Structures of thermolabile mutants of human glutathione transferase P1-1

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.     

Cardioprotective efficacy of dinitrosyl iron complex with L-cysteine in rats in vivo

The effect of dinitrosyl iron complex (DNIC) with L-cysteine on the hemodynamic indices and the size of myocardial infarction, which was induced by 40-min regional ischemia and subsequent 60-min reperfusion, have been studied in rats in vivo. Intravenous bolus injection of DNIC (3.1 μmol/kg body weight in 0.5 ml saline) was performed before regional ischemia; the control group was administered the same volume of saline. DNIC administration significantly decreased the mean blood pressure throughout the experiment. DNIC reduced the duration of cardiac arrhythmias to 170 ± 10 s as against 445 ± 30 s in control. The myocardial infraction size significantly decreased in the DNIC group compared to control (38.0 ± 1.4 and 48.0 ± 3.9% of the area at risk, respectively; p < 0.05). A combination of the vasodilatory effect of DNIC with the reduction of the damaging effect of cardiac ischemia and reperfusion encourage the development of hypotensive and antiischemic drugs on this class of NO donors.     

Structural characterization of acid-induced intermediates of human glutathione transferase P1-1

The acid denaturation of human glutathione transferase P1-1 (hGSTP1-1) has been performed to investigate the unfolding intermediates of the protein and their possible involvement in the refolding mechanism. The acid-induced structures of GSTP1-1 have been characterized by activity, gel filtration, intrinsic fluorescence and far-u.v. circular dichroism (CD) techniques. Because of the non-identity of the different transitions monitored, the acid denaturation of hGSTP1-1 appears to be a multistep process during which several intermediates coexist in equilibrium. The dependence of inactivation on the protein concentration, as well as gel-filtration experiments, indicate that the inactivation transition, centred at about pH 4.0, corresponds to the monomerization of the protein. At pH 2.0, when the enzyme is completely inactive, the protein retains a small, but significant, amount of secondary structure. This means that the dimeric arrangement of the molecule is important for maintaining the native-like secondary structure of the monomer. The results show that, at low pH, the compact state of the GST monomer, even upon the addition of salts, does not possess native-like secondary structure as described for many monomeric proteins (molten globule). In the presence of physiological concentrations of salts, the protein solution at pH 2.0 leads to a dead-end aggregation process, suggesting that this compact state cannot represent a productive intermediate of the refolding pathway.     

Antitumor activity of dinitrosyl iron complexes with glutathione

The antitumor dose-dependent effect of binuclear dinitrosyl iron complexes with glutathione as NO donors on a solid tumor in the mouse, Lewis lung carcinoma, was detected. The complexes being injected at doses of 21, 42, 105 mg/kg daily for 10 days blocked completely the development of the tumor for the first week after tumor cell implantation into animals. After that, the part of tumor cells which remained in intact alive state began to grow at a rate equal to that for control animals. The effect was proposed to be caused via formation of an antinitrosative defense system in the cells as a response to NO attack on cells. It was also hypothesized that this system can be inactivated by higher doses of dinitrosyl iron complexes. Data were obtained which were in line with the hypothesis.     

The antitumor activity of the S-nitrosoglutathione and dinitrosyl iron complex with glutathione: Comparative studies

The antitumor activity of the binuclear form of dinitrosyl iron complexes with glutathione against Lewis lung carcinoma was found earlier with intraperitoneal administration of the complexes. This activity was also observed when this preparation was injected subcutaneously. The complex inhibited the tumor growth by 43% upon subcutaneous injection at a daily dose of 100 µM/kg (as calculated per one iron atom in the binuclear dinitrosyl iron complex) for 10 or 15 days. The effect was observed during the first 2 weeks after tumor transplantation. After this, the tumors began to grow at a rate that was equal to or even higher than that for the control animals. The mean survival time for the treated mice exceeded the control values by 30%. Binuclear dinitrosyl iron complexes were also effective against Ca-755 adenocarcinoma with intraperitoneal administration. In this case, however, the mean survival time for the treated animals only increased by 7%. It was also shown that S-nitrosoglutathione inhibited the growth of Lewis lung carcinoma and Ca-755 adenocarcinoma by 70 and 90%, respectively. However, in contrast to binuclear dinitrosyl iron complexes, the antitumor effect of S-nitrosoglutathione decreased with an increase in the daily dose of the compound from 200 to 400 µM/kg. The initial antitumor effect of binuclear dinitrosyl iron complexes and S-nitrosoglutathione is suggested to be due to NO that is released from both compounds. The subsequent suppression of the effect is caused by the activation of antinitrosative and antioxidant defense systems in tumors.     

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 µM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 µM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 μM. The IC₅₀ value of 1 was 6.8 μM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with K_i values of 8.8 and 0.21 µM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.     

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 μM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 μM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 μM. The IC(50) value of 1 was 6.8 μM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with K(i) values of 8.8 and 0.21 μM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.     

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.     

Effects of dinitrosyl iron complex with glutathione and its components on ischemic rat heart during reperfusion

The effects of a complex of dinitrosyl iron with glutathione (DNIC-GS) lyophilized on dextran, its hydrolysis products (glutathione, nitrosoglutathione, dextran), as well as nitric oxide released from the drug on the energy metabolism and functional recovery of isolated perfused rat heart subjected to global ischemia and reperfusion have been studied. Infusion of 100 nM DNIC-GS after ischemia substantially enhanced the recovery of coronary flow, cardiac contractile and pump functions during reperfusion, with simultaneous preservation of myocardial high-energy phosphates and cell membrane integrity. It was shown by EPR that these effects were associated with transfer of Fe⁺(NO⁺)₂ groups from DNIC-GS to thiol-containing proteins of cardiomyocytes and coronary vessels. Combined infusion of 100 nM DNIC-GS and 25 μM 2-(phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, after ischemia profoundly reduced the metabolic and functional recovery of reperfused hearts. After postischemic administration of an equivalent amount of DNIC-GS hydrolysate (completely decomposed complex), most of the indices did not differ from those in control or were significantly lower. Thus, inclusion of Fe⁺(NO⁺)₂ groups into myocardial tissue and spontaneous release of nitric oxide trigger the protective mechanisms in the ischemic heart.     

Folding and assembly of dimeric human glutathione transferase A1-1

Glutathione transferases function as detoxification enzymes and ligand-binding proteins for many hydrophobic endogenous and xenobiotic compounds. The molecular mechanism of folding of urea-denatured homodimeric human glutathione transferase A1-1 (hGSTA1-1) was investigated. The kinetics of change were investigated using far-UV CD, Trp20 fluorescence, fluorescence-detected ANS binding, acrylamide quenching of Trp20 fluorescence, and catalytic reactivation. The very early stages of refolding (millisecond time range) involve the formation of structured monomers with native-like secondary structure and exposed hydrophobic surfaces that have a high binding capacity for the amphipathic dye ANS. Dimerization of the monomeric intermediates was detected using Trp fluorescence and occurs as fast and intermediate events. The intermediate event was distinguished from the fast event because it is limited by a preceding slow trans-to-cis isomerization reaction (optically silent in this study). At high concentrations of hFKBP, dimerization is not limited by the isomerization reaction, and only the fast event was detected. The fast (tau = 200 ms) and intermediate (tau = 2.5 s) events show similar urea-, temperature-, and ionic strength-dependent properties. The dimeric intermediate has a partially functional active site ( approximately 20%). Final reorganization to form the native tertiary and quaternary structures occurs during a slow, unimolecular, urea- and ionic strength-independent event. During this slow event (tau = 250 s), structural rearrangements at the domain interface occur at/near Trp20 and result in burial of Trp20. The slow event results in the regain of the fully functional dimer. The role of the C-terminus helix 9 (residues 210-221) as a structural determinant for this final event is proposed.     

Molecular dynamics simulations of human glutathione transferase P1-1: conformational fluctuations of the apo-structure.

We have investigated by molecular dynamics simulations the conformational fluctuations of the monomer of human apo-glutathione transferase P1-1. After attainment of steady-state dynamics, the structural fluctuations involve mainly the protein segments that participate also in the holo-apo transition discussed in the accompanying article (Stella et al., 1999:37:1-9.). The most mobile region is the C-terminal segment of helix 2. In contrast, helices 1, 6, 7, and 8 constitute a relatively rigid protein core. An "essential dynamics" analysis of the simulation shows that the largest fluctuations involve specific regions of glutathione transferases. In such regions, atomic motions are correlated. Motions of helix 2 are accounted for by the second most prominent principal component, which reveals a fluctuation between two distinct conformations. The residues that constitute the H-site undergo a breathing motion, possibly relevant during the binding of hydrophobic cosubstrates. Based on our simulation, several experimental findings can be rationalized, including the viscosity-dependent reactivity of Cys 47 and Cys 101 as well as the selective proteolysis of the peptide bond between Lys 44 and Ala 45. We have also modeled the structural changes that lead to the formation of an intrachain disulfide bridge between cysteines 47 and 101 and to the inactivation of the enzyme. The resulting structure maintains essentially the native fold except for helix 2, which closes the G-site. Proteins 1999;37:10-19.     

Effect of dinitrosyl iron complex with glutathione as a nitric oxide donor on blood circulation in healthy animals

It has been shown that the hypotensive action of the nitric oxide donor, the dinitrosyl complex of iron with glutathione, on the organism of healthy rats, which is caused by a decrease in the general peripherical immunity, does not impair the microcirculation and is accompanied by an enhancement of the contractile activity of the myocardium. In hypotension caused by the dinitrosyl iron complex, neither the tension of oxygen and nitrogen in the blood nor its basic-acidic status changes. Thus, the possible inhibitory action of this complex on some enzymes and proteins in the animal organism does not affect the functioning of the heart, vessels, and blood. The dinitrosyl iron complex with glutathione only causes a decrease in arterial pressure. It is assumed that these complexes as well as dinitrosyl complexes of iron with other thiol ligands may be considered as the basis for designing a novel type of drugs for the treatment of cardiovascular diseases.     

Disorder-to-order transition of the active site of human class Pi glutathione transferase, GST P1-1

Glutathione transferases comprise a large family of cellular detoxification enzymes that function by catalyzing the conjugation of glutathione (GSH) to electron-deficient centers on carcinogens and other toxins. NMR methods have been used to characterize the structure and dynamics of a human class pi enzyme, GST P1-1, in solution. Resonance assignments have been obtained for the unliganded enzyme and the GSH and S-hexylglutathione (GS-hexyl) complexes. Differences in chemical shifts between the GSH and GS-hexyl complexes suggest more extensive structural differences between these two enzyme-ligand complexes than detected by previous crystallographic methods. The NMR studies reported here clearly show that an alpha-helix (alpha2) within the GSH binding site exists in multiple conformations at physiological temperatures in the absence of ligand. A single conformation of alpha2 is induced by the presence of either GSH or GS-hexyl or a reduction in temperature to below 290 K. The large enthalpy of the transition ( approximately 150 kJ/mol) suggests a considerable structural rearrangement of the protein. The Gibbs free energy for the transition to the unfolded form is on the order of -4 to -6 kJ/mol at physiological temperatures (37 degrees C). This order-to-disorder transition contributes substantially to the overall thermodynamics of ligand binding and should be considered in the design of selective inhibitors of class pi glutathione transferases.     

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.     

Thermodynamic description of the effect of the mutation Y49F on human glutathione transferase P1-1 in binding with glutathione and the inhibitor S-hexylglutathione

The thermodynamics of binding of both the substrate glutathione (GSH) and the competitive inhibitor S-hexylglutathione to the mutant Y49F of human glutathione S-transferase (hGST P1-1), a key residue at the dimer interface, has been investigated by isothermal titration calorimetry and fluorescence spectroscopy. Calorimetric measurements indicated that the binding of these ligands to both the Y49F mutant and wild-type enzyme is enthalpically favorable and entropically unfavorable over the temperature range studied. The affinity of these ligands for the Y49F mutant is lower than those for the wild-type enzyme due mainly to an entropy change. Therefore, the thermodynamic effect of this mutation is to decrease the entropy loss due to binding. Calorimetric titrations in several buffers with different ionization heat amounts indicate a release of protons when the mutant binds GSH, whereas protons are taken up in binding S-hexylglutathione at pH 6.5. This suggests that the thiol group of GSH releases protons to buffer media during binding and a group with low pKa (such as Asp98) is responsible for the uptake of protons. The temperature dependence of the free energy of binding, DeltaG0, is weak because of the enthalpy-entropy compensation caused by a large heat capacity change. The heat capacity change is -199.5 +/- 26.9 cal K-1 mol-1 for GSH binding and -333.6 +/- 28.8 cal K-1 mol-1 for S-hexylglutathione binding. The thermodynamic parameters are consistent with the mutation Tyr49 --> Phe, producing a slight conformational change in the active site.     

Tocotrienols inhibit human glutathione S-transferase P1-1

Tocopherols and tocotrienols are food ingredients that are believed to have a positive effect on health. The most studied property of both groups of compounds is their antioxidant action. Previously, we found that tocopherols and diverse tocopherol derivatives can inhibit the activity of human glutathione S-transferase P1-1 (GST P1-1). In this study we found that GST P1-1 is also inhibited, in a concentration-dependent manner, by alpha- and gamma-tocotrienol. The concentration giving 50% inhibition of GST P1-1 is 1.8 +/- 0.1 microM for alpha-tocotrienol and 0.7 +/- 0.1 microM for gamma-tocotrienol. This inhibition of GST P1-1 is noncompetitive with respect to both substrates CDNB and GSH. We also examined the 3D structure of GST P1-1 for a possible tocopherol/tocotrienol binding site. The enzyme contains a very hydrophobic pit-like structure where the phytyl tail of tocopherols and tocotrienols could fit in. Binding of tocopherol and tocotrienol to this hydrophobic region might lead to bending of the 3D structure. In this way tocopherols and tocotrienols can inhibit the activity of the enzyme; this inhibition can have far-reaching implications for humans.     

Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences

We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.     

In vivo distribution and behavior of paramagnetic dinitrosyl dithiolato iron complex in the abdomen of mouse

It has been shown that a dinitrosyl dithiolato iron complex is formed under physiological conditions and that it functions as an NO transporter. In the present study, a diglutathionyl dinitrosyl iron complex [DNIC-(GS)₂] was injected into mice and its abdominal distribution and behavior were examined by using electron paramagnetic resonance (EPR) spectroscopy. The X-band EPR signal intensity of the blood, liver, kidney, and spleen decreased with time but signals from the liver and kidney were readily detectable even 24h after the injection. The time courses of signal intensity were quite similar when the agent was administered via intravenous and subcutaneous injection routes, suggesting that DNIC-(GS)₂ can penetrate readily and rapidly through the membranes. Real-time detection of DNIC-(GS)₂ in the upper abdomen of the living mice was performed by employing an in vivo EPR spectroscopy. These results suggest that DNIC-(GS)₂, an endogenous NO carrier, has an excellent membrane permeability and has a relatively high affinity for the liver and kidney.     

Modulation of glutathione transferase P1-1 activity by retinoic acid in neuroblastoma cells.

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.     

Effects of the donor of nitric oxide,dinitrosyl iron complex with glutathione,on blood circulation in healthy animals

We have found that the hypotensive effect of the nitric oxide donor dinitrosyl iron complex with glutathione was caused by a decrease in general peripheral resistance in healthy rats. This effect did not impair microcirculation and was accompanied by an increase in the myocardial contractile activity. Under the hypotension condition induced by dinitrosyl iron complex with glutathione, we did not find any changes in the oxygen or carbon dioxide tensions in the blood as compared to the control or any change in the acidic-basic blood state. Thus, the possible inhibitory influence of this complex on some enzymes and proteins in the animal body was not accompanied by effects on the heart, vessels, or blood. The dinitrosyl iron complex with glutathione induced a decrease in the arterial pressure only. We hypothesize that a new type of drugs for the treatment of cardiovascular diseases can be developed on the basis of such complexes and complexes with other thiol-containing ligands.