Biochemical Characterization of Yeast Mitochondrial Grx5 Monothiol Glutaredoxin*
Jordi Tamarit,
Gemma Bellí,
Elisa Cabiscol,
Enrique Herrero and
Joaquim Ros
From the
Departament de Ciències Mèdiques Bàsiques, Facultat
de Medicina, Universitat de Lleida, Rovira Roure 44, 25198 Lleida, Spain
Received for publication, April 3, 2003
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ABSTRACT
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Grx5 is a yeast mitochondrial protein involved in iron-sulfur biogenesis
that belongs to a recently described family of monothiolic glutaredoxin-like
proteins. No member of this family has been biochemically characterized
previously. Grx5 contains a conserved cysteine residue (Cys-60) and a
non-conserved one (Cys-117). In this work, we have purified wild type and
mutant C60S and C117S proteins and characterized their biochemical properties.
A redox potential of 175 mV was calculated for wild type Grx5. The
pKa values obtained by titration of mutant
proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for
Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed
disulfide was formed between glutathione and the cystein 60 of the protein
because of its low pKa. Binding of glutathione to
Cys-60 promoted a decrease in the Cys-117 pKa
value that triggered the formation of a disulfide bond between both cysteine
residues of the protein, indicating that Cys-117 plays an essential role in
the catalytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced
by GSH but at a rate at least 20 times slower than that observed for the
reduction of glutaredoxin 1 from E. coli, a dithiolic glutaredoxin.
This slow reduction rate could suggest that GSH may not be the physiologic
reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a
glutathiolated protein used as a substrate indicated that Grx5 may act as a
thiol reductase inside the mitochondria.
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INTRODUCTION
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Glutaredoxins
(Grx)1 are small
proteins with thiol reductase activity that are required for maintaining
protein cysteines in reduced form. In contrast to thioredoxins, glutaredoxins
require the reduced form of glutathione, GSH, as the electron donor
(13).
Previously characterized glutaredoxins contain an active site that includes
two conserved cysteine residues with two non-conserved residues between them
(46).
Mutagenic studies have shown that both residues are required for reducing
protein disulfides. However, only the amino-terminal cysteine may be essential
for the reduction of mixed disulfides of proteins with glutathione
(68).
In Saccharomyces cerevisiae, five different glutaredoxins have been
described. Two of them (Grx1/2) are classic dithiolic glutaredoxins containing
both conserved cysteine residues and have already been biochemically
characterized
(911).
On the basis of sequence analysis, a new family of monothiolic glutaredoxins
has been described recently. These proteins are highly homologous to
glutaredoxins but contain only one cysteine residue in its putative active
site (12). Members of this
family are found elsewhere, from bacteria to mammals, including human
(13). To date, none of them
has been biochemically characterized properly.
Three monothiolic glutaredoxins are found in yeast (Grx3/4/5). No clear
phenotypes have been described in yeast cells lacking Grx3 and Grx4 and,
consistent with this, no specific role has been assigned to any of these
proteins. In contrast, the absence of Grx5 induces severe growth defects
(12). Cells lacking Grx5 are
not able to grow on minimal medium or in the presence of non-fermentable
carbon sources; they accumulate iron in the mitochondria and show decreased
activities of iron-sulfur-containing enzymes. These characteristics are common
to other genes involved in the synthesis and assembly of Fe-S clusters such as
SSQ1, JAC1, ATM1, NFU, YAH1, ARH1, ISU12
(14), ISA12
(15), NFS1, YFH1
(16), and ERV1
(17). Recently, we have shown
that Grx5 is a mitochondrial protein involved in iron-sulfur biogenesis
(18).
A three-dimensional model of Grx5 was recently presented based on the known
structure of several dithiolic glutaredoxins
(13). Grx5 shows a classic
thioredoxin fold structure, with the putative catalytic cysteine (Cys-60)
lying opposite to another conserved motif that could be involved in the
formation of a glutathione cleft. Beside this motif, another non-conserved
cysteine is found (Cys-117). Site directed mutagenesis studies suggest that
this cysteine is not essential for the biological activity of the protein
(13).
Despite these observations, there is no evidence that Grx5 works as a thiol
reductase. Also, the specific role of Grx5 in iron-sulfur biogenesis is still
not clear. Shenton et al.
(19) showed that, in cells
lacking Grx5, the cytosolic enzyme glyceraldehyde-3-phosphate-dehydrogenase
was glutathiolated, and they suggested that Grx5 could work as a
deglutathiolase. However the recent finding that Grx5 is a mitochondrial
enzyme (18) suggests that this
glutathiolation may be related to the oxidative stress conditions generated by
iron accumulation in
grx5 cells rather than to the direct
effect of Grx5, a mitochondrial protein, on
glyceraldehyde-3-phosphate-dehydrogenase, a cytosolic enzyme. In this work we
address the biochemical characterization of Grx5, including determination of
the cysteine pKa value and redox potential. Based
on these results, we propose a mechanism of action for the Grx5 protein. This
is the first characterization of a monothiolic glutaredoxin and constitutes
the first evidence that these proteins can work as thioloxidoreductases.
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EXPERIMENTAL PROCEDURES
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MaterialsGSH, GSSG, cystine, dehydroascorbate,
iodoacetamide, glutathione reductase, thioredoxin, and trifluoroacetic acid
were from Sigma. Glutaredoxin 1 (Grx1) from Escherichia coli was from
Calbiochem, and 2-hydroxyethyl disulfide (HED) was from Aldrich. Rat carbonic
anhydrase III was a kind gift of Dr. Rod Levine (National Institutes of
Health, Bethesda, MD).
Strains and PlasmidsPlasmid pMM192 contains the
GRX5 open reading frame without the region coding from amino acids
229 (PCR-amplified from S. cerevisiae genomic DNA), cloned
between the NdeI and BamH I unique sites of the E.
coli expression vector pET-21a (Novagen). Point mutations in
GRX5 that yielded the different amino acid replacements were
constructed by the ExSite method
(20), using pMMM192 as
template. Oligonucleotides for the introduction of the point mutations were
designated in such a way that a restriction site that did not alter the
translation product was introduced near to the desired point mutation and used
as a marker for the DNA sequencing. Plasmids were maintained and amplified in
E. coli BL21 cells (Novagen).
Purification of Grx5 Wild Type and Mutant ProteinsE. coli
cells carrying the previously described plasmids coding for Grx5 wild type and
mutant proteins were grown at 30 °C in Luria-Bertani medium with 100
µg/ml ampicillin. When the A600 reached a value of 0.4,
expression of Grx5 was induced with 0.5 mM isopropyl
thio-
-D-galactoside. After 4 h of growth, the cells were
centrifuged, washed twice with 50 mM Tris-HCl, pH 8.0, and frozen
in liquid nitrogen. Purification of the enzyme was made at 4 °C. The cells
(3 g) were suspended in 5 ml of 50 mM Tris-HCl, pH 8.0, 100
mM NaCl, and 1 mM phenylmethanesulfonyl fluoride and
sonicated. After centrifugation at 14,000 rpm for 30 min, the supernatant
solution (5 ml, 35 mg of protein/ml) was applied on a Sephacryl S-100 HR
column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH
8.0, plus 100 mM NaCl. After void volume, 4-ml fractions were
analyzed for the presence of Grx5 by SDS-polyacrylamide gel electrophoresis.
Fractions containing Grx5 were pooled and applied to a DEAE-15HR column
(Waters Associates, Milford, MA) equilibrated with 50 mM Tris-HCl,
pH 8.0, and 100 mM NaCl. After a washing step of 20 min with the
same buffer, elution was carried out by a linear gradient from 100 to 500
mM NaCl over 40 min at a flow rate of 5 ml/min. Grx5 eluted at 300
mM NaCl. Salt was diluted 30 times by several steps of
concentration/dilution of the protein using an Amicon 8010 ultrafiltration
cell. Protein was stored at 80 °C at concentrations above 20 mg/ml.
Protein was 99% pure as examined by SDS-polyacrylamide gel
electrophoresis.
AnalysesProtein concentration was determined by the
Bradford method (21).
Titration of free sulfhydryl groups with 5,5'-dithiobis-2-nitrobenzoic
acid (DTNB) was performed as described
(22). Briefly, 2050
µg of protein were incubated for 10 min in a solution containing 200
µM DTNB in 100 mM Tris-HCl buffer, pH 8.0, in a final
volume of 0.1 ml. A molar extinction coefficient of 14,150
M1 cm1
was used to calculate the number of titrated sulfhydryl groups.
ActivitiesReduction of the mixed disulfide formed between
HED and glutathione (low molecular weight mixed disulfide reduction assay or
HED assay) was assayed as described in Ref.
2. Dehydroascorbate reductase
activity and glutathione peroxidase activities were performed according to
Refs. 23 and
24.
Determination of Thiol pKa
ValueThe rate of carboxymethylation of Grx5 was determined by
incubation of reduced Grx5 (25 µM), either wild type or mutant
proteins, with 0.6 mM iodoacetamide in 23 µl of 10 mM
Tris, 10 mM potassium acetate, 10 mM MOPS, 10
mM MES, and 0.2 M KCl at pH values between 3 and 10. At
desired incubation times, the reaction was stopped by the addition of 2 µl
of 100 mM dithiothreitol. Reduced and carboxymethylated Grx5
proteins were separated by HPLC in a DeltaPak HPI C18 column (Waters
Associates, Milford, MA). Proteins were eluted by a linear gradient from 40 to
50% acetonitrile in 0.05% trifluoroacetic acid over 20 min at a flow rate of
0.2 ml/min. Proteins were detected and quantified from their corresponding
peak areas at 276 nm.
Reaction of Grx5 with GSSGPreparations (23 µl)
containing 25 µM of either wild type or mutant proteins in 100
mM Tris-HCl (pH 8.0) buffer were incubated with different
concentrations of GSSG at 20 °Cin sealed tubes under nitrogen. The
reaction was stopped by the addition of 2 µl of 10% trifluoroacetic acid.
The pH dependence of the rate of glutathiolation was assayed under the same
conditions, except that GSSG was always present at 250 µM, and
Tris-HCl buffer was replaced by a mixture containing 10 mM Tris, 10
mM potassium acetate, 10 mM MOPS, 10 mM MES,
and 0.2 M KCl at pH values between 3 and 10. Reaction products were
separated and quantified by HPLC as described above.
Preparation of Oxidized ProteinsThe fully oxidized form of
WTGrx5 (disulfide bond) was prepared by incubation of 1 mg of reduced protein
(150 µM concentration) with 0.5 mM GSSG for 30 min at
20 °C in Tris-HCl buffer, pH 8.0. Excess glutathione was removed by size
exclusion chromatography using a PD10 column (Amersham Biosciences) and
concentration of the protein using a Centricon 10K (Amicon). After this
treatment, HPLC analysis showed that 95% of the protein was in the oxidized
form (disulfide bond, see "Results"), and the remaining 5% was in
the reduced form. WT-Grx5 glutathiolated at Cys-60 was obtained after
incubation of 30 µg of reduced protein (90 µM concentration)
with 1 mM GSSG at pH 5.0. This preparation was separated by HPLC,
and the peak corresponding to protein glutathiolated at Cys-60 (see
"Results") was collected and dried in a Speed Vac.
Molecular Weight Determination of Modified Forms of
Grx5Peaks obtained by incubation of Grx5 with GSSG were separated
by HPLC as described above, collected, dried in a Speed Vac, and solubilized
in 20 µl of 0.1% trifluoroacetic acid. Proteins were mixed 1:1 with matrix
solution (saturated 3,5-dimethoxy-4-hydroxycinnamic acid in 33% aqueous
acetonitrile and 0.1% trifluoroacetic acid). A 0.7-µl aliquot of this
mixture was deposited onto a stainless steel MALDI probe and allowed to dry at
room temperature. Samples were measured on a Bruker Reflex IV MALDI-TOF mass
spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with the SCOUT
source in positive ion linear mode using delayed extraction (200 ns). A
nitrogen laser (337 nm) was employed for desorption/ionization, and the ion
acceleration voltage was 20 kV. The equipment was externally calibrated
employing protonated mass signals from horse cytochrome c.
Determination of the Redox PotentialThe redox potential of
Grx5 was determined by direct protein-protein redox equilibration
(25). Briefly, 300 µl of a
preparation containing 50 µM reduced S. cerevisiae Grx5
and 50 µM oxidized E. coli Grx1 (and vice versa) in 0.1
M sodium phosphate buffer, pH 7.0, plus 1 mM EDTA were
incubated in small plastic tubes attached to a manifold and purged with a
constant flux of moist nitrogen. At different times, 23 µl of the
preparation were removed through the septum of the tubes using a degassed
25-µl Hamilton gas-tight syringe and mixed with 2 µl of 10%
trifluoroacetic acid to stop the reaction. Protein mixtures were analyzed by
HPLC as described above, except that a linear gradient of 3050%
acetonitrile was used.
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RESULTS
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Expression and Purification of Grx5 ProteinsIn a previous
work we had shown that Grx5 isolated from yeast cells was a processed form
lacking the first 29 amino acids cleaved during import of the protein into
mitochondria (18). To obtain
enzymes resembling as much as possible the mature form of Grx5, all proteins
used in this study were prepared without their mitochondrial targeting
signals. It should be noted, however, that for a better comprehension and
comparison with previous articles, original sequence number positions have
been maintained throughout the text. The wild type and mutant Grx5 proteins
were overexpressed in E. coli cells and purified by a two-step method
that includes size exclusion and ionic exchange chromatography. The resulting
proteins were >99% pure and showed an apparent molecular mass of 15.5 kDa.
The same size was determined for the mature form present in yeast extracts
detected by Western blot (18).
The theoretical mass of the protein (13,478 Da) was used for the determination
of the molar concentration of the proteins. After purification, proteins were
obtained in reduced form, as indicated by cysteine titration with DTNB
(Table I).
Grx5 Is Not Active in the HED AssaySeveral reactions can be
catalyzed by dithiolic glutaredoxins. The most widely used form to asses
glutaredoxin activity is the glutathione:HED transhydrogenase assay. In this
assay, glutaredoxin catalyzes the reduction of a mixed disulfide between
glutathione and HED (2).
Dithiolic glutaredoxins lacking one of the two conserved cysteines are still
capable of catalyzing this reaction
(6). Glutaredoxin activity of
WT Grx5, C117S Grx5, and C60S Grx5 was assayed with the HED assay. No activity
could be detected even when a wide range of pH values (79.5) and GSH
concentrations (0.640 mM) were used. Additionally,
dehydroascorbate reductase and glutathione peroxidase activities, which have
been described for dithiolic glutaredoxins
(23,
24), were also tested. None of
the Grx5 variants showed detectable activity in these assays.
Determination of Cys-60 and Cys-117 pKa
ValueReactivity of thiol groups in proteins depends highly on
its pKa value. Active cysteines from dithiolic
glutaredoxins have pKa values close to 4
(9,
26,
27). To determine the
pKa value of both cysteine residues in Grx5, we
measured the rate of alkylation of Grx5 with iodoacetamide at different pHs.
This reaction occurs only when cysteines are in the ionized thiolate anion
state (28). Thus, reduced WT,
C117S, and C60S Grx5 proteins were incubated with 0.6 mM
iodoacetamide at pH values between 3 and 11. Reaction was stopped at different
times by the addition of 10 mM dithiothreitol, and samples were
analyzed by HPLC. The concentrations of reduced (Grx5red) and
carboxymethylated glutaredoxin (Grx5cmc) were calculated from the
peak area of the HPLC profile (shown in
Fig. 1A). Plots of
1/[Grxred] versus time yielded straight lines, indicating
that the reaction of iodoacetamide with Grx5 follows a second order reaction
with a single rate constant. The second order rate constants were calculated
according to Equation 1,
 | (Eq. 1) |
and plotted against pH (Fig.
1B) ([Grx0] = initial concentration of Grx5).
Reaction rates showed a sigmoidal dependence on pH value at pH values of
5 (WT and C117S) and 8 (WT and C60S). From these data it can be deduced
that the increases in reaction rates at low and high pH were respectively a
consequence of the ionization of Cys-60 and Cys-117. Using the
Henderson-Hasselbalch equation
(28,
29), thiol
pKa values of 5.0 ± 0.1 and 8.2 ±
0.1 were calculated for Cys-60 and Cys-117, respectively
(Table I).
Reaction of Grx5 with GSSGReactivity of reduced Grx5 with
GSSG was tested at pH 8.0 in 0.1 M Tris-HCl buffer, because
mitochondrial pH is close to this value
(30). WT Grx5 was incubated
with increasing concentrations of GSSG for 15 min at 20 °C, and the
products of the reaction were separated by HPLC.
Fig. 2A shows that
four new peaks corresponding to oxidized forms of the protein appeared. When
the mutant proteins were incubated with GSSG, only one new peak appeared
(Fig. 2, B and
C). The characterization of these peaks is summarized in
Table II. From mass
spectrometry data it can be deduced that two additional glutathione molecules
were present in peak 1 compared with peak 5 (reduced form), whereas only one
additional glutathione molecule was present in both peak 3 and peak 4. Peak 1
corresponded to a protein glutathiolated at both cysteines, whereas peak 2 was
a protein presenting a disulfide bond between both cysteines. This was deduced
from the following observations. (i) No free thiols were detected when the
peak 2 protein was incubated with DTNB. (ii) Peak 2 presented a mass of 13,484
Da (as the reduced form). (iii) Peak 2 was also the major peak obtained when
reduced Grx5 was incubated with several oxidants such as
H2O2, cystine, or oxidized proteins. (iv) Peak 2 was the
end product obtained either from peak 3 or peak 4 when these peaks were
collected, dried, and solubilized at pH 8.0; and (v) reduced Grx5 (peak 5) was
obtained by incubation of peak 2 with dithiothreitol. Concerning the
monoglutathiolated forms of the protein, our results indicate that peak 3
corresponded to a protein glutathiolated at Cys-60, whereas peak 4
corresponded to a protein glutathiolated at Cys-117. According to the
pKa values previously calculated for Cys-60 and
Cys-117, the pH dependence of the appearance of peaks 3 and 4 was consistent
with this assumption (Fig.
3A). This was confirmed by analyzing the rate of
glutathiolation of C117S and C60S mutant proteins at different pHs
(Fig. 3B).
One interesting result was the observation that the rates of
carboxymethylation and glutathiolation did not follow the same pH dependence
in the WT protein, although in mutant proteins they were nearly the same
(compare Figs. 1 and
3). Thus, introduction of the
first glutathione in the WT protein may increase the reactivity of the
Cys-117. This suggested the idea that one glutathione molecule could be
transferred from Cys-60 to Cys-117 in the wild type protein during the
reaction, and glutathiolation of Cys-117 would be an intermediate step before
formation of the disulfide bond. To test this hypothesis peak 4 was collected,
dried, rehydrated with Tris-HCl buffer at pH 8.0, and incubated at 4 °C.
Fig. 4 shows the percentage of
each peak found at different incubation times as determined by HPLC analysis.
It can be observed that peak 3 appeared mainly at short incubation times, as
would be expected for an intermediary product of the transformation of peak 4
to peak 2.
Finally, it should be noted that the presence of both cysteines resulted in
a higher reactivity of the protein toward GSSG. When WT Grx5 and mutant
proteins were incubated for 15 min at 20 °C with increasing GSSG
concentrations at pH 8.0, oxidation of half of the WT protein required 99
µM GSSG, whereas oxidation of half of the C117S and C60S mutant
proteins required GSSG concentrations of 206 µM and 1.34
mM, respectively (Fig.
2D). These results reinforced the idea that interaction
between both cysteines occurred and that the presence of Cys-117 enhanced Grx5
reactivity. Nevertheless reactivity of Cys-117 alone (in the C60S protein) was
very poor.
Reduction of Grx5 by GSHOn the basis of their results with
Plasmodium falciparum GLP1 monothiolic glutaredoxin, Rahlfts et
al. (31) suggested that
GSH was unable to reduce monothiolic glutaredoxins. This fact would explain
the absence of activity of both Grx5 and GLP1 in the HED assay. From the above
results it was clear that GSSG strongly reacted with Grx5, promoting the
formation of a disulfide bond between both cysteines of the protein. To study
whether this disulfide bond could be reduced by GSH, oxidized Grx5 was
prepared by incubating 1 mg of protein with 0.5 mM GSSG for 30 min
as described under "Experimental Procedures." This preparation was
incubated with increasing amounts of GSH for 15 min at 20 °C. Reduction of
half of the protein required 1.4 mM GSH
(Fig. 5A). In
addition, we compared the rate of reduction of the oxidized Grx5 with that of
Grx1 from E. coli, a dithiolic glutaredoxin active on the HED assay.
Both proteins were incubated at fixed concentrations of 1 and 2 mM
GSH for different times. Fig.
5B shows that, even at the shorter incubation times (30
s), the reaction of Grx1 with GSH reached equilibrium. On the other hand, the
reaction of Grx5 with GSH required 1 h to reach equilibrium. Thus, the
reduction rate of Grx5 was at least 20 times slower than that of Grx1. These
results indicated that reduction of Grx5 by GSH can be a limiting step for its
thiol reductase activity. The absence of detectable HED activity in
monothiolic glutaredoxins may thus be related to the inefficient reduction of
these proteins.
Determination of the Redox Potential of Grx5The redox
potential of Grx5 was determined by direct protein-protein equilibration with
E. coli Grx1 (25).
Reduced Grx5 and oxidized Grx1 were incubated at 25 °C under anaerobic
conditions. HPLC separation and quantification of the four protein species was
performed after incubation for 1, 2, 4, 8, and 12 h
(Fig. 6). The redox equilibrium
was obtained after 4 h of incubation as indicated by a stable ratio of the
four protein species. The same results were obtained when oxidized Grx1 and
reduced Grx5 were used as the starting material. As shown in
Equation 2,
 | (Eq. 2) |
the standard state redox potential of Grx5 at
25°C(E°'Grx5) was calculated from a Nernst
equation. The standard redox potential of E. coli Grx1 is 233
mV (25). Analysis of different
mixtures of oxidized and reduced Grx5 and Grx1 resulted in an standard redox
potential of 175 ± 3 mV for Grx5. This result placed Grx5 in an
intermediate position among thiol-disulfide oxidoreductases. Members of this
family show very diverse redox potentials that range from the oxidizing
124 mV of E. coli DsbA to the strong reducing 270 mV of
E. coli thioredoxin
(25).

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FIG. 6. HPLC profile of the separation of the reduced (red) and
oxidized (ox) S. cerevisiae Grx5 and E. coli
Grx1. Reduced Grx5 and oxidized Grx1 were incubated for 4 h at 25 °C
in 200 µl of 100 mM sodium phosphate, pH 7.0, plus 1
mM EDTA. The proteins were separated by HPLC as described under
"Experimental Procedures."
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Reduction of Mixed Disulfides in Proteins by Grx5To test
whether Grx5 could participate in the deglutathiolation of cysteine residues,
rat carbonic anhydrase III was used as a substrate. This protein contains five
cysteines. Two of them, Cys-186 and Cys-181, can be easily glutathiolated
in vitro when purified protein is incubated with GSSG
(32). Glutathiolation has also
been described to occur in vivo
(33,
34). A mutant carbonic
anhydrase with cysteine 181 substituted for serine was used for this study.
When this mutant protein is incubated with GSSG, only cysteine 186 becomes
glutathiolated.2
Reduced and monoglutathiolated carbonic anhydrase were easily separated by
HPLC. Furthermore, these forms of carbonic anhydrase did not interfere in the
chromatographic separation with any of the forms of Grx5. The glutathiolated
form of C181S carbonic anhydrase was prepared by incubating the purified
protein with 250 µM GSSG for 3 h at 37 °C. Excess
glutathione was removed by extensive dialysis against 50 mM
Tris-HCl buffer, pH 7.5. Equimolar amounts of reduced WT Grx5 and
glutathiolated carbonic anhydrase were incubated at 20 °C and separated by
HPLC in a C18 column (Fig.
7A). As shown in Fig.
7B carbonic anhydrase was deglutathiolated in a
time-dependent manner. Grx5 was converted to the complete oxidized form
(disulfide bond). The same experiment was performed with C117S and C60S Grx5
proteins. Both mutants were almost unable to reduce carbonic anhydrase.
 |
DISCUSSION
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Grx3, 4 and 5 from S. cerevisiae were the first members of a new
family of proteins with glutaredoxin signature to be described. These proteins
contain one conserved cysteine residue at the putative active site
(12), and they have been found
in all types of organisms from bacteria to humans
(13). Very few of them have
been studied, and only two of them have an assigned function. The human PICOT
protein has been proposed to be a modulator of the protein kinase C-
pathway (35). We have recently
shown that Grx5 from yeast is located in the mitochondria and involved in the
maturation of Fe-S cluster-containing proteins
(18). The glutaredoxin-like
protein GLP1 from P. falciparum has also been cloned and purified but
has no specific assigned role
(31).
Despite these observations, there was no consistent biochemical data
supporting the involvement of monothiolic glutaredoxins in thiol redox
reactions and, consequently, no mechanism of action had been proposed for the
members of this family. Bushweller et al.
(6) reported that mutant
dithiolic glutaredoxins lacking the second conserved cysteine residue were
still able to catalyze the reduction of the HED-GSH mixed disulfide. The
mechanism proposed for this reaction (summarized in
Fig. 8A) involved the
formation of a mixed disulfide between glutathione and the cysteine located at
the active site. This mixed disulfide could be cleft by GSH, yielding reduced
glutaredoxin and GSSG. It has been suggested that monothiolic glutaredoxins
could follow this same scheme
(3). However, this was a
controversial issue. First, Rahlfs et al.
(31) purified and partially
characterized PfGLP1 from P. falciparum and concluded that it could
not be reduced by GSH. However, this was probably because PfGLP1 was already
reduced after purification, as occurs with Grx5. Second, neither Grx5 nor
PfGLP1 are active in the HED assay, although dithiolic glutaredoxins lacking
the C-terminal cysteine are still active in this assay
(31,
19).

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FIG. 8. Proposed catalytic mechanism of action of Grx5. A,
mechanism of action of mutant dithiolic glutaredoxins lacking the C-terminal
cysteine in the active site, as proposed by Bushweller et al.
(6). B, proposed
mechanism of reaction of Grx5 with glutathiolated proteins. C,
hypothetic mechanism of action for the reduction of protein disulfide bonds by
Grx5 based on the mechanism of action of dithiolic glutaredoxins (described in
Ref. 6).
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The results from this work demonstrate that Grx5 is a thiol reductase that
can participate in thiol redox reactions. Several pieces of evidence support
this idea. First, Cys-60 presents a low pKa,
close to the pKa values of reactive cysteines in
dithiolic glutaredoxins (9,
26,
27); second, Grx5 has the
potential to form a mixed disulfide with glutathione with high affinity; and
finally, Grx5 has the ability to reduce a glutathiolated protein such as
carbonic anhydrase, indicating that its redox potential is low enough to act
as an electron donor in redox reactions involving oxidized proteins. We
propose a mechanism of action for the reduction of mixed disulfides by Grx5
based on the reaction of Grx5 with GSSG (summarized in
Fig. 8B). First, a
mixed disulfide will be formed between Cys-60 and glutathione. This would
induce a decrease in the Cys-117 pKa value that
will trigger the formation of a disulfide bond between both cysteines and
yield reduced glutathione. However, it is not clear how Grx5 may be reduced
in vivo, because the reduction rate of Grx5 by GSH may not be fast
enough to allow the efficient reduction of oxidized Grx5. Thus, involvement of
other mitochondrial reducing agent(s) in this last step should be considered
in further investigations. In this context it is interesting to note that
E. coli thioredoxin efficiently reduces Grx5 (data not shown).
Finally, it should be noted that the absence of activity of Grx5 in the HED
assay may be a consequence of its inefficient reduction by GSH but also of its
redox potential, which would not be low enough to efficiently reduce the mixed
HEDGSH disulfide. The redox potential of Grx5 (175mV) is higher than
that of dithiolic glutaredoxins, which range from 198 to 233 mV
(25). However, it can be low
enough to reduce other disulfide bonds, as indicated by our results with
glutathiolated carbonic anhydrase.
Another important conclusion derived from this work is the relevance of
Cys-117 for Grx5 reactivity and the formation of a disulfide bond between both
cysteine residues in the polypeptide chain. The influence of this cysteine
residue on Grx5 reactivity is clearly observed in the experiments with
carbonic anhydrase. The involvement of a second cysteine in the mechanism of
action would allow Grx5 to perform the reduction of disulfide bonds in
proteins, increasing the number of potential substrates. A hypothetic
mechanism for this reaction, based on the mechanism of action of dithiolic
glutaredoxins, is presented in Fig.
8C. Another interesting point arises from the observation
that Cys-117 is only conserved in about half of the monothiolic glutaredoxins
identified so far. As a consequence, monothiolic glutaredoxins should be
separated in two different classes, depending on the presence of this second
cysteine residue. The relevance of this non-conserved residue is a nice
example of how a single mutation can modulate the reactivity of a polypeptide
chain and allow a member of a family of proteins to develop new specific
functions in cell metabolism. The relevance of Cys-117 was not identified in a
previous work (13) in which
the functional complementation of Grx5 by several mutant forms of the protein
(including the C117S variant) expressed in
grx5 yeast cells
was investigated. Although it is less efficient than WT Grx5, the C117S
protein may display enough activity to suppress the severe growth defects
found in a
grx5 strain by the monothiolic mechanism described
in Fig. 8A.
Genetic and biochemical results obtained with yeast cells depleted in Grx5
have linked this protein to the process of iron-sulfur assembly
(18). Now it is clear that its
role may be related to its thiol reductase activity. However, its
physiological substrate remains unknown. Several steps in the process of Fe-S
assembly may require the presence of a thiol reductase. Recent works in this
field indicate that the bacterial proteins IscU and IscA (homologous to Isa
and Isu proteins in yeast) serve as scaffolds for the assembly of iron-sulfur
clusters (36,
37). The first step in this
process is a sulfur transfer from the cysteine desulfurase IscS (NifS in
yeast) to IscU or IscA (38,
39). Later, iron is
incorporated, and a transient [2Fe2S] center is formed in IscA/U proteins.
Although the exact mechanism is still controversial, it seems clear that
reducing the equivalents required for this process would be provided by the
formation of a disulfide bond between two cysteines in IscA/U and/or IscS
proteins (40,
41). Grx5 would be required
for the reduction of these cysteine residues and constitute an essential
enzyme for the turnover of the whole process. Another possibility may be that
Grx5 would act as a general mitochondrial thiol reductase. Its absence would
affect the assembly of iron-sulfur centers more dramatically than any other
biological process. However, it is important to note that Grx5 is not the most
abundant thiol reductase in mitochondria, wherein the presence of thioredoxin
3 and Grx2 have also been described
(11,
42). Thus, a specific role for
Grx5 seems quite possible. Further research will determine whether this
specificity is a consequence of Grx5 redox potential or the recognition by
Grx5 of specific regions in target proteins.
 |
FOOTNOTES
|
---|
* This work was funded by Generalitat de Catalunya Grants 2000 SGR 0042 (to
J. R.) and 2001 SGR 00305 (to E. H.) and Ministerio de Ciencia y
Tecnología (Spain) Grants BMC2001-0874 (to J. R.), and
BMC2001-1213-C02-01 (to E. H.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 34-973-702407; Fax:
34-973-702426; E-mail:
joaquim.ros{at}cmb.udl.es.
1 The abbreviations used are: Grx, glutaredoxin; DTNB,
5,5'-dithiobis-2-nitrobenzoic acid; HED,
-hydroxyethyl disulfide;
MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid;
WT, wild type; HPLC, High performance liquid chromatography; MALDI-TOF,
matrix-assisted laser desorption/ionization-time of flight. 
2 R. L. Levine, personal communication. 
 |
ACKNOWLEDGMENTS
|
---|
We appreciate the assistance of Vanessa Guijarro throughout this work. We
thank the proteomics facility at Centro Nacional de Biotecnología,
Madrid, Spain, for assistance in molecular mass determination. We also thank
Silvia Esteve for editorial assistance.
 |
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