©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Thioredoxin Reductase Directly Reduces Lipid Hydroperoxides by NADPH and Selenocystine Strongly Stimulates the Reaction via Catalytically Generated Selenols(*)

Mikael Björnstedt (1), Mats Hamberg (2), Sushil Kumar (1), Jiyan Xue (1), Arne Holmgren (1)(§)

From the (1) Medical Nobel Institute for Biochemistry and the (2) Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
REFERENCES

ABSTRACT

Human placenta thioredoxin reductase (HP-TR) in the presence of NADPH-catalyzed reduction of (15S)-hydroperoxy-(5Z),(8Z),11(Z),13(E)-eicosatetraenoic acid ((15S)-HPETE) into the corresponding alcohol ((15S)-HETE). Incubation of 50 nM HP-TR and 0.5 mM NADPH with 300 µM 15-HPETE for 5 min resulted in formation of 16.5 µM 15-HETE. After 60 min, 74.7 µM 15-HPETE was reduced. The rate of the reduction of 15-HPETE by the HP-TR/NADPH peroxidase system was increased 8-fold by the presence of 2.5 µM selenocystine, a diselenide amino acid. In this case, 15-HPETE was catalytically reduced by the selenol amino acid, selenocysteine, generated from the diselenide by the HP-TR/NADPH system. To a smaller extent, selenodiglutathione or human thioredoxin also potentiated the reduction of 15-HPETE by HP-TR. Hydrogen peroxide and 15-HPETE were reduced at approximately the same rate by HP-TR, thioredoxin, and selenocystine. In contrast, t-butyl hydroperoxide was reduced at a 10-fold lower rate. Our data suggest two novel pathways for the reduction and detoxification of lipid hydroperoxides, hydrogen peroxide, and organic hydroperoxides, i.e. the human thioredoxin reductase-dependent pathway and a coupled reduction in the presence of selenols or selenide resulting from the reduction of selenocystine or selenodiglutathione.


INTRODUCTION

Polyunsaturated fatty acids can be oxygenated into hydroperoxides, either non-enzymatically (1) or in the presence of specific lipoxygenases (2) . Thus, arachidonic acid, an abundant polyunsaturated fatty acid in tissues, can provide (15R,S)-HPETE() and its regioisomers by non-enzymatic oxygenation as well as (15S)-HPETE when exposed to arachidonic acid 15-lipoxygenase (3). Further conversion of 15-HPETE, enzymatically and non-enzymatically, leads to the formation of epoxy alcohols and trihydroxyeicosatrienoic acids (4) , to dioxygenated derivatives such as 5,15-diHPETE, 8,15-diHETE, and 14,15-diHETE (3) and to lipoxins (5). In addition, glutathione peroxidase-catalyzed reduction of 15-HPETE into the corresponding hydroxy acid (15-HETE) is likely to have importance for in vivo detoxification of 15-HPETE and other fatty acid hydroperoxides.

Thioredoxin (Trx) is a ubiquitous 12-kDa protein with the conserved active-site sequence -Cys-Gly-Pro-Cys- located on a protrusion in its three-dimensional structure (6) . Oxidized thioredoxin with an active-site disulfide is reduced by NADPH and thioredoxin reductase (TR), and reduced thioredoxin is a powerful general protein disulfide reductase (6, 7) . Since its original isolation from Escherichia coli as a hydrogen donor for the enzyme ribonucleotide reductase essential for the synthesis of deoxyribonucleotides and DNA (8) , many new functions have been discovered (6, 7) . Thus, thioredoxin can regulate the activity of enzymes, receptors, or transcription factors via thiol-redox control (7) . Adult T-cell leukemia-derived factor is a secreted form of human thioredoxin operating as a cytokine (9) . Thioredoxin reductase is a FAD-containing dimeric enzyme that has been purified and characterized from a variety of species (10) . Mammalian TR has a broad substrate specificity and reacts not only with its homologous Trx but also with E. coli Trx (11) , 5,5`-dithiobis(2-nitrobenzoic acid) (11) , GS-Se-SG (12) , selenite (13) , vitamin K (14) , alloxan (15) , and the active-site selenocysteine residue in glutathione peroxidases (GSH-Px) (16) .

Selenium is an essential trace element that is known to have antioxidant properties. The most established mechanism behind the antioxidant function is the formation of selenocysteine from selenide and the incorporation of this amino acid in the active site of glutathione peroxidases (17) . These enzymes detoxify hydrogen peroxide, lipid hydroperoxides, and organic hydroperoxides (18) . Selenite and GS-Se-SG are both efficiently reduced directly by mammalian TR or the complete thioredoxin system (12, 13) . In these reactions selenide (HSe) is formed and recycles with oxygen giving rise to a non-stoichiometric large oxidation of NADPH. Recently we showed that thioredoxin reductase with or without reduced thioredoxin is an electron donor to human plasma GSH-Px in the reduction of hydroperoxides (16) .

The aim of the present study was to investigate whether thioredoxin reductase and thioredoxin can reduce lipid hydroperoxides and if low molecular weight selenium compounds could act as charge transfer catalysts. This could be an important alternative pathway for the detoxification of hydroperoxides in addition to GSH-Px-mediated reduction.


EXPERIMENTAL PROCEDURES

(15S)-HPETE (purity, in excess of 96%) was obtained by incubation of arachidonic acid (Nu-Chek-Prep, Inc., Elysian, MN) with soybean lipoxygenase at 0 °C (19) . (15R,S)-HPETE was prepared by autoxidation of arachidonic acid followed by isolation by silicic acid chromatography and straight-phase HPLC (cf. Ref. 20). (15S)-HETE was prepared in quantitative yield by reduction of (15S)-HPETE with triphenylphosphine in diethyl ether. t-Butyl hydroperoxide and yeast GR were from Sigma. DL-Selenocystine was from Serva. TR from human placenta and calf thymus was purified essentially as described by Luthman and Holmgren (14) . Enzyme activities were standardized by using 5,5`-dithiobis(2-nitrobenzoic acid) as a substrate (14) . E. coli TR was a homogenous preparation as described previously (13) . Recombinant human Trx was prepared as described by Ren et al.(21) and was reduced prior to use with dithiothreitol followed by desalting over a Sephadex G-25 column. GS-Se-SG was prepared as described by Björnstedt et al.(12) .

Spectrophotometric Measurements

The reduction of selenocystine was performed in 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, with 200 µM NADPH. The reduction was followed at 340 nm as oxidation of NADPH using a mM extinction coefficient of 6.2. To both the reference and the sample cuvette was added HP-TR. The reactions were started by addition of selenocystine to the sample cuvette and an equal volume of buffer to the reference cuvette. The final volume was 0.50 ml.

Spectrophotometric Determination of Peroxidase Activity

The reactions were performed in 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, with 500 µM NADPH. The reduction of hydroperoxides was followed as oxidation of NADPH at 340 nm. To the cuvettes were added HP-TR, Trx, and selenocystine to final concentrations of 50 nM, 2.0 µM, and 2.5 µM, respectively. The reactions were started by addition of peroxide (150 µM) to the sample cuvette and an equal volume of 99.5% ethanol to the reference cuvette.

Determination of Peroxidase Activity by HPLC Analysis of (15S)-HPETE and (15S)-HETE

To a reaction mixture composed of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, and 0.5 mM or 1 mM NADPH was added either TR, TR + Trx, TR + selenocystine, TR + GS-Se-SG, or GR. The reactions were started by addition of (15S)-HPETE to a final concentration of 300 µM. After incubation at room temperature for 15 min (if not otherwise indicated) the reactions were terminated by rapid freezing in dry ice/ethanol. Incubation mixtures of 15-HPETE (0.5 ml) were acidified to pH 4 and rapidly extracted with 7 ml of diethyl ether. The ether phase was washed three times with water and taken to dryness. The residue was analyzed by straight-phase HPLC using a column of Nucleosil 50-5 (250 4.6 mm; Macherey-Nagel (Düren, Germany)) and 2-propanol/hexane/acetic acid (1.5:98.5:0.02, v/v/v) at a flow rate of 1.5 ml/min. The absorbance at 235 nm of the effluent was monitored and the digitized signal integrated using a Macintosh SE/30. The retention volumes of (15S)-HETE and (15S)-HPETE were 10.2-10.9 and 12.8-13.5 ml, respectively.

RESULTS

Reduction of Lipid Hydroperoxides by Mammalian Thioredoxin Reductase

(15S)-HPETE (300 µM) incubated with HP-TR or CT-TR and NADPH was reduced to (15S)-HETE (Fig. 1). In order to investigate possible stereospecificity, (15R,S)-HPETE was incubated with CT-TR/NADPH and the 15-HETE produced (32% reduction) as well as the remaining 15-HPETE (68%) were subjected to steric analysis (22). Both compounds were found to be virtually racemic (ratio of S to R enantiomers, 49.9:50.1 (15-HETE) and 50.8:49.2 (15-HPETE)). Thus there was no discrimination between enantiomers in the CT-TR-promoted reduction of 15-HPETE. The initial part of the reaction was faster using CT-TR compared with HP-TR (Fig. 1). There was an almost linear relation between the concentration of (15S)-HPETE and product formed up to 500 µM concentration of (15S)-HPETE (Fig. 2). Addition of Trx to HP-TR increased the rate of the reduction of (15S)-HPETE by 60% (Fig. 3) indicating that both proteins of the human Trx system can reduce lipid hydroperoxides. In contrast, neither E. coli thioredoxin reductase nor yeast glutathione reductase had any significant peroxidase activity (Fig. 3).


Figure 1: Time course of reduction of (15S)-HPETE in the presence of TR and NADPH. (15S)-HPETE (300 µM) was added to 0.5 ml of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, containing NADPH (0.5 mM) and either 50 nM) HP-TR () or 50 nM CT-TR (). The mixtures were kept at room temperature, and at the times indicated, the reactions were terminated by freezing in dry ice/ethanol. The hydroperoxide and its corresponding alcohol were extracted with diethyl ether and analyzed by straight-phase HPLC as described in the text.




Figure 2: Reduction of (15S)-HPETE as a function of substrate concentration. (15S)-HPETE (0-500 µM) was added to 0.5 ml of HP-TR (50 nM), NADPH (0.5 mM), and EDTA (1 mM) in 50 mM Tris-HCl buffer, pH 7.5, and kept at room temperature for 30 min. The reaction mixtures were extracted with diethyl ether and analyzed by straight-phase HPLC.




Figure 3: Reduction of (15S)-HPETE under various conditions. (15S)-HPETE (300 µM) was added to the agents indicated dissolved in 0.5 ml of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5. The mixtures were kept at room temperature for 15 min. A, buffer control; B, 1 mM NADPH; C, 50 nM HP-TR; D, 50 nM HP-TR plus 1 mM NADPH; E, 50 nME. coli thioredoxin reductase (EC-TR) plus 1 mM NADPH; F, 50 nM CT-TR plus 1 mM NADPH; G, 1 unit of yeast GR plus 1 mM NADPH; H, 50 nM HP-TR, 2 µM Trx, and 1 mM NADPH; I, 50 nM HP-TR, 5 µM GS-Se-SG, and 1 mM NADPH; J, 50 nM HP-TR, 2.5 µM selenocystine, and 1 mM NADPH; K, 50 nM HP-TR, 2.5 µM selenocystine, 2 µM Trx, and 1 mM NADPH. The reaction mixtures were extracted with diethyl ether and analyzed by straight-phase HPLC. The values are averages of at least two individual experiments.



Reduction of Selenocystine by Mammalian Thioredoxin Reductase

Addition of HP-TR (50 nM) to selenocystine resulted in a very rapid oxidation of a stoichiometric amount of NADPH consistent with cleavage of each molecule of selenocystine into two molecules of selenocysteine (Fig. 4). Addition of GS-Se-SG or selenite to mammalian TR results in redox cycling with oxygen and a large non-stoichiometric oxidation of NADPH (12, 13) . In the reaction between TR and selenocystine, especially at high concentrations of selenocystine, the oxidation of NADPH continued after oxidation of a stoichiometric amount (Fig. 4). This is consistent with redox cycling with oxygen. Thus, selenocysteine is autoxidized by oxygen, and the resulting selenocystine is again reduced by TR.


Figure 4: Reduction of selenocystine by TR. The reactions were performed in 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, with 200 µM NADPH. To both the reference and the sample cuvette was added selenocystine to final concentrations of 10 µM (), 20 µM (), 30 µM (), and 40 µM (). The reactions were started by addition of TR to the sample cuvette and an equal volume of buffer to the reference cuvette (final volume, 500 µl). The reactions were followed at 340 nm.



Reduction of Lipid Hydroperoxides by Catalytically Regenerated Selenols

The presence of selenocystine (2.5 µM) increased the peroxidase activity of HP-TR 8-fold (Fig. 3). After 15 min of incubation of HP-TR and selenocystine 72.5% of (15S)-HPETE (or 108.8 nmol) was reduced. Similar results were obtained with two other fatty acid hydroperoxides, i.e. the -linolenic acid-derived (13S)-hydroperoxy-(9Z),(11E),(15Z)-octadecatrienoic acid and (9S)-hydroperoxy-(10E),(12Z),(15Z)-octadecatrienoic acid (data not shown). The presence of Trx only marginally increased the rate of the selenocysteine-coupled reaction (Fig. 3). Even when used in 0.5 µM concentration, selenocystine approximately doubled the rate of the TR/Trx-dependent peroxidase reaction (data not shown). Hydrogen peroxide and (15S)-HPETE were reduced at approximately the same rate by HP-TR, Trx, and selenocystine (Fig. 5). However, the rate of reduction of t-butyl hydroperoxide was slower. Addition of GS-Se-SG (5 µM) to HP-TR increased the rate of the reaction by 60% (Fig. 3). GS-Se-SG will react with TR and form selenide (12) . The results show that selenide can act as a charge transfer catalyst between TR and hydroperoxides. As a control, selenocystine (2.5 µM) without TR and NADPH was added to (15S)-HPETE (300 µM). The extent of reduction (0.6%) of the hydroperoxide did not differ from that observed with buffer (Fig. 3).


Figure 5: Reduction of different hydroperoxides. The reactions were performed in 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, with 500 µM NADPH. To the reference as well as the sample cuvette were added HP-TR (50 nM), Trx (2 µM), and selenocystine (2.5 µM). The reactions were started by addition of t-butyl hydroperoxide (), (15S)-HPETE (), or HO () to the sample cuvette in a final concentration of 150 µM and an equal volume of buffer or ethanol to the reference cuvette (final volume, 500 µl). The reduction of hydroperoxide was followed as oxidation of NADPH at 340 nm.



DISCUSSION

Our results show that lipid hydroperoxides can be reduced by TR. The mechanism of TR-dependent peroxidase activity is likely as shown in Fig. 6A. Trx has previously been shown to slowly reduce hydrogen peroxide and other non-disulfide reagents if coupled with NADPH and thioredoxin reductase (14, 15, 23, 24) .


Figure 6: Proposed mechanism for the TR-dependent reduction of hydroperoxides. A, TR peroxidase reaction; B, selenolate (Cys-Se)-coupled peroxidase reaction.



Selenocystine is a diselenide amino acid (25, 26) . Upon reduction selenocystine is decomposed into two molecules of the selenol amino acid selenocysteine (26) . This amino acid is an essential component of several proteins including GSH-Px (17) . In mammalian cells selenocysteine is incorporated in proteins by a selenocysteinyl-tRNA that recognizes the nonsense codon UGA (27) . Free selenocysteine is produced in mammalian tissues by decomposition of selenoproteins and trans-sulfuration of selenomethionine (28) . Selenocysteine is catabolized by selenocysteine -lyase into L-alanine and selenide (29) . However, the Kof this enzyme is high (0.83 mM) (29) , indicating that it is important only at rather high concentrations of selenocysteine. In the present study it was shown that selenocystine was efficiently reduced by TR and NADPH. Experiments with low concentrations of TR indicated that the kinetic parameters for the reaction with selenocystine are similar to that of the natural substrate thioredoxin, i.e. a low Kand a high turnover number.() The very fast reduction of selenocystine by TR suggests that selenocystine is kept reduced intracellularly.

Rotruck et al.(30) in 1973 reported the presence of selenium as a component of GSH-Px. The field of selenium and antioxidant functions have expanded to include intracellular and extracellular GSH-Px (28) as well as synthetic selenium compounds like ebselen (31) . GSH has been considered to be the only electron source for selenium-dependent hydroperoxide reduction, but human plasma contains essentially no free GSH (32) . Recently we showed that the human Trx system is an efficient electron donor to human plasma GSH-Px (16) . In this reaction TR or TR + Trx regenerates the charge of the active-site selenocysteine, which is oxidized during the peroxidase reaction. To investigate if this selenium-coupled peroxidase activity of TR was dependent on selenium inserted in a protein, i.e. GSH-Px, we added free selenocystine and GS-Se-SG to TR. The selenium-coupled reaction involves regeneration of Cys-Se from Cys-SeOH (Fig. 6B), in accordance with the regeneration of the active-site charge of GSH-Px (16) . Cys-Se reduces hydroperoxides to the corresponding alcohol. At high concentrations of selenocystine, reduction of Cys-SeOH by Cys-Se and formation of Cys-Se-Se-Cys will occur. A possible mechanism for the potentiation of TR-dependent peroxidase activity by GS-Se-SG is the regeneration of HSe from HSeOH. One explanation for the higher activity of selenocystine compared with selenide is that the nucleophilicity of the selenium atom in selenocysteine should be greater than that of selenium in selenide.

Selenocystine was previously reported to have a low GSH-Px activity together with 2 mM GSH and GR (25) . However, this activity is unlikely to be of physiological significance since the pH optimum for the reaction was 8.0 and the reaction was observed with 10.0 µM selenocystine and the maximum turnover based on the V was only 0.01 min(25) . In fact sodium selenite was found to be more active than selenocystine. In the present study we found that the turnover of the selenol with hydroperoxide in the reaction catalyzed by TR was 6 min.

Unsaturated fatty acid hydroperoxides serve as intermediates in the formation of biologically important compounds such as leukotrienes and lipoxins. In other situations, lipid hydroperoxides can accumulate in tissues and exert harmful effects. Specifically, 15-HPETE, the hydroperoxide used in the present study, can oxidatively modify low density lipoprotein and has been implicated in atherosclerosis (33) . One pathway for the elimination of lipid hydroperoxides involves reduction in the presence of glutathione peroxidase and glutathione. The present work gives evidence for another mechanism for detoxification of lipid hydroperoxides, i.e. the reduction into alcohols in the presence of thioredoxin reductase and NADPH. This pathway and its high capacity when operating together with catalytic amounts of selenocysteine could serve as an important alternative to glutathione peroxidase.


FOOTNOTES

*
This investigation was supported by grants from the Karolinska Institute, the Swedish Cancer Society (961), the Swedish Medical Research Council (13X-3529 and 13X-05170), and Knut och Alice Wallenbergs Stiftelse. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-8-728 76 86; Fax: 46-8-728 47 16.

The abbreviations used are: HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; TR, thioredoxin reductase; Trx, thioredoxin; HP-TR, human placenta thioredoxin reductase; CT-TR, calf thymus thioredoxin reductase; HPLC, high performance liquid chromatography; GS-Se-SG, selenodiglutathione; GR, glutathione reductase; GSH-Px, glutathione peroxidase.

M. Björnstedt, M. Hamberg, and A. Holmgren, manuscript in preparation.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.