From the
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.
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
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
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.
(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) .
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
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
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.
(
)
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.
) 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) .
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 H
O
(
) 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 K
of
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 K
and a high turnover
number.
(
)
The very fast reduction of
selenocystine by TR suggests that selenocystine is kept reduced
intracellularly.
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.
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
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.