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 k cat /K m 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 (EH 4 ) and two-electron reduced (EH 2 ) states in quinone reduction. The redox potential of the EH 2 /EH 4 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 K i ؍ 6.3 ؎ 1 M). 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.Thioredoxin reductase (TrxR, 1 EC 1.8.1.9) catalyzes NADPHdependent reduction of the redox-active disulfide in thioredoxin (Trx), which serves a wide range of functions in cellular proliferation and redox control (1, 2). Thioredoxin reductases are homodimeric proteins that differ in properties between different classes of organisms. Low M r (34-kDa subunit) TrxRs of prokaryotes, plants, or yeast contain FAD and a redox-active disulfide/dithiol active site and display narrow substrate specificities. High M r (54 -58 kDa) TrxRs of animals have in contrast remarkably wide substrate specificities, explained by an additional easily accessible C-terminal redox center. This redox center is either a disulfide/dithiol as in TrxR of Plasmodium falciparum or Drosophila melanogaster or a selenocysteinecontaining selenenylsulfide/selenolthiol motif as found in TrxRs of mammals (3-6). In recent years, the catalytic mechanism of mammalian TrxR has been unraveled in significant detail. The three-dimensional crystal structure of rat TrxR is similar to that of glutathione reductase, including conserved FAD and NADP(H)-binding domains, but TrxR has a 16-residue C-term...
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 k cat of 1.8 and 2.8 s ؊1 , respectively, at pH 7.0 and 25°C using 50 M NADPH. As a nitroreductase, TrxR cycled between four-and twoelectron-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-Nmethylnitramine, 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.
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