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The results of a study of the effect of a mononuclear dinitrosyl iron complex (DNIC7) with functional sulfur-containing ligands (NO donor) on the cell viability of multiple myeloma cells are presented. It has been shown that DNIC7 decreased cell viability and inhibited the proliferation of cells of multiple myeloma, i.e. possesses cytotoxic properties. Fluorescent analysis revealed that the DNIC7 compound lowers the level of intracellular glutathione and increases the level of reactive oxygen species in cells of multiple myeloma. It is assumed that DNIC7 has the therapeutic potential for the treatment of cancer.

The effect of 10 racemic spirocyclic hydroxamic acids (CHA 1–10, derivatives of 1-hydroxy-1,4,8-triazaspiro[4,5]decan-2-one), containing pharmacophore imidazolidinone and piperidine fragments with different substituents, on the activity of enzyme histone deacetylase (HDAC) was studied. It was shown that CHA (1–10) inhibit HDAC activity in cultured breast cancer cells. It was shown that CHA (1–10) as a part of polychemotherapy with cisplatin and cyclophosphane have a pronounced chemosensitizing antitumor activity in vivo. The results obtained on tumor models in vivo showed that CHA can be considered as potential medicinal components of tumor polychemotherapy.

The effect of synthetic analogues of dinitrosyl mononuclear iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the activity of myeloperoxidase (MPO) was studied, and their efficiency was evaluated. It was shown that the enzyme MPO is the molecular target of DNICs. It was found that six DNICs inhibited the activity of MPO and one compound potentiated it. The evaluation of their efficiency showed that two DNICs effectively inhibited the activity of MPO by 50% at IC50 = 2 × 10–4 M and IC50 = 5 × 10–7 M.

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.

Decomposition of binuclear neutral iron nitrosyl complex [Fe2(S2C7H4N)2(NO)4]0 (I) of μ-N–C–S structural type in aqueous solution has been studied. Effect of glutathione GSH on the decomposition of complex I has been studied.

Nanoparticles from chitosan and hyaluronic acid were obtained using ionotropic gelation technology. The size of the nanoparticles was investigated using electron microscopy and dynamic light scattering. Nanoparticles were obtained of an optimal size of ∼ 100 nm. A physical association method has been developed of encapsulating nanoparticles with doxorubicin, a well-known antitumor drug, and dinitrosyl iron complex (donor of nitric oxide). The surface potential of nanoparticles was determined by dynamic light scattering. It was shown that HA-DOX: CS {6: 1} nanoparticles were stable and had a potential of -45.6 meV. The localization of nanoparticles in the cancer cells has been studied by confocal and FLIM microscopy. It was found that nanoparticles with doxorubicin are located in the cell near and inside the nucleus. It was shown that the encapsulation of DNIC in the composition of nanoparticles significantly increases the stability of DNIC, prolongs the formation and increases the yield of nitric oxide. A unique nano-system has been created for the delivery of anticancer drugs into the cell.

The results of the study of the effect of a mononuclear dinitrosyl iron complex (DNIC7) with functional sulfur-containing ligands (NO donors) on the viability of multiple myeloma cells are presented. It was shown that DNIC7 decreased cell viability and inhibited the proliferation of multiple myeloma cells, i.e., exhibits cytotoxic properties. Fluorescent analysis showed that the DNIC7 compound decreases the level of intracellular glutathione and increases the level of reactive oxygen species in multiple myeloma cells. It is assumed that DNIC7 has a therapeutic potential for the treatment of cancer.

In this paper, the kinetics of the interaction of the nitrosyl iron complex with the ligands penicillamine [Fe2(SC5H11NО2)2(NO)4]SO4·5H2O (I) with deoxyhemoglobin (Hb) was studied. The kinetic modeling method defined the number of binding (I) molecules and equilibrium constant of the coupling reaction of (Biomedicine, Iron-Sulfur Cluster, Ligand Binding, Heme, Nitric Oxide ) with Hb (Ks). At equimolar concentrations of (I) and Hb (2 × 10−5 M), the Hb molecule binds only one (I) with Ks equal to 4.3 × 107 M−1. When increasing the (Biomedicine, Iron-Sulfur Cluster, Ligand Binding, Heme, Nitric Oxide ) concentration, the number of binding sites of Hb increases and Ks decreases. These results are analyzed in accordance with the data on the existence of cations binding sites in Hb.

This paper describes the comparative study of the decomposition of two iron nitrosyl complexes (NICs) with a cysteamine thiolate ligand {Fe2[S(CH2)2NH3]2(NO)4}SO4·2.5H2O (I) and a glutathione (GSH)-ligand, [Fe2(SC10H17N3O6)2(NO)4]SO4·2H2O (II), which spontaneously evolve NO in aqueous medium. NO formation was measured by using a spectrophotometric method by the formation of a hemoglobin (Hb)–NO complex. Spectrophotometry and mass-spectrometry methods have firmly shown that the cysteamine ligands are exchanged for 2 GS− during decomposition of 1.5 × 10−4 M (I) in the presence of 10−3 M GSH, with 77% yield at 68 h. As has been established, such behaviour is caused by the resistance of (II) to decomposition due to the higher affinity of iron towards GSH in the complex. The discovered reaction may impede S-glutathionation of the essential enzyme systems the presence of (I) and is important for metabolism of NICs, connected with their anti-tumor activity.

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.

NO-donating ability of nitrosyl [Fe–S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S 2 O 3 ) 2 (NO) 2 ]-( I ) and neutral [Fe 2 (SL 1 ) 2 (NO) 2 ] with L 1 = 1 H -1,2,4-triazole-3-yl ( II ); tetranitrosyl binuclear neutral complexes [Fe 2 (SL 2 ) 2 (NO) 4 ] with L 2 = 5-amino-1,2,4-triazole-3-yl ( III ); 1-methyl-1 H -tetrazole-5-yl ( IV ); imidazole-2-yl ( V ) and 1-methyl-imidazole-2-yl ( VI ) has been studied. In addition, Roussin’s “red salt” Na 2 [Fe 2 S 2 (NO) 4 ]·8H 2 O ( VII ) and Na 2 [Fe(CN) 5 NO]·H 2 O ( VIII ) have been investigated. The method for research has been based on the formation of Hb–NO adduct upon the interaction of hemoglobin with NO generated by complexes I – VIII in aqueous solutions. Kinetics of NO formation was studied by registration of absorption spectra of the reaction systems containing Hb and the complex under study. For determination of HbNO concentration, the experimental absorption spectra were processed during the reaction using standard program MATHCAD to determine the contribution of individual Hb and HbNO spectra in each spectrum. The reaction rate constants were obtained by analyzing kinetic dependence of Hb interaction with NO donors under study. All kinetic dependences for complexes I – VI were shown to be described well in the frame of formalism of pseudo first-order reactions. The effective first-order rate constants for the studied reactions have been determined. As follows from the values of rate constants, the rate of interaction of sulfur-nitrosyl iron complexes ( I – VI ) with Hb is limited by the stage of NO release in the solution.

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.

This paper describes a comparative study of the decomposition of two nitrosyl iron complexes (NICs) with penicillamine thiolic ligands [Fe2(SC5H11NO2)2(NO)4]SO4·5H2O (I) and glutathione- (GSH-) ligands [Fe2(SC10H17N3O6)2(NO)4]SO4·2H2O (II), which spontaneously evolve to NO in aqueous medium. NO formation was measured by a sensor electrode and by spectrophotometric methods by measuring the formation of a hemoglobin- (Hb-) NO complex. The NO evolution reaction rate from (I) k1= (4.6 ± 0.1)·10−3 s−1and the elimination rate constant of the penicillamine ligandk2= (1.8 ± 0.2)·10−3 s−1at 25°C in 0.05 M phosphate buffer, pH 7.0, was calculated using kinetic modeling based on the experimental data. Both reactions are reversible. Spectrophotometry and mass-spectrometry methods have firmly shown that the penicillamine ligand is exchanged for GS−during decomposition of 1.5·10−4 M (I) in the presence of 10−3 M GSH, with 76% yield in 24 h. As has been established, such behaviour is caused by the resistance of (II) to decomposition due to the higher affinity of iron to GSH in the complex. The discovered reaction may impede S-glutathionylation of the essential enzyme systems in the presence of (I) and is important for metabolism of NIC, connected with its antitumor activity.

The kinetics of interaction between organic nitrates (3,3-bis(nitroxymethyl)oxetane) and cysteine were evaluated by the rate of nitrite ion formation at various concentrations of reagents and pH. The activities of natural reducing agents, including cysteine, glutathione, and NADH, in generating the nitrite ion from organic nitrates (3,3-bis(nitroxymethyl)oxetane) were compared. Cysteine was shown to be the most potent reducing agent. Studying the effectiveness of nitrates (trinitroglycerol, 3,3-bis(nitroxymethyl)oxetane, and nicorandil) at a concentration of 3 mM showed that the rate of nitrite ion accumulation in the reaction with 10 mM cysteine is 1.66, 0.37, and 0.02 microM/min, respectively.

The kinetics of transfer of two electrons from a photodonor (a system containing eosin and NADH or 4;,5;-dibromofluorescein and NADH) to Fe-protein (Av2) and the kinetics of transfer of the first and second electrons from Av2 to Mo-Fe-protein (Av1) were studied by kinetic laser spectroscopy of nitrogenase from Azotobacter vinelandii. The effects of the substrates of nitrogenase (nitrogen, acetylene, and protons) on the intramolecular electron transfer in nitrogenase were studied. Analysis of the effect of photodonor excitation radiation intensity on the rate of electron transfer was used to determine the transfer rate constants for the first (k1) and second (k2) electrons from Av2 to Av1. In the presence of MgATP, two electrons are sequentially transferred from Av2 to Av1, and no delay between these reactions was detected. The first electron transferred from Av2 to Av1 is not targeted to the substrate; k1 = 154 +/- 15 sec-1 at 23 degrees C for the system 4;,5;-dibromofluorescein-NADH; k2 = 53 +/- 5 sec-1, 95 +/- 9 sec-1, and 24 +/- 2 sec-1 at 23 degrees C in the presence of nitrogen, acetylene, and argon, respectively. An unidentified slow step (k3 = 18 +/- 2 sec-1 at 23 degrees C) may be associated with electron transfer within Av1.

The ability of xanthene dyes to mediate photoinduced reduction of nitrogenase was tested. In addition to eosin, which was studied in the preceding work (Biochemistry (Moscow), 1996, 61, 2165-2172), 4', 5'-dibromofluorescein (DBF), cyanosine, and erythrosin are effective photodonors of an electron in the presence of NADH. Fluorescein, rhodamine B, rhodamine 6G, and porphyrins are unable to mediate photoinduced reduction of nitrogenase. The mechanism underlying different efficiency of xanthene dyes in this reaction was studied. At high concentrations, all xanthene dyes tested were shown to inhibit the intramolecular electron transfer in nitrogenase. The inhibiting concentration of DBF is 1.5.10-4 M, whereas for other dyes, the inhibiting concentrations are less than 1.5.10-4 M. Under otherwise identical conditions, the ATPase activity was inhibited by xanthene dyes to a lesser extent than the nitrogenase activity. DBF, the most effective photodonor, was also studied by differential kinetic pulse laser spectroscopy. Photoinduced reduction of nitrogenase, (Fe-proteinox.Mo-Fe-protein).MgATP or (Av2ox.Av1).MgATP, was studied within the time range from 0 to 100 msec. Two initial stages of the nitrogenase turnover were detected: photoinduced reduction of Av2 and electron transfer from Av2red to Av1. The kinetics of the photoinduced reduction of Av2.MgADP was studied in the presence of DBF (up to 1.3.10-4 M) both in solution and the complex with Av1. The apparent second-order rate constants of the photoinduced reduction of Av2.MgADP in solution and the complex with Av1 were determined as 9.7.107 +/- 106 and 1.2.108 +/- 1.2.107 M-1. sec-1, respectively. The rate constant of the second reaction in the presence of another donor (dithionite) is 2500 times less. In complexes with Av1, the photochemical donor system DBF--NADH reduces Av2 more effectively than in free state in solution. In the presence of the photochemical donor system, neither photoreduction of Av2 in complexes with Av1 nor electron transfer from Av2red to Av1 are the rate-limiting stages of nitrogenase turnover.

Despite a great deal of research on the field of nitrogenase, the individual role(s) of the two types of metal-sulphur clusters (P and M) found in Av1 (MoFe protein) is not clear and the mechanism of charge separation following electron transfer against the potential from Av2 (Fe protein) to Av1 is unknown. One of the most promising ways of gaining this information appears to be photoreduction of nitrogenase over a microsecond range. An alternative electron donor for nitrogenase was developed (Druzhinin S. et al., 1993) based on photoexcited eosin with NADH. Some xanthene dyes were also studied as possible photodonors. Eosin, 4′,5′-dibromofluorescein, cyanosine, erythrosin are effective photodonors for nitrogenase, fluorescein and rhodamine B or 6G are not. The main difference between these active and inactive compounds was the high quantum yield of phosphorescence for active photodonors versus high quantum yield of fluorescence for inactive photodonors. All studied photodonors can inhibit electron transfer within nitrogenase following substrate reduction at high dye concentration. For 4′,5′-dibromofluorescein this concentration is 0,1 mM, for other dyes it is less. Under the same conditions the ATPase activity was inhibited to a significantly less degree. Optimal concentration of each xanthene dye as photodonor depends on its inhibition characteristics and varies in the range of 0,03–0,1 mM.