S2cnrs

complex II -i-y complex I

Fe2+

Scheme 1.

It was found that anions of cyanide or cysteine do not act directly on these NO—Fe—S complexes but by binding free ferrous iron in solution. The depletion of free ferrous ions subsequently favors the shift towards complex I. Phrased otherwise, these anions act on the equilibrium indirectly by modulating the status of free iron. The addition of the reagents for persulfide, phosphites (RO)3P induced the degradation of complex II. This points to the presence of persulfide (polysulfide) ligands in the complex.

The proposition is in line with the investigation of the complex II formed from a model iron-sulfur center in serum albumin. The latter was synthesized by serum albumin treatment with ferrous iron and sodium sulfide as described elsewhere [68]. Subsequent exposure to NO gas induced the formation of complex II in the preparation. Consequent addition of triethyl phosphite or cysteine destroyed this complex II and induced the formation of protein-bound DNIC with thiol-containing ligands. This transformation was attributed to the removal of persulfide ligands from complex II, and transfer of the Fe(NO)2 moiety to one of the thiols in the protein.

The formation of complex II was observed when solutions of isolated nitrogenase enzyme treated with exposure to NO gas in the presence of 8 M urea for 10 min (Fig. 15, curve f) [68]. The EPR signal of DNIC-thiol was observed in the absence of urea at NO treatment (Fig. 15, curve d). The complex II appeared at gaseous NO treatment of the enzyme solution for 20 h (Fig. 15, curve e).

It is reasonable to suggest that these observation apply to other iron-sulfur proteins also, and that exposure to NO transforms the iron-sulfur clusters of other proteins to complex II as well due to the formation of persulfide bond in the reaction between thiol groups of protein and inorganic sulfur atoms from iron-sulfur complexes. Experimental support is provided by the shape of the EPR signal of nitrosyl-iron complexes formed if the [4Fe—4S] cluster of transcription factor FNR protein is exposed to the NO-donor proline NONOate [69]. The EPR spectrum appears as the superposition of a narrow signal from complex II and that of a DNIC with thiolate ligands (Fig. 3A in Ref. [69]).

Neocuproine (2,9-dimethyl-phenanthroline) is often referred to as a selective bidentate chelator for monovalent Cu+ ions. Recent investigations with EPR have shown that the situation with neocuproine is far less simple. In fact, neocuproine is able to bind ferrous iron as well, and it can be even incorporated as an iron ligand in DNIC complexes [70]. Stable species of paramagnetic DNIC with neocuproine were observed under the following conditions: after the addition of neocuproine to a solution of DNIC with phosphate ligands; after exposure in an aqueous solution of Fe2+ and neocuproine at pH 6.5-8 to NO gas; and after the addition of Fe2+ -citrate and neocuproine to a solution of Cys-NO [70]. Two different species of DNIC with neocuproine ligands could be distinguished from their EPR spectra. The first species had rhombic symmetry with g-factors 2.087, 2.055 and 2.025 (Fig. 16, curve d). In liquid solution at ambient temperature, the EPR spectrum simplifies to a single line at g = 2.05. The second species of DNIC with neocuproine can be recorded in frozen solution only (T ~ 77 K) and has a rhombic EPR spectrum with g-factors 2.042, 2.02 and 2.003 (Fig. 16, curve c).

The participation of neocuproine in DNIC allows us to argue against the consideration of neocuproine just as a selective bidentate chelator for Cu+. This has important implications for the presence of S-nitrosothiols in biological systems. It is often observed that neocuproine

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