Henrikas Nivinskas

Interactions of Quinones with Thioredoxin Reductase A CHALLENGE TO THE ANTIOXIDANT ROLE OF THE MAMMALIAN SELENOPROTEIN

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single- and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the kcat/Km value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH4) and two-electron reduced (EH2) states in quinone reduction. The redox potential of the EH2/EH4 couple of TrxR calculated according to the Haldane relationship with NADPH/NADP+ was –0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate Ki = 6.3 ± 1 μm). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones.

Interactions of Nitroaromatic Compounds with the Mammalian Selenoprotein Thioredoxin Reductase and the Relation to Induction of Apoptosis in Human Cancer Cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a kcat of 1.8 and 2.8 s–1, respectively, at pH 7.0 and 25 °C using 50 μM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5′-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5′-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.

Structure-Activity Relationships in Two-Electron Reduction of Quinones

Publisher Summary This chapter analyzes the structure-activity relationships in two-electron reduction of quinines. Quinones may accept electrons from various flavoenzymes, iron-sulfur proteins and photosynthetic reaction centers. The energetics of the quinine reduction are studied extensively by pulse-radiolysis, electron spin resonance, and electrochemical techniques. The single-electron reduction of quinones by flavoenzyme dehydrogenases-electrontransferases may be treated according to an ‘‘outer-sphere electron transfer’’ model. In general, the reaction rates increase with an increase in quinine single-electron reduction potential. In this chapter, it is demonstrated that multiparameter regression analysis, by using redox potential and several simple structural parameters of quinones, may provide important information on the mechanisms of two-electron enzymatic reduction. In view of the simplicity of the single-electron reduction mechanism and the presumable involvement of single-electron transfers in two-electron reduction, the single-electron reduction of quinones is analyzed. Single-electron reduction of quinones by flavoenzymes is analyzed. The chapter presents the outer-sphere electron transfer model in single-electron reduction of quinones. Structure-activity relationships in single-electron reduction of quinones by NADPH:Cytochrome P-450 reductase and ferredoxin:NADP + reductase are explored. Two-electron reduction of quinones by flavoenzymes is explained in the chapter. The chapter describes the mechanism of two-electron (Hydride) transfer.

Reduction of aliphatic nitroesters and N-nitramines by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships.

Enterobacter cloacae PB2 NADPH:pentaerythritol tetranitrate reductase (PETNR) performs the biodegradation of explosive organic nitrate esters via their reductive denitration. In order to understand the enzyme substrate specificity, we have examined the reactions of PETNR with organic nitrates (n = 15) and their nitrogen analogues, N‐nitramines (n = 4). The reactions of these compounds with PETNR were accompanied by the release of 1–2 mol of nitrite per mole of compound, but were not accompanied by their redox cycling and superoxide formation. The reduction rate constants (kcat/Km) of inositol hexanitrate, diglycerol tetranitrate, erythritol tetranitrate, mannitol hexanitrate and xylitol pentanitrate were similar to those of the established PETNR substrates, PETN and glycerol trinitrate, whereas the reactivities of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine and octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine were three orders of magnitude lower. The log kcat/Km value of the compounds increased with a decrease in the enthalpy of formation of the hydride adducts [ΔHf(R–O–N(OH)O−) or ΔHf(R1,R2 > N–N(OH)O−)], and with an increase in their lipophilicity (octanol/water partition coefficient, log Pow), and did not depend on their van der Waals’ volumes. Hydrophobic organic nitroesters and hydrophilic N‐nitramines compete for the same binding site in the reduced enzyme form. The role of the hydrophobic interaction of PETNR with glycerol trinitrate was supported by the positive dependence of glycerol trinitrate reactivity on the solution ionic strength. The discrimination of nitroesters and N‐nitramines according to their log Pow values seems to be a specific feature of the Old Yellow Enzyme family of flavoenzymes.

DT-diaphorase catalyzes N-denitration and redox cycling of tetryl

Rat liver DT‐diaphorase (EC 1.6.99.2) catalyzed reductive N‐denitration of tetryl (2,4,6‐tri‐nitrophenyl‐N‐methylnitramine) and 2,4‐dinitrophenyl‐N‐methylnitramine, oxidizing the excess of NADPH. The reactions were accompanied by oxygen consumption and superoxide dismutase‐sensitive reduction of added cytochrome c and reductive release of Fe2+ from ferritin. Quantitatively, the reactions of DT‐diaphorase proceeded like single‐electron reductive N‐denitration of tetryl by ferredoxin:NADP+ reductase (EC 1.18.1.2) (Shah, M.M. and Spain, J.C. (1996) Biochem. Biophys. Res. Commun. 220, 563–568), which was additionally checked up in this work. Thus, although reductive N‐denitration of nitrophenyl‐N‐nitramines is a net two‐electron (hydride) transfer process, DT‐diaphorase catalyzed the reaction in a single‐electron way. These data point out the possibility of single‐electron transfer steps during obligatory two‐electron (hydride) reduction of quinones and nitroaromatics by DT‐diaphorase.

Antiplasmodial activity of quinones: Roles of aziridinyl substituents and the inhibition of Plasmodium falciparum glutathione reductase

Although quinones have been the subject of great interest as possible antimalarial agents, the mechanism of their antimalarial activity is poorly understood. Flavoenzyme electrontransferase-catalyzed redox cycling of quinones, and their inhibition of the antioxidant flavoenzyme glutathione reductase (GR, EC 1.8.1.7) have been proposed as possible mechanisms. Here, we have examined the activity of a number of quinones, including the novel antitumor agent RH1, against the malaria parasite Plasmodium falciparum strain FcB1 in vitro, their single-electron reduction rates by P. falciparum ferredoxin:NADP(+) reductase (PfFNR, EC 1.18.1.2), and their ability to inhibit P. falciparum GR. The multiparameter statistical analysis of our data implies, that the antiplasmodial activity of fully-substituted quinones (n=15) is relatively independent from their one-electron reduction potential (E(7)(1)). The presence of aziridinyl groups in quinone ring increased their antiplasmodial activity. Since aziridinyl-substituted quinones do not possess enhanced redox cycling activity towards PfFNR, we propose that they could act as as DNA-alkylating agents after their net two-electron reduction into aziridinyl-hydroquinones. We found that under the partial anaerobiosis, i.e., at the oxygen concentration below 40-50 microM, this reaction may be carried out by single-electron transferring flavoenzymes present in P. falciparum, like PfFNR. Another parameter increasing the antiplasmodial activity of fully-substituted quinones is an increase in their potency as P. falciparum GR inhibitors, which was revealed using multiparameter regression analysis. To our knowledge, this is the first quantitative demonstration of a link between the antiplasmodial activity of compounds and GR inhibition.

Cytotoxicity of Natural Hydroxyanthraquinones: Role of Oxidative Stress

In order to assess the role of oxidative stress in the cytotoxicity of natural hydroxyanthraquinones, we compared rhein, emodin, danthron, chrysophanol, and carminic acid, and a series of model quinones with available values of single-electron reduction midpoint potential at pH 7.0 (E17), with respect to their reactivity in the single-electron enzymatic reduction, and their mammalian cell toxicity. The toxicity of model uinones to the bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK), and HL-60, a human promyelocytic leukemia cell line, increased with an increase in their E17. A close parallelism was found between the reactivity of hydroxyanthraquinones and model quinones with single-electron transferring flavoenzymes ferredoxin: NADP+ reductase and NADPH: cytochrome P-450 reductase, and their cytotoxicity. This points to the importance of oxidative stress in the toxicity of hydroxyanthraquinones in these cell lines, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine, by the potentiating effects of 1,3-bis-(2-chloroethyl)-1-nitrosourea, and an increase in lipid peroxidation.

The protective effects of dihydrolipoamide and glutathione against photodynamic damage by Al-phtalocyanine tetrasulfonate.

In spite of well‐known ability of lipoamide/dihydrolipoamide (LipS2NH2/Lip(SH)2 NH2) and oxidized/reduced glutathione (GSSg/GSH) couples to scavenge singlet oxygen (1O2), the possible protective effects of these compounds against photodynamical damage by Al‐phtalocyanine tetrasulfonate (Al‐PcS4) were examined. Using erythrocyte glutathione reductase, pig heart lipoamide dehydrogenase and hamster kidney fibroblast culture as model systems, we have found that protective effects of Lip(SH)2NH2 and LipS2NH2 were close to that of azide, far exceeding the effects of GSH and GSSG, and paralleling the rates of Al‐PcS4‐sensitized photooxidation of these compounds. We have failed to observe a previously described (Devasagayam, T.P.A., et al. (1991) Biochim. Biophys. Acta 1088, 409‐412) enhancement of damaging action of 1O2 by GSH. These findings point out to the possibility of LipS2NH2/Lip(SH)2NH2 to neutralize the side‐effects of photodynamic therapy, and to a minor but definitely protective role of GSH.

Prooxidant Cytotoxicity of Chromate in Mammalian Cells: The Opposite Roles of DT-Diaphorase and Glutathione Reductase

Abstract The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 ± 4) μᴍ. The cytotoxicity was partly prevented by desferri- oxamine, the antioxidant N(N′-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-l-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.

Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin.

Abstract We have found that the binding of NADP+ (Kd = 0.86 ±0.11 μᴍ) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH :thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 μᴍ) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 μᴍ TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of A ADP+.