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The decomposition of two neutral binuclear nitrosyl iron complexes (NICs) of the µ-S structural type and general composition [Fe2(SR)2(NO)4]0 was studied in comparison. The exchange reaction of the thiophenol or 2-aminothiophenol thiolate ligand by glutathione (GSH) in neutral NICs was studied. The reaction system was analyzed by spectrophotometry to prove the presence of a new NICs with the GS– ligand in it. It was found that, unlike the earlier studied binuclear cationic NICs of the µ-S type and general composition [Fe2(µ-SR)2(NO)4]2+SO4•nH2O with cysteamine and penicillamine ligands in which both thiolate ligands exchange by GS–, in these neutral complexes both thiolate ligands are de-tached by only one GSH ligand is attached. A water molecule is inserted into the second free site. It is assumed that the antitumor activity of the neutral NICs can be determined not only by their NO-donor activity but also by their ability to exchange the thiolate ligand by GS–, i.e., "to remove" GSH from the medium as in the case of cationic NICs. The discovered reaction can prevent, most likely, the S-glutathionylation of important metabolites in the presence of GSH and is very significant for metabolism of NICs.

Two decomposition mechanisms of nitrosyl iron complexes (NICs) [Fe2(μ-SR)(NO)4] in aqueous medium are known. One mechanism (for instance, in the case of complex [Fe2(μ-SC4H3N2)2(NO)4]) involves irreversible and rapid hydrolysis of NIC with the NO release accompanied with the formation of the products of further NO transformations. In the other mechanism (for instance, in the case of complexes [Fe2(μ-S(CH2)2NH3)2(NO)4]SO4• •2.5H2O and [Fe2(μ-SC5H11NO2)2(NO)4]SO4•5H2O), no hydrolysis occurs but NICs reversibly dissociate to release both NO and thiolate ligand into the medium. In the present work, the difference in the mechanisms of the NIC decomposition is explained by the difference in the NIC redox potentials. The experimental evidences of this fact are given.

Deoxyhemoglobin (Hb) stabilizes the cationic nitrosyl iron complex with cysteamine {Fe2[S(CH2)2NH3]2(NO)4}SO4·2.5H2O (CysAm), by slowing down its hydrolysis. In the absence of Hb, the electrochemical detection of NO release in the course of the hydrolysis using a sensor electrode gave the rate constant of (5.2±0.2)·10−5 s−1. The release of NO is a reversible process, and the amount of released NO is 1.4% of the CysAm concentration. In the presence of Hb, NO is released much more slowly, and the reaction is more intense than that in the absence of Hb. The adsorption of CysAm by an Hb molecule results in NO release from the CysAm-Hb complex with a rate constant of 1·10−8 s−1. The analysis of the Hb surface revealed the possible location of the cation-binding sites, which reversibly bind the cationic CysAm complex. The kinetic parameters of NO release from CysAm in the absence and in the presence of Hb were studied by the kinetic modeling.

The hydrolysis of the iron nitrosyl complex [Fe2(μ2-SC4H3N2)2(NO)4](C4H3N2S− is pyrimidine-2-thiolate) in the presence of hemoglobin (Hb) is accompanied by the NO release into a solution. In the absence of Hb, the starting complex is oxidized by nitric oxide that is released into a solution, which leads to further transformations of NO, nitric oxide being present in the solution only partially. The effective rate constant for the decomposition of the complex is high and depends on its concentration. On the one hand, in the presence of Hb, NO molecules rapidly and irreversibly bind to Hb to form HbNO, which is the intermediate in the nitric oxide metabolism. On the other hand, the reversible binding of the iron nitrosyl complex to the surface functional groups of Hb leads to a decrease in its concentration in a solution and deceleration of the formation of NO. Therefore, Hb can ensure the complete and more prolonged assimilation of NO.

By an example of the iron cysteamine nitrosyl complex {Fe2[S(CH2)2NH3]2(NO)4}SO4··2.5H2O (CAC), it was shown for the first time that the hydrolysis of this NO donor in the presence of ferrocytochrome c (cyt c2+) affords the iron nitrosyl complex NO-cyt c2+, which serves as the NO depot. The rate constant of NO release from CAC was determined from the kinetics of the formation of NO-cyt c2+. At pH 3.0 the rate constant is (2.7±0.1)·10−3 s−1. Ferrocytochrome c produces a less stabilizing effect on CAC than deoxyhemoglobin (Hb). Thus in the presence of cyt c2+, the reaction is completed in 1 h, whereas NO is released from a solution of CAC (2·10−4 mol L−1) in the presence of Hb during 40 h. The previously unknown stabilization of iron nitrosyl complexes by hemoglobin was found.

By an example of cysteamine iron nitrosyl complex {Fe2[S(CH2)2NH3]2(NO)4}SO4 • 2.5H2O (CAC) it was shown for the first time that the NO donor hydrolysis in the presence of ferricytochrome c (cyt c3+) affords the iron nitrosyl complex NO—cyt c3+. It was found that cyt c3+ can serve as a depot for NO evolved during the hydrolysis of CAC. In the presence of CAC, the rate of NO—cyt c3+ complex decomposition to NO and cyt c3+ depends on the molar ratio [cyt c3+]: [CAC] and at [cyt c3+]: [CAC] = 0.3 it was found to be lower than that in decomposition of CAC in the absence of cyt c3+. As a result, the total NO evolving process becomes 5.6 times more prolonged. The number of NO groups evolved from CAC can be determined by the reaction of CAC with cyt c3+ in the presence of ferricyanide: at most one NO group is evolved to a solution in the spontaneous hydrolysis of CAC (pH 7.0), and no less than three of them are evolved from oxidized CAC.

The NO-donor ability of new binuclear tetranitrosyl complexes of the μ-N—C—S type, namely, bis(5-methylbenzimidazol-2-ylthio)- (1), bis(benzimidazol-2-ylthio)- (2), and bis(benzothiazol-2-ylthio)(tetranitrosyl)diiron (3), was studied in aqueous solutions by spectrophotometry. All kinetic regularities obtained for complexes 1–3 are well described in terms of formalism of pseudo-first-order reactions. The apparent first-order reaction rate constants for NO evolution by the complexes to solution were determined. Complexes 1–3 are good donors of NO. The structures of the complexes and the effect of their stabilization by hemoglobin were compared. The stabilization effect is explained by different basicities of the sulfur-containing ligands in the complexes studied.

The reaction of hemoglobin (Hb), oxyhemoglobin (HbO2), and methemoglobin (metHb) with the tetranitrosyl iron complex of the fu2-S type [Fe2(SC4H3N2)2(NO)4] (1) was studied. The reaction results in the nitrosylation of the free SH group of 93-β-cysteine in these forms of hemoglobin. The change in the Hb, HbO2, and metHb concentrations was monitored by spectrophotometry, recording the difference absorption spectra of the experimental systems with these forms of hemoglobin and the buffer containing complex 1 in the same concentration. The absorption spectra were processed to obtain the components using the MATHCAD method. The nitrosothiol concentration was determined by the Saville reaction. In a protic medium containing 3.3% DMSO, complex 1 spontaneously generates NO due to hydrolysis (k = 3.7 · 10-4 s-1). Oxyhemoglobin reacts with evolved NO to form metHb. Complex 1 reduces metHb with a high rate to yield Hb (k = 6.7 · 10-3 s-1) followed by the formation of HbNO (k = 6.5 · 10-3 s-1). Oxidized complex 1 yields NO with a higher rate than the starting complex does. The reaction of HbO2 and metHb (0.02 mmo1 L-1) with complex 1 affords nitrosothiols in micromolar concentration during 5 min, and no nitrosothiol is formed in the case of Hb.

The tetranitrosyl iron complex with pyridine-2-yl [Fe2(SC5H4N)2(NO)4] (1) has higher NO-donating activity in 3% aqueous solutions of DMSO (pH 7.0, 25 °C) than the organic NO donor, viz., adduct of NO with diethylenetriamine (NO-adduct). The NO concentration was determined by the spectrophotometric method based on the formation of an NO complex with hemoglobin (Hb). The apparent first-order rate constants of the studied reactions are (6.15±0.6)·10−1 s−1 and (0.8±0.08)·10−1 s−1 for complex 1 and the NO-adduct, respectively, at an Hb concentration of 2·10−1 mol L−1 and the ratio [NO donor]/[Hb] = 10. The effect of Hb and [NO donor]/[Hb] ratio on the rate of NO generation from a solution of complex 1 was studied. For a fourfold decrease in the concentration of complex 1 the reaction rate constant decreases to 0.5·10−4 s−1, whereas the fourfold increase in the Hb concentration results in the stabilization of complex 1.

Hemoglobin (Hb) reduces 3,3-bis(nitroxymethyl)oxetane (NMO) only in the presence of cysteine (Cys) via intermediate cysteine thionitrate. The kinetics of the reaction of NMO with Cys and the kinetics and mechanism of formation of NO in the ternary system Hb-NMO-Cys were studied. The formation rate of Hb-NO in the ternary system is higher than that of Hb-NO in the reaction of Hb only with NO 2 − generated in the binary system NMO-Cys. The second-order rate constants of the main reaction steps in the system Hb-NMO-Cys were estimated by simulating the kinetics of the reactions with a system of equations taking into account equilibria between all components of the reaction mixture. Hemoglobin reduces cysteine thionitrate, the intermediate in the reaction of NMO with Cys, to NO.

Structural data on the nitrogenase complex of Azotobacter vinelandii, Av1·(Av2)2, stabilized by MgADP·AlF4− and, in particular, the structure and properties of a P-cluster involved in the nitrogenase ATPase reaction, were analyzed. The ATP-binding site and all nitrogenase metal clusters are arranged in one plane, the distances between the closest partners being 14–15 A. The ATP-binding site in the Fe-protein, which decreases the half-reduction potential (Em) of the [4Fe-4S]-cluster in Av2 to −0.43 V, does not affect the potentials of the P-cluster and Fe-Mo cofactor (FeMoco). Amino acids 74–95 in the β-subunit of Av1 “envelop” the P-cluster in Av1; therefore, the phosphate intermediate of the ATPase reaction of nitrogenase occurs apparently in the direct contact with the P-cluster. By increasing the acceptor properties of the P-cluster, this intermediate may favor the electron transfer from the Fe-protein to the P-cluster, thus bringing it into the super-reduced state.

The influence of glycerol on the ATPase reaction of nitrogenase and reduction of the substrate (acetylene) is studied. Glycerol inhibits the ATPase nitrogenase reaction dependent on an electron donor. The reaction rate is halved at a glycerol concentration of 11% in the medium when the solution viscosity increases only 1.31 times. The electron donor–independent (decoupled) ATPase reaction of nitrogenase is inhibited to a lesser extent. The activation energies (Ea) of reactions studied in the presence of glycerol are determined. Despite the inhibition effect, glycerol in a concentration of 7.5% does not affect the Ea of acetylene reduction. The introduction of glycerol significantly decreases the Ea of the electron donor-dependent ATPase reaction. In the absence of glycerol, this reaction limits the nitrogenase reaction: Ea = 14 ± 1.4 kcal/mol at temperatures higher than 21°C and Ea = 50 ± 10 kcal/mol at temperatures below 21°C, which are close to the Ea of acetylene reduction. In the presence of 7.5% glycerol, the Ea = 0.7 ± 0.6 kcal/mol at temperatures above 21°C and the Ea = 2.4 ± 0.6 kcal/mol at temperatures below 21°C. This indicates that the reactions of substrate-binding and ATPase sites are decoupled in the presence of glycerol, and the step of substrate reduction becomes the limiting step of the nitrogenase reaction. Glycerol also has a noticeable effect on the Ea of the electron donor-independent ATPase reaction and the shape of the plot of logw vs. 1/T for this reaction. The data obtained indicate the specific interaction of glycerol with nitrogenase in the region of the ATPase site perhaps due to the distortion of the structure of hydrogen bonds, and this interaction changes the limiting step of the nitrogenase reaction.