[A rapid method to evaluate potential vasodilatory activity of organic nitrates].

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.

Reactions of sulfur-nitrosyl iron complexes of "g=2.03" family with hemoglobin (Hb): kinetics of Hb-NO formation in aqueous solutions.

NO-donating ability of nitrosyl [Fe-S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S2O3)2(NO)2]-(I) and neutral [Fe2(SL1)2(NO)2] with L1=1H-1,2,4-triazole-3-yl (II); tetranitrosyl binuclear neutral complexes [Fe2(SL2)2(NO)4] with L2=5-amino-1,2,4-triazole-3-yl (III); 1-methyl-1H-tetrazole-5-yl (IV); imidazole-2-yl (V) and 1-methyl-imidazole-2-yl (VI) has been studied. In addition, Roussin's "red salt" Na2[Fe2S2(NO)4] x 8H2O (VII) and Na2[Fe(CN)5NO] x H2O (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.

Kinetics of elementary steps of electron transfer in nitrogenase in the presence of a photodonor.

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.

Xanthene dyes as photochemical donors for the nitrogenase reaction.

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.

Kinetics of all stages of electron transfer in nitrogenase in the presence of a photodonor.

A photodonor is considered as an alternative electron donor for nitrogenase. The kinetic mechanism of nitrogenase turnover is discussed. The turnover is initiated by the transfer of an electron to the enzyme and results in formation of a substrate molecule. The effective rate constant of concerted transfer of the first and the second electron from Av2 (Fe-protein) to Av1 (Mo-Fe-protein) and the rate constant of transfer of the second electron are 70 +/- 7 and 116 +/- 10 sec-1, respectively. The rate constant of the rate-limiting reaction--MgADP release during formation of the superreduced state of Av1 (*Av12-)--is 12 +/- 2 sec-1. Nitrogenase (E) states in complex E.N2 on binding and reduction of nitrogen are: E2, E4, E6 (2, 4, and 6 electrons).

The photoreduction of nitrogenase.

The photoreduction, without reductant dithionite, of N2 to NH3 or acetylene to ethylene catalysed by nitrogenase in the presence of Mg2+. ATP, eosin and NADH in the light has been established. There is an optimum NADH concentration for each particular eosin concentration. When the ratio of the iron protein component of nitrogenase from Azotobacter vinelandii (Av2)/the molybdenum-iron protein component of nitrogenase from A. vinelandii (Av1) is equal to 3 for 4 x 10(-5) M eosin the optimum NADH concentration is 5 x 10(-4) M. The rate of photoreduction (per one electron) of acetylene or N2 under identical conditions was shown to be similar. The photoreductant-dependent ATPase activity, in the presence of a given photochemical system in the light, was revealed. Eosin is shown to be the inhibitor of the coupling site. Concentrations of 8 x 10(-6) -1 x 10(-4) M eosin do not inhibit the ATPase activity. The inhibition of substrate-reduction activity depends on the ratio of the nitrogenase components. Under conditions where the Av2/Av1 ratio is equal to 1 the rate of photochemical reduction is higher than in the presence of dithionite: the total electron flux through nitrogenase being increased 2.2-fold. We suggest that in this case the nitrogenase complex (1:1) works without dissociation.

[Localization of the ATPase site of nitrogenase by isotopic oxygen exchange [180]-Pi in equilibrium with H20]. / Issledovanie lokalizatsii ATPaznogo tsentra nitrogenazy metodom izotopnogo obmena kisloroda [180]-Pi v ravnovesii H20.

The components of the nitrogenase complex, MoFe-protein and FeMo-cofactor, possessing no ATPase or nitrogen-fixing activity, maintain the 18O-exchange at the level of 1 atom of 18O per molecule of Pi, which is inhibited by ATP. The Fe-protein complex does not catalyze the 18O-exchange. The nitrogenase components do not hydrolyze the substrates for phosphatase (p-nitrophenylphosphate, beta-glycerophosphate, glucose 1-phosphate and ribose 5-phosphate). The artificial albumin-containing MoFe- and Fe-proteins and the carboxyl group-containing proteins (albumin, hemoglobin, lysozyme) as well as sodium molibdate do not catalyze the 18O-exchange. It is assumed that the site of the ATPase center which is subjected to phosphorylation, is located on the MoFe-protein.

[Role of adenosine triphosphatase on nitrogenase function]. / O roli adenozintrifosfatazy v funktsionirovanii nitrogenazy.

The decoupling possibility of ATPase reaction with electron transfer process in the time of nitrogenase photolysis by lambda 435 nm light has been established. The COOH and the possibility of imidazole groups have been revealed in nitrogenase ATPase centre by methods of chemical modification. The reaction of direct 18O-exchange between inorganic phosphate and medium water was discovered, proceeding under reverse hydrolysis of acylphosphate bond, formed by phosphorylation of COOH-group in ATPase centre. Direct 18O-exchange was shown to be stimulated by ATP and ADP, but to be insensitive to GTP, CTP, AMP-nucleotide which are not ATPase centre substrates. The coupling mechanism of ATPase reaction with electron transfer is suggested; it is based on the possibility of compulsory protonation of Fe-S-cluster at the expense of proton transfer from the imidazole site, facilitating additional electron transfer under "superreduction" of nitrogenase component of the Mo-Fe-protein. It is assumed that this protonation is initiated by COOH-group of charge relay transfer with imidasole fragment.

Structure and mechanism of catalytic action of active sites of nitrogenase.

A review of the data on the macromolecular structure of nitrogenase and its individual fragments, the electronic structure of iron- and molybdenum-containing components of the active site, and the functional groups of the ATPase site of the enzyme is given. Reactions of N2 reduction, ATP hydrolysis, and H2 evolution, inhibitory processes, and electron transport reactions catalyzed by the enzyme are analyzed within the framework of a general kinetic model. The results of an investigation of the location of the iron-containing cluster system of electron transport, the ATPase site, and the N2-binding and reducing site on the nitrogenase macro-molecule with the aid of a new complex approach including methods of spin, luminescent, and electron-dense labeling are described. On the basis of a number of physicochemical and kinetic data a model of the structure and mechanism of action of the active site of nitrogenase is proposed, which assumes four-step electron transfer from an external reducing agent along the chain of ferredoxin-like iron-containing clusters of the enzyme and an increase in the reducing potential of the iron clusters through the energy of ATP hydrolysis and four-electron reduction in a binuclear molybdenum-containing complex.