Interactions of Quinones with Thioredoxin Reductase

Abstract

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...

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

“…The Cys-59 thiolateand charge-transfer-containing active site is thereby instead available for other types of reactions, including one-(or two-) electron reduction of quinones such as juglone. This line of reasoning is supported by the known high capacity of native, Sec-derivatized as well as Sec-lacking truncated forms of TrxR in reduction of juglone directly through the N-terminal redox active motif (38,39,68). Hence, without resonance effects of Tyr-116 and therefore a lack of an efficient reductive half-reaction, the result would easily be the observed increased propensity for juglone reduction but diminished activity with Trx as substrate.…”

“…However, considering that juglone can be efficiently reduced by either the C-terminal selenolthiol motif or directly by the N-terminal FAD/dithiol motif of TrxR (38,39,68) and considering the plausible roles of Tyr-116 in the reductive and oxidative half-reactions, as described above, the effects of Tyr-116 mutations become explainable. Hence, we propose that the Tyr-116 substitutions diminish the possibility for the Cys-59 thiolate to attack Sec-498 of the selenenylsulfide, thereby mainly leaving the selenenylsulfide in the inactive oxidized state.…”

“…mutants of mouse TrxR2 (33)(34)(35)(36), human TrxR1 (37) (also as directly deposited PDB entry 2CFY), and the non-selenoprotein orthologue of Drosophila (34,35). These structures as well as additional modeling studies and several extensive biochemical analyses (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) have generated a generally accepted model for the major features of the catalytic mechanism of mammalian TrxRs. This model is summarized in Fig.…”

Conditional Expression of 15-Lipoxygenase-1 Inhibits the Selenoenzyme Thioredoxin Reductase

“…The catalytic mechanism of mammalian TrxR is well understood (5). However, less is known about endogenous cellular factors that modulate its activity.…”

Effects of Tetrahydropterines on the Generation of Quinones Catalyzed by Tyrosinase

“…A possible mechanism for o-quinone reduction is the thioredoxin reductase/thioredoxin (TR/ T) system. 3) In human keratinocytes and melanocytes, the presence of the TR isoform has been demonstrated, 4) while 6BH 4 can reduce o-quinone L-dopaquinone back to L-dopa. 5) It has been found that keratinocytes and melanocytes have a capacity for de novo synthesis, recycling, and regulation of 6-tetrahydrobiopterin (6BH 4 ).…”