Narimantas Č≐nas

NITROREDUCTASE REACTIONS OF ARABIDOPSIS THALIANA THIOREDOXIN REDUCTASE

Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) catalyzed redox cycling of aromatic nitrocompounds, including the explosives 2,4,6-trinitrotoluene and tetryl, and the herbicide 3,5-dinitro-o-cresol. The yield of nitro anion radicals was equal to 70-90%. Redox cycling of tetryl was accompanied by formation of N-methylpicramide. Bimolecular rate constants of nitroaromatic reduction (kcat/Km) and reaction catalytic constants (kcat) increased upon an increase in oxidant single-electron reduction potential (E(1)7). Using compounds with an unknown E(1)7 value, the reactivity of TR increased parallelly to the increase in reactivity of ferredoxin:NADP+ reductase of Anabaena PCC 7119 (EC 1.18.1.2). This indicated that the main factor determining reactivity of nitroaromatics towards TR was their energetics of single-electron reduction. Incubation of reduced TR in the presence of tetryl or 2,4-dinitrochlorobenzene resulted in a loss of thioredoxin reductase activity, most probably due to modification of reduced catalytic disulfide, whereas nitroreductase reaction rates were unchanged. This means that on the analogy of quinone reduction by TR (D. Bironaite, Z. Anusevicius, J.-P. Jacquot, N. Cenas, Biochim. Biophys. Acta 1383 (1998) 82-92), FAD and not catalytic disulfide of TR was responsible for the reduction of nitroaromatics. Tetryl, 2,4,6-trinitrotoluene and thioredoxin increased the FAD fluorescence intensity of TR. This finding suggests that nitroaromatics may bind close to the thioredoxin-binding site at the catalytic disulfide domain of TR, and induce a conformational change of enzymes (S.B. Mulrooney, C.H. Williams Jr., Protein Sci. 6 (1997) 2188-2195). Our data indicate that certain nitroaromatic herbicides, explosives and other classes of xenobiotics may interfere with the reduction of thioredoxin by plant TR, and confer prooxidant properties to this antioxidant enzyme.

Inhibition of phthalocyanine-sensitized photohemolysis of human erythrocytes by polyphenolic antioxidants: description of quantitative structure–activity relationships

Polyphenolic antioxidants protected against Al-phthalocyanine tetrasulfonate-sensitized photohemolysis of human erythrocytes. A quantitative structure-activity relationship has been obtained to describe the protective effects of di- and trihydroxybenzenes: log cI(50) (microM)=(1.8620+/-1.5565)+(3.6366+/-2.8245) E(1)(7) (V)-(0. 4034+/-0.0765) log P (r(2)=0.8367), where cI(50) represents the concentrations of compounds for the 2-fold increase in the lag-phase of hemolysis, E(1)(7) represents the compound single-electron oxidation potential, and P represents the octanol/water partition coefficient. The cI(50) for quercetin and taxifolin were close, and cI(50) for morin, kaempferol and hesperetin were lower than might be predicted by this equation. The protection from hemolysis by azide, a quencher of singlet oxygen ((1)O(2)) was accompanied by increase in cI(50) of polyphenols, indicating that azide and polyphenols competed for the same damaging species, (1)O(2). These findings point out to two factors, determining the protective efficiency of polyphenols against (1)O(2), namely, ease of electron donation and lipophilicity.

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.

Interaction of quinones with Arabidopsis thaliana thioredoxin reductase

In view of the ubiquitous role of the thioredoxin/thioredoxin reductase (TRX/TR) system in living cells, the interaction of Arabidopsis thaliana NADPH-thioredoxin reductase (EC 1.6.4.5) with quinones, an important class of redox cycling and alkylating xenobiotics, was studied. The steady-state reactions of A. thaliana TR with thioredoxin (TRX) and reaction product NADP+ inhibition patterns were in agreement with a proposed model of E. coli enzyme (B.W. Lennon, C.H. Williams, Jr., Biochemistry, vol. 35 (1996), pp. 4704-4712), that involved enzyme cycling between four- and two-electron reduced forms with FAD being reduced. Quinone reduction by TR proceeded via a mixed single- and two-electron transfer, the percentage of single-electron flux being equal to 12-16%. Bimolecular rate constants of quinone reduction (kcat/km) and reaction catalytic constants (kcat) increased upon an increase in quinone single-electron reduction potential. E(1)7. In several cases, the kcat of quinone reduction exceeded kcat of TRX reduction, suggesting that quinones intercepted electron flux from TR to TRX. Incubation of reduced TR with alkylating quinones resulted in a rapid loss of TRX-reductase activity, while quinone reduction rate was unchanged. In TRX-reductase and quinone reductase reactions of TR, NADP+ exhibited different inhibition patterns. These data point out that FAD and not the catalytic disulfide of TR is responsible for quinone reduction, and that quinones may oxidize FADH2 before it reduces catalytic disulfide. Most probably, quinones may oxidize the two-electron reduced form of TR, and the enzyme may cycle between two-electron reduced and oxidized forms in this reaction. The relatively high rate of quinone reduction by A. thaliana thioredoxin reductase accompanied by their redox cycling, confers pro-oxidant properties to this antioxidant enzyme. These factors make plant TR an attractive target for redox active and alkylating pesticide action.