in vitro from thiols with low molecular weight [3].

Large quantities of DNIC were formed in tissues of animals after the addition of sodium nitrite (0.3%) to drinking water of mongrel mice or rats [11,120-126]. Particularly high yields were found in liver. If the animals were maintained on the nitrite diet, the 2.03 signal in the mouse liver became observable after ca 3 days. After 7 days, the yield reached the steady state maximum of ca 8-10 ^M/kg liver tissue. The intensity and narrow linewidth made

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