Purification and Characterization of a Chimeric Enzyme from
Haemophilus influenzae Rd That Exhibits
Glutathione-dependent Peroxidase Activity*
Frederik
Pauwels,
Bjorn
Vergauwen,
Frank
Vanrobaeys,
Bart
Devreese, and
Jozef J.
Van
Beeumen
From the Laboratory of Protein Biochemistry and Protein
Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium
Received for publication, January 7, 2003, and in revised form, February 17, 2003
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ABSTRACT |
While belonging to the same family of antioxidant
enzymes, members of the peroxiredoxins do not necessarily employ one
and the same method for their reduction. Most representatives become reduced with the aid of thioredoxin, whereas some members use AhpF,
tryparedoxin, or cyclophilin A. Recent research on a new peroxiredoxin
isoform (type C) from Populus trichocarpa has shown that
these particular types may also use glutaredoxin instead of
thioredoxin. This finding is supported by the occurrence of chimeric
proteins composed of a peroxiredoxin and glutaredoxin region. A gene
encoding such a fusion protein is enclosed in the Haemophilus
influenzae Rd genome. We expressed the H. influenzae protein, denoted here as PGdx, in Escherichia coli and
purified the recombinant enzyme. In vitro assays
demonstrate that PGdx, in the presence of dithiothreitol or
glutathione, is able to protect supercoiled DNA against the metal
ion-catalyzed oxidation-system. Enzymatic assays did, indeed,
characterize PGdx as a peroxidase, requiring the glutathione redox
cycle for the reduction of hydrogen peroxide
(kcat/Km 5.01 × 106 s
1 M
1) as well
as the small organic hydroperoxide tert-butylhydroperoxide (kcat/Km 5.67 × 104 s
1 M
1).
Enzymatic activity as function of the glutathione concentration deviated from normal Michaelis-Menten kinetics, giving a sigmoidal pattern with an apparent Hill coefficient of 2.9. Besides the formation
of a disulfide-linked PGdx dimer, it was also shown by mass
spectrometric analysis that cysteine 49, which is equivalent to the
active site cysteine of the peroxiredoxins, undergoes glutathionylation during purification under nonreducing conditions. Based on these results, we propose a model for the catalytic mechanism.
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INTRODUCTION |
Aerobic organisms intrinsically encounter reactive oxygen
species, such as hydrogen peroxide
(H2O2), the superoxide anion radical
(O
), and the hydroxyl radical (OH·), during some stage
of the four-electron reduction of O2 to water or following
exposure to environmental factors (1, 2). The unrestrained accumulation
of these species gives rise to oxidative stress and can lead to cell
damage, mutations, or even death. One issue of the reactive oxygen
species detoxification concerns the decomposition of hydroperoxides.
Most pro- and eukaryotic cells rely on the action of heme-containing
enzymes called catalases, which disproportionate
H2O2 to water and O2. Some
eukaryotic cells may also use glutathione peroxidases to remove
H2O2 as well as organic and lipid
hydroperoxides. In addition, research over the past decades has led to
the characterization of a new family of peroxidases, collectively
called "peroxiredoxins"
(Prxs)1 (3). They decompose
organic hydroperoxides and H2O2 by means of
thiol-containing electron donors such as thioredoxin (Trx), AhpF,
cyclophilin A, tryparedoxin, or, as recently reported, also the redox
protein glutaredoxin (Grx) (4-8).
Haemophilus influenzae is an important, opportunistic,
Gram-negative human pathogen. The bacterium resides in the upper
respiratory tract of humans, where it generally grows aerobically,
although facultative anaerobic growth is also possible (9). Besides oxidative stress from its aerobic respiratory metabolism or as a result
of the high O2 tension at the nasopharynx, H. influenzae may also be exposed to high levels of oxidants produced
by the host's immune system, which uses the destructive power of
reactive oxygen species to eliminate bacterial infections (10).
Moreover, experimental data indicate that H. influenzae has
to deal with H2O2 secreted by peroxidogenic
Streptococci (11). Whereas the existence of an
H2O2-inducible catalase (HktE) has been
described in H. influenzae Rd, the enzyme seems to be
redundant (12, 13). As yet, no other antioxidant enzyme has been
identified that acts against hydroperoxides, making the ways in which
the bacterium deals with hydroperoxide stress an interesting topic for
future research.
Previously, we described a glutathione amide-dependent
peroxidase from the phototrophic purple sulfur bacterium
Chromatium gracile, capable of reducing both
H2O2 and tert-butylhydroperoxide (t-BOOH) at comparable high rates (14). By means of a BLAST search using its deduced amino acid sequence, we were able to identify
several homologs in different bacterial species, including one encoded
by an open reading frame (HI0572) enclosed in the H. influenzae Rd genome. The comparisons revealed the fusion of an
N-terminal Prx region to a C-terminal Grx region, a unique feature
typical for this novel family of homologs. This structure suggests that
a thioltransferase reaction by the Grx moiety may be involved in the
reduction of the Prx moiety (8, 15, 16). Grxs are small, ubiquitous
thioltransferases that are specifically designed to use GSH for their
reduction (17, 18). They catalyze the reduction of protein disulfide
groups and GSH-containing mixed disulfide groups either via a dithiol
or monothiol mechanism (18).
In this paper, we expand our knowledge of the chimeric enzyme from
H. influenzae Rd (aptly named PGdx, as in
Peroxiredoxin/Glutaredoxin) by demonstrating its abilities to protect supercoiled DNA from oxidative damage and to catalyze the in vitro reduction of
H2O2 and t-BOOH using GSH as
electron donor. Furthermore, we noticed the formation of a homodimer
and a glutathionylated monomer during purification under nonreducing
conditions. Surprisingly, kinetic studies of PGdx revealed sigmoidal
kinetics with respect to GSH, normally attributed to the phenomenon of cooperativity.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction endonucleases were obtained from New
England Biolabs (Beverly, MA). DNA purification from gel or solution
was carried out using either the Qiaquick DNA Extraction or PCR
Purification Kit (Qiagen, Crawley, UK). Ligations were performed using
Ready-To-Go T4 DNA ligase (Amersham Biosciences). Plasmid
DNA was prepared by the alkaline lysis method on either a small scale
(19) or a 30-ml scale using the Qiagen plasmid purification kit.
Chromatographic protein purification steps were performed on an
ÄKTA-design fast protein liquid chromatography system (Amersham
Biosciences) with chromatographic equipment from the same manufacturer
or with materials from Bio-Rad. Spectrophotometric measurements were
taken using a Uvikon 943 double beam UV-visible spectrophotometer
(Kontron Instruments, Watford, UK). Reduced GSH,
H2O2, t-BOOH, DTT, ascorbate, and
NADPH were obtained from Sigma. GSH reductase (GR; type IV from
bakers' yeast) was from Fluka (Glossop, UK). Escherichia coli Trx and Trx reductase (TR) were from Sigma.
Bacterial Strains, Media, and Growth Conditions--
H.
influenzae Rd (KW20) was obtained from ATCC (Manassas, VA; catalog
no. 51907). E. coli MC1061 and E. coli B834(DE3)
were used as host for cloning and expression of PGdx, respectively. All
E. coli strains were cultured at 37 °C in Luria-Bertani
(LB) medium on an orbital shaker rotating at 200 rpm. H. influenzae Rd was grown at 37 °C under a 3% CO2
atmosphere (candle extinction jar method) on an orbital shaker rotating
at 100 rpm. H. influenzae Rd medium consisted of brain heart
infusion liquid (Difco) supplemented with
-NAD and hemin (Fluka,
Glossop, UK). Solid media for all strains were prepared by adding agar
to the liquid media to a final concentration of 1.8%. When
appropriate, 100 µg of carbenicillin/ml was added to the E. coli culture medium.
Cloning of H. influenzae PGdx--
The gene HI0572
(GenBankTM accession number AAC22230) was amplified by PCR
from H. influenzae Rd genomic DNA (prepared as described
elsewhere) (20) using Gold Star DNA polymerase (Eurogentec, Seraing,
Belgium) and the following primers: forward primer (5'-TC CAT
ATG TCT AGT ATG GAA GG-3') containing an
NdeI (underlined) site and the initiation codon (boldface) and reverse primer (5'-CGC GGA TCC TTA TGC AAA
GTA T-3') containing a BamHI site (underlined) and the stop
codon (boldface). The PCR product obtained was purified and subcloned
into a pGEM-T vector (Promega, Madison, WI) prior to digestion with
NdeI/BamHI. The digested fragment was cloned into
an NdeI/BamHI-digested pET-11a expression vector
(Novagen, Madison, WI).
Expression and Purification of Recombinant PGdx--
An E. coli B834(DE3) expression strain was transformed with the
expression construct, cultured overnight in 100 ml of LB medium supplemented with carbenicillin, and then transferred to fresh medium
up to a ratio of 1:100. Cells were grown to an optical density at 600 nm of 0.8, after which
isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM. After induction for 4 h,
cells were harvested by centrifugation, resuspended in 10 mM Tris-HCl buffer, pH 7.5, containing the Complete
Protease Inhibitor mix (Roche Applied Science), and stored at
80 °C.
Frozen cells were thawed and then disrupted by sonication. The
supernatant was cleared by centrifugation at 14,000 rpm for 30 min.
Clear supernatant was loaded onto a Q-Sepharose Fast Flow column
(10 × 200 mm) equilibrated with 10 mM Tris-HCl, pH
7.8. Proteins were eluted with a continuous gradient of 0-1
M NaCl, at a flow rate of 3 ml/min. Fractions containing
the protein were pooled and dialyzed against 50 mM
phosphate buffer, pH 6.8. The sample was applied to a CHT-2 ceramic
hydroxyapatite column from Bio-Rad equilibrated with 50 mM
sodium phosphate buffer, pH 6.8. The column was washed with
equilibration buffer before eluting the protein with increasing
concentrations of phosphate, pH 6.8. Protein fractions were pooled and
concentrated using a Millipore Corp. (Bedford, MA) concentrator. For
the ultimate purification step, we used a MonoQ column. Purified PGdx
was stored at
80 °C until further use. Aliquots of the peak
fractions were analyzed using SDS-PAGE (15%) performed as described by
Laemmli (21). Protein concentration was measured in 20 mM
phosphate buffer (pH 6.5)/6 M guanidine hydrochloride on
basis of the molar extinction coefficient computed from the amino acid
composition using software at ExPASy (available on the World Wide Web
at www.expasy.org;
280 = 23,470 M
1 cm
1).
Mass Spectrometric Analysis of PGdx and PGdx Peptides--
Mass
determinations were performed on a hybrid quadrupole-time of flight
mass spectrometer (Micromass, Manchester, UK), equipped with a
nanoelectrospray source. For precise measurements, the samples were
first desalted by ultrafiltration on a Millipore 0.5-ml concentrator
(Bedford, MA) with a molecular mass cut-off of 5 kDa. The sample was
then dissolved and diluted in 50% acetonitrile, 0.1% formic acid to a
final concentration of ~2 pmol/µl.
Tryptic digestion of PGdx was carried out by first dissolving 1 µl of
the purified PGdx (10 µg/µl) in 50 mM ammonium
bicarbonate (pH 7.8) and then adding trypsin up to an enzyme/PGdx ratio
of 1:50 (w/w). After 4 h of incubation (37 °C), 1 µl of the
digested protein was diluted in 20 µl of 50% acetonitrile, 0.1%
formic acid and analyzed by quadrupole-time of flight mass spectrometry.
DNA Supercoiling Assay--
The ability of PGdx to protect
supercoiled DNA from oxidative degradation was assayed with either a
DTT or GSH metal ion-catalyzed oxidation system (DTT or
GSH/Fe3+/O2; thiol MCO system) or an ascorbate
metal ion-catalyzed oxidation system
(ascorbate/Fe3+/O2; nonthiol MCO system).
Assays were done in a 20-µl reaction mixture containing 50 mM HEPES-NaOH, pH 7.3, 3 µM freshly prepared FeCl3, 10 µM PGdx (except for the control,
where PGdx was replaced with 10 mM EDTA), and 10 mM DTT, GSH, or ascorbate. The reactions were incubated for
40 min before adding ~1 µg of plasmid BlueScript DNA. After
incubation for 30 min at 37 °C, samples were analyzed on a 1%
agarose gel stained with ethidium bromide.
Enzyme Assays--
GSH/GR/NADPH-dependent peroxidase
activity of PGdx was determined using a continuous assay. Assays were
performed at 25 °C in a 0.1 M sodium/potassium phosphate
buffer, pH 7.8, with 0.15 mM NADPH, 0.1 mM
EDTA, and the following products each in turn added as a last
component, with the others already having been added: 50 nM
PGdx, 10 mM GSH, 100 µM hydroperoxide, and 3 units of GR. Trx/TR/NADPH-dependent peroxidase activity was
assayed by adding 100 µM hydroperoxide to a reaction
mixture containing 50 nM PGdx, 8.5 µM Trx,
and 2 µM TR in 0.1 M sodium/potassium phosphate buffer, pH 7.8, with 0.15 mM NADPH and 0.1 mM EDTA. Final reaction volumes were 500 µl each. The
decrease in NADPH absorbance was continuously monitored at 340 nm.
Velocity versus substrate curves for GSH,
H2O2, and t-BOOH were determined
using a coupled assay consisting of 0.15 mM NADPH, 3 units
of GR, and 50 nM PGdx in 0.1 mM
sodium/potassium phosphate buffer, pH 7.1, with 0.1 mM
EDTA. Concentrations of GSH were varied while keeping those of
t-BOOH or H2O2 constant, and
vice versa. Hydroperoxides were added last. Peroxidase
activity was monitored at 340 nm against a blank containing no enzyme
and expressed as µM NADPH oxidized per min using the
molar extinction coefficient of 6,200 M
1
cm
1 for NADPH. All measurements were done in triplicate,
and the mean and S.E. were calculated. Data were analyzed by fitting
them to the Michaelis-Menten equation (v = Vmax[S]/Km + [S]), the
Hill equation (v = Vmax[S]n/K' + [S]n), or the Eadie-Scatchard equation
(v/[S]n =
(1/K')v + (Vmax/K')) using the nonlinear least
squares method.
 |
RESULTS |
Similarity of PGdx to Type C Prx and Grx3--
A BLAST search with
the deduced amino acid sequence of the C. gracile Prx/Grx
protein (14) identified the highly homologous open reading frame HI0572
(63% identity, 79% similarity) in the H. influenzae Rd
genome sequence data base (available on the World Wide Web at
www.tigr.org/) (22). This open reading frame, annotated as
"hypothetical protein" and/or "membrane protein," based on its
hydrophobic character, encodes a 241-amino acid polypeptide chain with
a theoretical mass of 26,742.5-Da and, in particular, three cysteine
residues located at positions 49, 180, and 183.
Alignments show the N-terminal region of Haemophilus PGdx
(amino acids 1-160) to share 30-40% identity and 50-60% similarity with members of the Prx family, particularly with members of type C. The term "type C Prx" is a recently adopted nomenclature by Rouhier
et al. (8). It includes those Prxs that are characterized by
their short length (averaging 160 amino acids), limited sequence similarity with type A (2-Cys) and B (1-Cys) Prxs (except for the
conserved sequence surrounding the strictly conserved N-terminal cysteines), and an extra cysteine residue located some 24 amino acids
further down the N-terminal cysteine. Whereas this extra cysteine is
lacking in PGdx, a situation also shared by the Oryza and
Actinobacillus homolog, identities of the N-terminal region are still higher with type C than with type A or B Prxs. Those Prxs
that show the highest identity with the Prx region of PGdx are depicted
in Fig. 1.

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Fig. 1.
Multiple sequence alignment of H. influenzae Rd PGdx with prokaryotic homologs and members of
the Prx and Grx family. The alignment was performed using ClustalX
software. Strictly conserved residues are marked with an
asterisk, functional homology with a colon, and
structural homology with dot. Symbols
above the PGdx sequence represent conserved positions for
the Prx and Grx alignment; symbols below the PGdx
sequence represent conserved positions for the alignment of the PGdx
homologs. Redox-active cysteines are shaded gray;
gaps are represented with a dash. Accession numbers or
references for the sequences aligned are as follows. Brara,
Brassica rapa, AF133302; Poptr1, P. trichocarpa Prx (9); Orysa, Oryza sativa,
AF203879; Homsa, Homo sapiens putative
peroxisomal antioxidant enzyme, P30044; Escco, E. coli glutaredoxin 3, P37687; Poptr2, P. trichocarpa Grx, AI166603; Haein, H. influenzae PGdx, AAC22230; Chrgr, C. gracile
(14); Borpe, Bordetella pertussis,
NC_002929; Actac, Actinobacillus
actinomycetemcomitans, NC_002924; Yerpe, Yersinia
pestis, CAC93382; Vibch, Vibrio cholerae,
AAF95778; Haedu, Haemophilus ducreyi, NC_002940;
Pasmu, Pasteurella multocida, NP_246286;
Neime, Neisseria meningitidis, NP_273984.
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Another distinguishable fact is the apparent ability of type C Prxs to
use Grx instead of Trx as electron donor (8). This especially merits
mention, since the C-terminal region of PGdx shares strong homology
(52% identity, 69% similarity) with Grx3 from E. coli.
This Grx-homologous domain contains cysteine residues 180 and 183, arranged in a characteristic CPFC disulfide motif, and is coupled to
the Prx region via a Gln-rich stretch starting with a Pro. Depicted in
Fig. 1 are the sequences of E. coli Grx3 and Populus
trichocarpa Grx. The latter reduces the poplar type C Prx, which
is also included in Fig. 1.
BLAST searches of the H. influenzae PGdx against the
Microbial Genomes data base revealed numerous as yet uncharacterized homologs, the majority of them in microorganisms implicated in human
disease (Fig. 1). Similarity extends over the entire sequence, especially in the Prx region, and ranges from 60 to 95% identity and
from 75 to 100% similarity.
Expression, Purification, and Physical Characteristics of
PGdx--
Recombinant PGdx, of which the expression level in the
E. coli cytosol was considerably high, was purified to
homogeneity as described under "Experimental Procedures." About 30 mg of pure PGdx was obtained from 1-liter cell culture. Apart from a
protein band at monomeric migration distance, nonreducing SDS-PAGE also clearly visualized a second band migrating as a homodimer (Fig. 2). This band disappeared with the
addition of
-mercaptoethanol, DTT, or GSH to the sample buffer but
did not with ascorbate, suggesting the possibility of two monomers
being linked by a disulfide bridge. Electrospray ionization mass spectrometry
measurements confirmed the existence of a completely oxidized dimer at
53,216.2 Da that disappeared when 10 mM GSH was added
to the sample solutions (Table I, Fig.
3).

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Fig. 2.
SDS-PAGE analysis of purified
recombinant PGdx from H. influenzae Rd.
Lane 1, Coomassie-stained 15% polyacrylamide gel
with purified recombinant protein in nonreducing sample buffer;
lane 2, PGdx with 5 mM ascorbate;
lane 3, PGdx with 5 mM GSH;
lane 4, PGdx with 5 mM DTT;
lane 5, molecular mass marker (protein ladder,
10-200 kDa; MBI Fermentas). Lanes 1-4 contain
about 3 µg of protein.
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Table I
Mass spectrometric analysis of purified PGdx
Predicted masses are those for PGdx lacking the N-terminal Met (NM;
26,742.5 131.2 Da), as confirmed by NH2-terminal amino acid
sequencing (data not shown).
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Fig. 3.
Mass spectrometric analysis of the monomeric
and dimeric form of PGdx. Electrospray ionization mass
spectrometry spectra of the PGdx protein under nonreducing conditions
(upper trace) and after reduction with 10 mM GSH (lower trace). The relative
intensities of the peaks are shown against the mass.
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When the nonreduced monomeric form was analyzed by electrospray
ionization mass spectrometry, we observed a peak with a mass of
26,915.1 Da, which is 305 Da higher than expected (Table I, Fig. 3).
This prompted us to investigate the possibility whether the enzyme was
modified by glutathionylation during the overexpression and subsequent
isolation from E. coli. The addition of GSH, indeed, did
reduce the mass to that of the fully reduced monomer (Table I, Fig. 3),
a phenomenon that was not observed when ascorbate was added (not
shown). In order to determine the location of the GSH moiety in the
peptide chain, we performed a tryptic digest and analyzed the digest
mixture of the reduced and nonreduced condition by mass spectrometry
(spectra not shown). The mass spectrum of the reduced PGdx digest
mixture contained a peak at 23,16.2 Da, corresponding to the
monoisotopic mass of the unmodified peptide Thr35-Arg56. Under nonreducing
conditions, this peak was absent and replaced by a peak at 2,621.2 Da,
which agrees with the monoisotopic mass of the modified peptide
Thr35-Arg56. To confirm this
observation and the location of the modification, the peptide was
subjected to collision-induced fragmentation mass spectrometry. As
shown in Fig. 4, the MS/MS spectrum was
found to be consistent with the sequence of the peptide. Fig. 4 also shows an increment in mass of 305 Da after Cys49 (note the
shift of the y" ions), which points to the GSH molecule being linked to
the Cys49 residue.

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Fig. 4.
MS/MS spectrometric analysis of the tryptic
fragment Thr35-Arg56 of
nonreduced PGdx. The peptide has a theoretical molecular mass of
2,621.2 Da. Note the shift of 305 Da due to a molecule of GSH
covalently linked to the cysteine residue at position 15 (position 49 of the protein).
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PGdx Protects Supercoiled DNA from Oxidative
Damage--
Supercoiled DNA is prone to nicking when exposed to
oxidative radicals such as those generated by the MCO system.
Therefore, PGdx was tested for its ability to protect supercoiled DNA
from degradation induced by the MCO system in the presence of DTT, GSH,
or ascorbate (Fig. 5). The absence of
PGdx resulted in open coiled or nicked DNA, whereas the addition of 10 mM EDTA completely inhibited degradation. PGdx, in
combination with DTT or GSH, was successful in protecting the DNA. When
DTT or GSH was replaced by ascorbate as electron donor, the enzyme was
unable to protect the DNA at a concentration that was sufficient to
provide full protection against degradation when a thiol was
present.

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Fig. 5.
Protection of supercoiled DNA against
oxidative degradation by PGdx. Experiments were performed as
described under "Experimental Procedures" and subsequently analyzed
on a 1% agarose gel stained with ethidium bromide. OC, open
coiled DNA; SC, supercoiled DNA. Open coiled DNA forms are
more distinctively present in the absence of PGdx or in the presence of
PGdx and the nonthiol MCO system. In the case of the DTT-MCO system
(lane 2), one can even observe a DNA smear.
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The GSH/GR/NADPH System Provides Electrons for PGdx-catalyzed
Hydroperoxide Reduction--
We set up a reconstitution assay by which
we demonstrated that PGdx can use the GSH/GR/NADPH system and that the
reduction of hydroperoxides depends on the presence of each component.
The high activity observed after the addition of GR (Fig.
6A) is due to the accumulation
of its substrate, GSSG. In contrast, no discernible peroxidase activity
was observed when GSH and GR were replaced by Trx and TR. For both
H2O2 (not shown) and t-BOOH (Fig. 6,
E and F), the background activity coincided with
the decrease in absorbance when PGdx was present.

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Fig. 6.
Continuous assay evaluating the
GSH/GR/NADPH- or Trx/TR/NADPH-dependent PGdx activity.
NADPH oxidation is coupled by GSH/GR or Trx/TR to the PGdx-mediated
reduction of t-BOOH. Reactions were performed as described
under "Experimental Procedures." Oxidation was measured at 340 nm;
the arrow indicates the addition of product. In the case of
E (background) and F, t-BOOH was added
as the last component to a reaction mixture containing the complete
Trx/TR/NADPH system.
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Kinetic Parameters of PGdx-catalyzed Peroxide
Reduction--
Measurements for H2O2 or
t-BOOH reduction at saturating GSH concentrations gave
normal Michaelis-Menten patterns with Km and
kcat/Km values of 2.29 µM and 5.01 × 106 s
1
M
1 for H2O2 reduction
(not shown) and 208.80 µM and 5.67 × 104 s
1 M
1 for
t-BOOH reduction (Fig.
7A). The
Vmax was in the range of 25.74 µmol/min/mg
PGdx for H2O2 reduction and 26.57 µmol/min/mg PGdx for t-BOOH reduction.

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Fig. 7.
Velocity versus substrate
curves of PGdx catalyzed t-BOOH reduction.
A, PGdx shows Michaelis-Menten kinetics with respect to
t-BOOH. B, PGdx shows positive cooperativity with
respect to GSH. The solid line represents the
best fit through all data points using either the Michaelis-Menten
(A) or the Hill (B) equation. The assays were
performed in triplicate, and values ± S.E. are shown.
Insets, Eadie-Scatchard plot.
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For a kinetic analysis with GSH, we chose t-BOOH as
substrate, since its spontaneous reaction at physiological pH with GSH is less pronounced compared with H2O2. In order
to avoid extensive background activity with GSH, we also used a pH of
7.1 instead of pH 7.8, the established pH optimum for PGdx (not shown).
Measurements revealed a sigmoidal substrate-velocity curve (Fig.
7B). By fitting our data into the Hill equation, we obtained
an apparent Hill coefficient (napp) of 2.9, indicating a phenomenon of strong cooperativity. Km(app) and
kcat/Km(app) were
3.11 mM and 3.01 × 103 s
1
M
1, respectively.
Vmax(app) was 20.98 µmol/min/mg PGdx. The
insets in Fig. 7 represent the data as an Eadie-Scatchard
plot. When n equals 1, the v/[S]
versus v is linear, as is the case for
t-BOOH, but when n is greater than 1, the plot is
curved, as shown for GSH (23).
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DISCUSSION |
We have recently shown that a subgenomic fragment from H. influenzae Rd, bearing the open reading frame HI0572, is capable of complementing t-BOOH and H2O2
sensitivity of an Ahp- and catalase-negative E. coli strain,
respectively, and of delivering GSH-dependent alkyl
hydroperoxide reductase activity to a naturally GSH peroxidase-negative E. coli (24). It was envisioned that these properties could be attributed to the unusual primary structure of the gene product, consisting of a peroxidase region at the N terminus, showing homology with type C Prx, and a C-terminal region, showing homology with Grx.
Studies described here demonstrate clearly that this chimeric protein
of H. influenzae Rd, denoted as PGdx, effectively catalyzes the GSH/GR/NADPH-dependent reduction of both
H2O2 and t-BOOH at high rates. The
Trx/TR/NADPH system was unable to support peroxidase activity. PGdx
protects supercoiled DNA against the thiol MCO-system, suggesting that
the protein can function as an effective antioxidant enzyme in
vivo. Assays with t-BOOH and
H2O2 as substrates indicated activities with
specificity constants of 104 and 106
s
1 M
1, respectively.
Interestingly, this latter value is comparable with that of the major
peroxidase system of E. coli, AhpR (25). Whereas the AhpR
system reduces both organic hydroperoxides and H2O2 with similar kinetic efficiencies, it was
proposed that organic hydroperoxides are pseudosubstrates and that the
only role of AhpR in nature is the decomposition of
H2O2 (25). Consequently, bearing in mind that
PGdx could fulfill a similar role as AhpR, the 2-order of magnitude
lower value of kcat/Km for
t-BOOH reduction compared with that of
H2O2, at least, supports this assumption.
Rouhier et al. (8, 15) previously described the
Grx-dependent reduction of a poplar phloem Prx. They
suggested a mechanism where the sulfenic acid of the oxidized Prx
becomes reduced by formation of a disulfide linkage with the N-terminal
cysteine residue of the CXXC motif from Grx. This disulfide
bond then becomes reduced either through the mono- or the dithiol
mechanism characteristic for Grx activity (Fig.
8). On the basis of our results, it
appears that Cys49 has an affinity for GSH. Therefore, we
propose another possible reaction mechanism for PGdx, in which the
reduction of hydroperoxides is accompanied by the formation of a
glutathionylated Prx-cysteine. The GSH-mixed disulfide is subsequently
reduced by the action of the C-terminal Grx region, following a
monothiol pathway. The mechanism can be summarized as follows and is
schematically given in Fig. 8, where E represents PGdx and
numbers represent the positions of the cysteine
residues.
Reaction 1 shows the formation of sulfenic acid (Cys-SOH) at
Cys49, with concomitant reduction of the hydroperoxide. In
Reaction 2, GSH forms a protein-mixed disulfide with Cys49.
Reaction 3 describes the dethiolation of the Prx region by
Cys180 of the Grx region. In Reaction 4, PGdx becomes
regenerated by GSH, forming GSSG, which in turn will be reduced by GR
in Reaction 5. Glutathionylation of Prxs has already been mentioned in
numerous cases (26-28), where it functions as a regulatory mechanism
in which the Prx gets inactivated and protected against further
oxidation of its active site cysteine into the more stable forms of
sulfinic (Cys-SO2H) or sulfonic acid
(Cys-SO3H). Other examples of such protection and
regulation are already known to occur in protein-tyrosine phosphatases,
where reactivation takes place with either GSH or Grxs (29-31).
Dethiolation via Grxs follows a monothiol mechanism, requiring only one
Cys residue of the redox-active disulfide motif. Besides the inability
of monocysteinic mutants of Trx to follow a monothiol pathway (32),
Grxs are also 10 times more effective, on a molar basis, than Trxs in
reducing GSH-mixed protein disulfides (31, 33). In addition, the
C-terminal Grx region of PGdx shares strong homology with Grx3 from
E. coli, which has a higher activity as reductant of
glutathionylated proteins than E. coli Grx1 and -2 (34).
Hence, from an evolutionary point of view, the mechanism we propose
provides a possible explanation for the fact that Grx homologs instead
of Trx homologs constitute these chimeric enzymes.

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Fig. 8.
Proposal for the mechanism of the
GSH-dependent reduction of PGdx. Dotted
lines represent steps as originally proposed by Rouhier
et al. (15) for a Grx-reducible poplar Prx but
adapted here for PGdx; see "Discussion" for a detailed
discussion of each reaction (1-5). The top black
part of the letter E corresponds to
the N-terminal Prx region, whereas the bottom
gray part of the letter represents the C-terminal
Grx region.
|
|
Most oxidized Prxs (e.g. bacterial AhpC and yeast
thiol-specific antioxidant) form disulfide-linked dimers that are
subsequently reduced by a separate constituent (e.g. AhpF
and Trx) (3). The existence, however, of a PGdx homodimer does not
necessarily imply such a form to be involved in the catalytic
mechanism. Given that PGdx is a hybrid protein with its reducing
partner actually a part of the enzyme, we are inclined to believe that
the dimeric form may be an artifact of purification or a way to prevent
irreversible oxidation, and consequently inactivation, in the absence
of significant levels of GSH. In order to validate this premise, we are
currently studying cysteinic mutants of PGdx. Note that poplar Prx
forms no dimers during its catalytic cycle (8, 15).
Since the precise reaction mechanism of PGdx awaits further
investigation, interpretation of the sigmoidal behavior observed during
kinetic measurements is not straightforward. Generally, velocity curves
displaying sigmoidal kinetics reflect allosteric enzymes in which
distal binding sites of an oligomeric complex communicate with each
other in a cooperative manner (23, 35). Besides covalent homodimers,
PGdx may also form multimeric complexes like the decameric structures
formed by some Prxs (36, 37). However, no sigmoidal kinetics were
mentioned in these cases. Therefore, although the situation of a
cooperative active oligomeric PGdx complex is possible, in which
binding of GSH induces an increase in peroxidase activity of the other
associated enzymes, we are not convinced that this is indeed the case.
Rather, we believe that other factors are responsible for the sigmoidal
features of the velocity curve. Hence, the Hill coefficient obtained
does not relate to the number of cooperative interacting sites but rather is the intrinsic result of the sigmoidal velocity curve. Substrate depletion seems unlikely because the decrease in activity sets in when GSH concentrations are still high, and, moreover, the
lowest GSH concentration used was still 12,000 times that of the total
enzyme concentration.
Kinetic measurements were performed with a nonreduced PGdx sample, thus
containing both the glutathionylated monomer and the homodimer.
Although sigmoidicity remained with a protein sample purified under
reducing conditions (not shown), we cannot exclude the possibility
that, in the presence of low GSH concentrations, formation of dimeric
PGdx through oxidation prevails. Since participation in our proposed
catalytic mechanism requires PGdx to remain monomeric, the homodimer
formed needs to be reduced. Hence, low GSH concentrations may lead to a
lower availability of active monomeric species and, therefore, to lower activity.
Given the proposed reaction scheme (Fig. 8), the unproductive side
reaction in the monothiol mechanism of a Grx may also be considered to
be responsible for the sigmoidicity. When concentrations of reduced GSH
are running low, the equilibrium shifts toward the formation of a
disulfide bridge between the Cys residues of the active motif, with
concomitant release of GSH from the N-terminal cysteine. Such an event
renders a Grx inactive, up to the moment the disulfide bridge becomes
reduced again by GSH, a situation that may also apply to PGdx. At low
GSH concentrations, only a small fraction of the total enzyme
population remains reduced and active. Enzyme activity gradually
increases with increasing GSH concentrations until the side reaction
(reaction 6 in Fig. 8) becomes insignificant. Although further
experiments are needed to prove this hypothesis, initial kinetic
measurements with a cysteinic 183 mutant of PGdx revealed a total
absence of sigmoidicity (data not shown). A full characterization of
the cysteinic mutants of PGdx is being carried out.
In conclusion, our initial characterization of the chimeric H. influenzae PGdx provides a basis for future studies of other homologs. Not only is PGdx the first heme-independent hydroperoxidase in H. influenzae ever characterized, it is also, to our
knowledge, the first prokaryotic peroxidase effectively using GSH for
its reduction, albeit in a manner different from eukaryotic GSH
peroxidases. Besides a more in depth investigation of the catalytic
role behind each of its three cysteine residues and their roles in
catalysis, regulatory as well as physiological studies are currently
under way to gain more insight into its in vivo functions.
Studies with the separate regions of the PGdx enzyme will provide new
insights into the catalytic mechanism and the interaction between its
two regions.
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. Brigé and Dr. L. De
Smet for helpful suggestions.
 |
FOOTNOTES |
*
This work was supported by Institute for the Promotion of
Innovation by Science and Technology in Flanders Grant 3072 (to F. P.)
and by Concerted Research Action 12050198 from Ghent University (to
J. V. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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: Laboratory of Protein
Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium. Tel.: 32-9-264-51-09; Fax:
32-9-264-53-38; E-mail: Jozef.vanbeeumen@rug.ac.be.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M300157200
 |
ABBREVIATIONS |
The abbreviations used are:
Prx, peroxiredoxin;
Trx, thioredoxin;
Grx, glutaredoxin;
DTT, dithiothreitol;
GR, glutathione reductase;
MS, mass spectrometry;
MCO, metal-catalyzed oxidation;
t-BOOH, tert-butylhydroperoxide;
AhpR, alkyl hydroperoxide
reductase;
AhpC, a 21-kDa component of AhpR;
AhpF, a 57-kDa component
of AhpR;
TR, Trx reductase.
 |
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