Introduction

Dinitrosyl-iron complexes (DNICs) with thiol-containing ligands were discovered in cells and tissues with EPR spectroscopy. The EPR spectrum of these paramagnetic (S = 1 /2) complexes has a characteristic anisotropic lineshape centered at gav = 2.03 (2.03 signal) which may be observed over a wide range of temperatures from liquid helium to room temperature. The g = 2.03 signal was recorded and discussed for the first time in cultured yeast cells and subsequently in the tissues of some animals [1-3] (Fig. 1A,C). Independently, Commoner and colleagues from USA (1965) observed weak signals at g = 2.03 in rat livers during the initial stages of chemically induced carcinogenesis [4] (Fig. 1D). Interestingly, some spectra published earlier by Mallard and Kent did show the presence of unidentified paramagnetic centers at g = 2.03 in rat liver in similar experiments (Fig. 1B) [5] but their nature was not discussed.

The identity of these paramagnetic centers at g = 2.03 was recognized when it was demonstrated that the shape and EPR spectroscopic parameters of 2.03 signal and EPR signals of low-molecular DNIC with cysteine in a frozen solution were similar (Fig. 2) [6,7]. Subsequent studies confirmed that the paramagnetic species at g = 2.03 in cells and tissues are really DNIC with protein or low-molecular thiol-containing ligands [3,6-12].

As a rule, at X-band the 2.03 signal is usually characterized by a g-factor with axial symmetry (gx = 2.04, g||= 2.014). At room temperature, the protein-bound DNICs may be distinguished easily from their low-molecular-weight analogs. At this temperature, the EPR spectrum from protein-bound DNIC retains the shape of a powder spectrum as would be recorded in frozen solution at low temperature (77 K) (Figs. 2, 3 and 5). In contrast, the low-molecular DNICs are rapidly tumbling and show a motionally narrowed isotropic line with a half-width of 0.7 mT and resolved 13-component hyperfine structure (HFS) [13-15] (Figs. 2-4).

Fig. 1. The first recordings of 2.03 signal; yeast (A) [1,2]; rat liver carcinoma induced by the hepatocar-cinogenic compound, P-dimethylamino-azobenzene (butter yellow) (panel B, spectrum b; (a) spectrum from normal liver) [5]; yeast and rabbit liver (panel C, spectra a, b and c, respectively) [3]; panel D: livers from rats maintaining 7, 14, 21, 35 and 49 days on a diet containing butter yellow [4]. Recordings were made at 77 K (A-C) or ambient temperature (D). (With permission.)

Fig. 1. The first recordings of 2.03 signal; yeast (A) [1,2]; rat liver carcinoma induced by the hepatocar-cinogenic compound, P-dimethylamino-azobenzene (butter yellow) (panel B, spectrum b; (a) spectrum from normal liver) [5]; yeast and rabbit liver (panel C, spectra a, b and c, respectively) [3]; panel D: livers from rats maintaining 7, 14, 21, 35 and 49 days on a diet containing butter yellow [4]. Recordings were made at 77 K (A-C) or ambient temperature (D). (With permission.)

Interest in DNIC complexes sharply increased in the 1990s after the discovery of the physiological roles of endogenous NO radicals in mammals and of the L-arginine/NO pathway catalyzed by the nitric oxide synthases. The L-arginine-dependent formation of DNIC was demonstrated in various cultured cells and tissues expressing inducible, high output, NO synthase activity. These presently include macrophages [16-19], fibroblasts [20], hepatocytes [21-25], vascular smooth muscle cells [26], isolated human islets of Langerhans [27], isolated rat aorta [28], different types of tumor cells [18,29,30] (all treated with lipopolysaccharides and/or cytokines in vitro), as well as liver of mice treated with Corynebacterium parvum [31], murine tumor transplants [30,32] and rat heart allografts [33,34] in vivo. Formation of DNIC via constitutive NO synthase was also demonstrated in isolated porcine endothelial cells stimulated with bradykinin or the ionophore A23187 [35].

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