In aerobic cells hydrogen peroxide is produced as a byproduct of respiration in mitochondria

42 Pages·2002·1.83 MB·English

JBC Papers in Press. Published on August 2, 2002 as Manuscript M206626200
Inactivation of Human Peroxiredoxin I During Catalysis as the Result of the Oxidation of
Catalytic Site Cysteine to Cys-Sulfinic acid*

Kap-Seok Yang§, Sang Won Kang§†, Hyun Ae Woo, Sung Chul Hwang‡, Ho Zoon
Chae¦, Kanghwa Kim**, and Sue Goo Rhee¶

Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland 20892, USA

* This work was partly supported by Korea Research Foundation Grant KRF-2001-015-
DP0482 (to HZC) and by the Korea Science and Engineering Foundation through the
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Center for Cell Signaling Research at Ewha Womans University, Korea (to HAW) ow
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§ Both authors contributed equally to this work. from
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† Present Address: Center for Cell Signaling Research and Division of Molecular Life ttp://w
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Sciences, Ewha Womans University, 11-1 Daehyun Dong, Seodaemoon-gu, Seoul 120- w
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750, Korea .o
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‡ On leave from the Department of Pulmonary and Critical Care Medicine, Ajou y g
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University School of Medicine, Suwon 442-749, Korea t o
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¦ On leave from the Department of Biological Sciences, College of Natural Sciences, nu
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Chonnam National University, Gwangju 500-757, Korea 10
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** Present Address: Department of Food and Nutrition, College of Home Economics, 19
Chonnam National University, Gwangju 500-757, Korea

¶ To whom correspondence should be addressed:

Building 50, Room 3523, South Drive, MSC 8015
Bethesda, MD 20892
Phone: [phone]; Fax: [phone]; Email: [email protected]

Running title: Oxidative inactivation of Prx I

1
ABSTRACT

By following peroxiredoxin I-dependent NADPH oxidation spectrophotometrically, we
observed that peroxiredoxin I activity decreases gradually with time. The decay in
activity was coincident with the conversion of peroxiredoxin I to a more acidic species as
assessed by 2-dimensional gel electrophoresis. Mass spectral analysis and studies with
Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic
site Cys51-SH to Cys51-SO H. Thus, the Cys51-SOH generated as an intermediate during
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catalysis appeared to undergo occasional further oxidation to Cys51-SO2H, which cannot ow
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be reversed by thioredoxin. The presence of H O alone was not sufficient to cause ed
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oxidation of Cys51 to Cys51-SO2H. Rather, the presence of complete catalytic http
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components (H O , thioredoxin, thioredoxin reductase, and NADPH) was necessary, w
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indicating that such hyper-oxidation occurred only when peroxiredoxin I was engaged in rg
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the catalytic cycle. Likewise, hyper-oxidation of Cys172/Ser172 mutant peroxiredoxin I es
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required not only H O but also a catalysis-supporting thiol, dithiothreitol. Kinetic nu
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analysis of peroxiredoxin I inactivation in the presence of a steady-state, low level (< 1 , 2
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µM) of H O indicated that peroxiredoxin I was hyper-oxidized at a rate of 0.072% per
2 2
turnover at 30 °C. Hyper-oxidation of peroxiredoxin I was also detected in HeLa cells
treated with H O .
2 2

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INTRODUCTION

Peroxiredoxins (Prx)1 are a family of peroxidases that reduce hydrogen peroxide
and alkyl hydroperoxides to water and alcohol, respectively, with the use of reducing
equivalents provided by thiol-containing proteins (1-3). The first Prx proteins to be
discovered were a 25-kDa yeast protein, initially called thiol-specific antioxidant (TSA)
enzyme (4-6), and a 21-kDa Salmonella typhimurium alkyhydroperoxide reductase,
termed AhpC (7-10). Subsequently a mammalian homolog of TSA/AhpC was purified
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and it, together with TSA and AhpC, defined a family of peroxidases that now includes ow
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six mammalian isoforms (Prx I-VI) and members identified in organisms from each ed
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kingdom (3,10,11). http
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All Prx proteins contain a conserved Cys residue, which corresponds to Cys51 in ww
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mammalian Prx I, in the NH -terminal portion of the molecule (10,11). The majority of rg
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Prx proteins, including four (Prx I-IV) of six mammalian Prxs, contain an additional es
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conserved Cys in the COOH-terminal region that corresponds to Cys172 in mammalian nu
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Prx I (2,3,12,13). The Prx enzymes containing two conserved Cys residues are thus , 2
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called 2-Cys Prx, in comparison to a small number of Prx proteins, termed 1-Cys Prx,
that contain only one conserved cysteine at NH -terminal domain (2,10). In 2-Cys Prx
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enzymes, the NH -terminal conserved cysteine is oxidized by H O to Cys-sulfenic acid
2 2 2
(Cys51-SOH), which then reacts with the Cys172-SH of the other subunit to produce an
intermolecular disulfide (1,14). Reduction of the disulfide intermediate of Prx I-IV is
specific in that it can be achieved by thioredoxin (Trx) but not by GSH or glutaredoxin
(15,16). Thus, the reducing equivalents for the peroxidase activity of Prx I-IV are
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ultimately derived from NADPH via thioredoxin reductase (TrxR) and Trx. Not all Prx
enzymes containing both conserved Cys residues are reduced by Trx: the bacterial Prx
AhpC is reduced by AhpF, which contains both Trx and TrxR domains (10,17). In the
absence of a physiological electron donor, the peroxidase activities of 2-Cys Prx enzymes
can be supported by small thiol molecules like dithiothreitol (DTT) and 2-
mercaptoethanol, but not by GSH (1,14,15). In 1-Cys Prx enzymes that include
mammalian Prx VI, the conserved cysteine is also the site of oxidation, but remains as a
sulfenic acid state upon oxidation because there is no nearby partner cysteine to form a
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disulfide (18,19). Trx cannot reduce this sulfenic acid-containing intermediate (18,20). ow
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GSH has been proposed to be the electron donor, but these data remain controversial ed
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(18,20-23). http
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The crystal structures of 2-Cys and 1-Cys Prx enzymes reveal that the catalytic w
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cysteine is located in a small pocket formed by the NH -terminal and COOH-terminal rg
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domains of the two subunits (19,24-27). The reactive cysteine is thus protected from es
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larger oxidant molecules that contain disulfide linkages. The structure also shows that nu
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the NH2-terminal conserved cysteine is surrounded by positively charged amino acid , 20
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residues, which stabilize thiolate (Cys-S–) anion. Thiolate anion is more readily oxidized
by peroxides than its protonated thiol counterpart (Cys-SH) (28). This provides the
mechanistic basis for the observed sensitivity of the active site cysteine to oxidation by
peroxides.
Previously, we reported that Prx purified from yeast is readily inactivated during
catalysis (1). We speculated that such inactivation occurred if the sulfenic acid moiety of
the reaction intermediate was further oxidized by H O to Cys-sulfinic acid (Cys-SO H)
2 2 2
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before disulfide formation with Cys172 could occur (1). Sulfinic acid cannot be reduced
by the Trx or DTT included in the assay mixture. Recently Mitsumoto et al. (29) used 2-
dimensional polyacrylamide gel electrophoresis (2-D PAGE) to compare proteins in
human umbilical vein endothelial cells before and after exposure of cells to H O . In
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H O -treated cells, a number of proteins including Prx I and Prx II demonstrated altered
2 2
migration consistent with decreased isoelectric pH (pI), suggesting that such oxidative
inactivation might also occur in cells. However, these acidic Prx enzymes were not
characterized in detail. We have now investigated the mechanism of human Prx I
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inactivation by H2O2. Here, we demonstrated that the enzymatic inactivation and ow
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concomitant acidic shift of Prx on 2-D gels are due in fact to the conversion of the active ed
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site cysteine to Cys-SO2H. Furthermore, we observed that only those Prx molecules http
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actively engaged in the catalytic cycle are vulnerable to oxidative inactivation. w
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MATERIALS AND METHODS nu
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Preparation of recombinant proteins --- The construction of a bacterial expression vector
for human Prx I (pETprxI-WT) has been described (15). Two Prx I mutants, C51S and
C172S, in which Cys51 and Cys172 were individually replaced by serine residues, were
generated by standard PCR-mediated site-directed mutagenesis with pETprxI-WT as the
template and complementary primers containing a single-base mismatch that converts the
codon for Cys to one for Ser. The final mutated PCR products were ligated into the pET
vector to generate pETprxI-C51S and pETprxI-C172S. E.coli BL21 (DE3) competent
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cells (Novagen) were transformed with pETprxI-WT, pETprxI-C51S or pETprxI-C172S,
cultured at 37 °C overnight in 100 ml of LB medium supplemented with ampicillin (100
µg/ml), and then transferred to 10 liters of fresh LB medium in a Microferm Fermentor
(New Brunswick Scientific). When the optical density of the culture at 600 nm reached
0.6 to 0.8, expression was induced by isopropyl-β-D-thiogalactopyranoside at a final
concentration of 0.4 mM. After incubation for an additional 3 h, the cells were collected
by centrifugation, frozen in liquid nitrogen, and stored at –70 °C until use. The
recombinant proteins were purified as described (30).
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Prx assay--- NADPH oxidation coupled to the reduction of H O was monitored at 30 °C ed
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as a decrease in A340 using a UV-visible Spectrophotometer (Hewlett Packard 8453) http
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equipped with a thermostable cell holder and a multicell transport. The reaction was w
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initiated by adding the indicated concentration of Prx I to a 200 µl reaction mixture rg
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containing 50 mM Hepes-NaOH (pH 7.0), 1 mM EDTA, 0.8 µM TrxR, and the indicated es
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concentrations of H O , NADPH, and Trx. nu
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Cell Culture--- HeLa S3 cells were adapted to suspension growth in Spinner minimum
essential medium (Quality Biological) supplemented with 10% (v/v) fetal bovine serum
(Life Technologies). The cells were grown to a density of 1 × 106 cells/ml and
maintained by dilution with fresh complete medium every 4 days.

Sample preparation, 2-D PAGE, and immunoblot analysis --- HeLa S3 cells were rinsed
three times with ice-cold PBS, and lysed in lysis buffer (8M urea, 4% CHAPS, 40 mM
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Tris base) by sonicating in sonic bath 3-4 times for 30 s. After removal of insoluble
materials by centrifugation at 14,000 × g for 30 min, cell lysates were mixed with 10 vol
of rehydration buffer (8M urea, 2% CHAPS, 0.5% IPG buffer, 20 mM DTT and 0.005%
bromophenol blue), and loaded on IPG strips (pH 3-10, nonlinear). Isoelectric focusing
on an IPGPhor isoelectrofocusing unit (Amersham Biosciences) and preparation
(reduction and alkylation) of the IPG strips for the second-dimension SDS-PAGE were
carried out according to the procedures recommended by the manufacturer. SDS-PAGE
was conducted on 12% gels using an SE600 Vertical Unit (Amersham Biosciences) and
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the protein spots were visualized by staining with silver nitrate. For immunoblot analyses ow
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of Prx enzymes, proteins on 2-D gels were transferred electrophoretically to a ed
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nitrocellulose membrane and the membrane was incubated with rabbit antibodies to Prx I. http
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Immune complexes were detected with horseradish peroxidase-conjugated secondary w
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antibodies and enhanced chemiluminescence reagents (Amersham Biosciences or Pierce). rg
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Silver-stained 2-D gels were scanned with Personal densitometer SI (Molecular es
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Dynamics). nu
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Reverse-phase HPLC chromatography --- Reverse-phase high-performance liquid
chromatography (HPLC) analyses were performed using an Agilent 1100 HPLC system
(Palo Alto, CA) with a Vydac 218TP54 column (0.47 mm × 25 cm, Vydac Corp.
Hesperia, CA). For protein analysis, injected samples were eluted at 1 ml/min with a 5-
35% (v/v) acetonitrile/water gradient containing 0.04% (v/v) trifluoroacetic acid over 15
min, and a 35-60% gradient over the next 15 min. Gradients of 5-10% over 10 min, 10-
40% over 30 min, and 40-60% over 30 min were used for peptide analysis.
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ESI-MS analysis of Prx I proteins and sequence analysis of tryptic peptides --- A
Finnigan LCQ electrospray ion-trap mass spectrometer (Finnigan MAT, San Jose, CA)
was used for the analysis of Prx I proteins and sequence analysis of Cys51-containing
peptides. Dried HPLC fractions dissolved in 75% acetonitrile/0.1% acetic acid were
introduced at 1 µl/min using a syringe pump. Data were collected for positive ions of
300-2000 m/z using the following settings: capillary temperature, 215 °C; maximum ion
inject time, 100 ms; full scan target, 9 × 107; and 3 microscans/full scan. For proteins,
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electrospray ionization-mass spectrometry (ESI-MS) spectra were obtained and used to ow
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calculate the masses of proteins by deconvolution using Finnigan BioWorks software. ed
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For the sequence analysis of peptides, once the parent ions were identified, the mass http
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spectrometer was setup to obtain collision-induced dissociation (CID) MS/MS spectra of w
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the parent ions. The instrument for MS/MS spectra was configured as follows: capillary rg
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temperature, 215 °C; maximum ion inject time, 100 ms; full scan target, 2 × 107; 3 es
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microscans/full scan; mass window, 2.5; and collision energy was used within a range of nu
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0% to 50%. , 2
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MALDI-TOF-MS --- Mass spectrometric analysis of the tryptic peptides was performed
on a Voyager-STR matrix-assisted laser desorption ionization time-of-flight (MALDI-
TOF) instrument (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen
laser. Samples were dissolved in 50% acetonitrile/5% formic acid, mixed with 10 mg/ml
3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid; Aldrich Chemicals) in 0.1%
trifluoroacetic acid/70% acetonitrile and spotted on a sample plate. The spectra were
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obtained in the positive-ion reflector or linear mode with delayed extraction using
standard conditions. Spectra were analyzed using DataExplorer (PerSeptive Biosystems)
software. Standard peptides were used for the calibration of peptides, while
apomyoglobin and carbonic anhydrase were used as internal standards for mass scale
calibration of proteins.

RESULTS

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Prx I inactivation in vitro --- The peroxidase activity of human Prx I was monitored by ow
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following the decrease in A attributable to the oxidation of NADPH in a reaction ed
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mixture containing NADPH, Trx, TrxR, and varying concentrations of H2O2. The initial http
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rate of NADPH oxidation (the slope at t=0) was independent of H O concentration at w
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saturating concentrations (0.1-1 mM) of H O [K for H O < 20 µM (31)] (Fig. 1A). rg
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However, the rate decreased with time, and the higher the H O concentration, the faster es
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the rate of decrease. The H O concentration-dependence of this decrease is shown more nu
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quantitatively by plotting the rate of NADPH oxidation (the first derivative of the , 2
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NADPH oxidation curve) versus time (Fig. 1B). Replenishment of Prx I was shown to
restore enzymatic rate, indicating that the markedly decreased NADPH oxidation rate
was not attributable to exhaustion of substrate or to product inhibition (not shown).
Reactions containing 200 µM H O were stopped at 0, 60, and 150 s, and the resulting
2 2
samples were subjected to 2-D PAGE analysis. A single spot at a position corresponding
to a pI value of 8.1 (the theoretical pI of Prx I is 8.2) was observed at 0 s. At 60 s,
however, a new spot, whose intensity is similar to that of the original spot, appeared at a
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more acidic position (pI=7.6). At 150 s, the intensity of the acidic spot was enhanced at
the expense of the original spot. The increasing proportion of the acidic species
compared to the original spot is consistent with the notion that the 50 and 80% decrease
in rate observed at 60 and 150 s, respectively, is related to the conversion of Prx I to a
more acidic derivative.

Selective hyper-oxidation of Cys51-SH to Cys51-SO H by H O ---To identify the
2 2 2
modification responsible for the acidic shift, Prx I was oxidized for 5 min in the presence
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of 1 mM H2O2 and 10 mM DTT. Reduced and oxidized enzymes were separated from ow
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the reaction mixture by reverse-phase HPLC (Fig. 2A). The molecular masses of the ed
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separated proteins were measured by ESI-MS (Fig. 2B). The masses calculated from m/z http
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and total charge of multiply charged ions of the reduced and oxidized enzymes were w
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21979 and 22011, respectively. The mass of the reduced enzyme was in good agreement rg
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with the theoretical mass of 21979.2. The difference of 32 mass units between the es
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reduced and oxidized Prx I suggests the presence of two additional oxygen atoms in the nu
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oxidized species. , 2
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To determine the site of oxidation, the reduced and oxidized Prx I separated in
Fig. 2A were digested with trypsin, and the resultant peptides were fractionated by
reverse-phase HPLC (Fig. 2C). The HPLC elution profiles of reduced and oxidized Prx I
peptides were nearly identical except between 48-56 min (Fig. 2C). Major peptide peaks
from the reduced and oxidized proteins were collected and analyzed by MALDI-TOF
MS, enabling the assignment of 12 such peaks to defined fragments of Prx I. In addition
to the disulfide-forming Cys51 and Cys172 residues, Prx I contains two additional cysteine
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