Paradoxically, if an excess of H2O2 is produced in chloroplasts as a result of oxidative stress such as drought or intense light, they are rapidly inactivated because the reaction interm
Trang 1of stromal ascorbate peroxidase in response to rapid
Sakihito Kitajima1, Taise Shimaoka2, Miyo Kurioka1and Akiho Yokota3
1 Graduate School of Science and Technology, Kyoto Institute of Technology, Japan
2 Research Institute of Innovative Technology for the Earth (RITE), Kyoto, Japan
3 Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Nara, Japan
Ascorbate peroxidase (APX; EC 1.11.1.11) isoforms of
chloroplasts play a central role in scavenging reactive
oxygen species such as O2–Æ and H2O2, which are
gen-erated in large amounts by photosystems when there is
an energy surplus Chloroplasts of higher plants have
two APX isoforms, one localized in the stroma and
the other bound to the stromal side of the thylakoid
membrane [1] APX first reacts with one molecule of
H2O2 and forms a porphyrin-based (compound I) and
then a protein-based (decay product of compound I
[2]) radical intermediate The intermediates are then
reduced back to the resting state through compound II
by interaction with two molecules of ascorbate
Paradoxically, if an excess of H2O2 is produced in
chloroplasts as a result of oxidative stress such as
drought or intense light, they are rapidly inactivated
because the reaction intermediate of the APX is attacked
by excess H2O2 instead of being reduced by ascorbate
[1,3,4] As a result of the inactivation of APX, H2O2 con-tinues to accumulate, resulting in cell damage In vitro experiments reveal that, under conditions of ascorbate depletion, inactivation of APX occurs within minutes [5,6] Other APX isoforms are localized in the cytosol [7] and microbodies [8] of plants These other isoforms and cytochrome c peroxidase (CCP) [9], a yeast homo-log of APX, are much more tolerant to H2O2 than chloroplast APXs, although they have similar amino-acid sequences and structures [10]
The aim of this study was to determine why chloro-plast APXs are more sensitive to H2O2than other per-oxidases Using a chimera of stromal APX and red algal H2O2-tolerant APX, we previously showed that a unique loop structure near the catalytic site is involved
in the rapid inactivation of stromal APX [6] In the present study, we examined the structural change asso-ciated with inactivation by H2O2
Keywords
ascorbate peroxidase; chloroplast; cross-link;
hydrogen peroxide; inactivation
Correspondence
S Kitajima, Graduate School of Science and
Technology, Kyoto Institute of Technology,
Sakyo-ku, Kyoto 606-8585, Japan
Fax: +81 75 724 7762
Tel: +81 75 724 7791
E-mail: sakito@kit.ac.jp
(Received 1 February 2007, revised 22
March 2007, accepted 16 April 2007)
doi:10.1111/j.1742-4658.2007.05829.x
Ascorbate peroxidase (APX) isoforms localized in the stroma and thyla-koid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is deple-ted We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from < 10 to 62 s In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2
Abbreviations
APX, ascorbate peroxidase; CCP, cytochrome c peroxidase; tsAPX, stromal APX from tobacco.
Trang 2Irreversible cross-linking of heme to apoprotein
in H2O2-inactivated stromal APX
We produced recombinant stromal APX from tobacco
(Nicotiana tabacum) (tsAPX) in Escherichia coli and
examined its elution on RP-HPLC before or after
treatment for 25 s with 20 mol H2O2, which reduced
the activity by 80% (Fig 1) Separate peaks for
heme (18.2 min) and apoprotein (25.4 min) were
observed for untreated tsAPX (Fig 2A), whereas
signi-ficant amounts of heme were coeluted with the
apo-protein (broad peaks at 26.4 and 27.0 min) when
tsAPX was treated with H2O2 (Fig 2B)
MALDI-TOF-MS of the H2O2-treated enzyme revealed a peak
for the apoprotein ( 32 720 m ⁄ z) and a second peak
( 33 360 m ⁄ z) roughly corresponding to apoprotein
plus heme (616.48 Da) and oxygen (Fig 2E) The mass
measurement error in these experiments was less than
250 p.p.m Although part of tsAPX was polymerized
by treatment with H2O2, detectable by SDS⁄ PAGE
(data not shown) as indicated in CCP [11], the
poly-mers were not detectable by MALDI-TOF-MS because
of low sensitivity in the higher mass range These
results indicate that, when inactivated by H2O2, heme
is irreversibly cross-linked to the APX apoprotein
Identification of the heme-binding amino-acid
residue in H2O2-inactivated stromal APX
To determine which amino-acid residue in the
apopro-tein was cross-linked to heme, the inactivated tsAPX
was digested with trypsin and subjected to RP-HPLC
We found at least four peaks that absorbed at 400 nm (38.0, 38.9, 40.5 and 50.9 min; Fig 3A) Because the retention time for the product eluted at 50.9 min was
0
20
40
60
80
100
time (sec)
tsAPXW35F, – H 2 O 2
tsAPXW35F, + H2O2
tsAPX, – H2O2 tsAPX, + H2O2
Fig 1 Remaining activities of tsAPX and tsAPXW35F treated with
H2O2 APXs were treated with or without 20 mol H2O2in O2-free
50 m M sodium phosphate, pH 7.0, at 25 C The concentration of
treated and untreated APXs was 1.9 l M Results are mean ± SD
from five measurements.
Fig 2 Inactivation and cross-linking of APXs by H2O2 APXs were untreated (A,C) or treated (B,D) with 20 mol H2O2in O2-free 50 m M
sodium phosphate, pH 7.0, at 25 C Enzymes were separated by HPLC on a C4 reversed-phase column Protein and heme were detected at 220 nm (thin line) and 400 nm (thick line), respectively (A) 2.0 l M untreated tsAPX; (B) 1.9 l M tsAPX treated with H 2 O 2 for
25 s; (C) 2.1 l M untreated tsAPXW35F; (D) 2.1 l M tsAPXW35F treated with H2O2for 120 s; (E) MALDI-TOF-MS spectra of tsAPX treated with or without 5 mol H 2 O 2 for 2 min The mass measure-ment error was less than 250 p.p.m mAU, Milliabsorbance units.
Trang 3identical with that for a commercial sample of hemin
(data not shown), we concluded that it represented a
free heme species The mass spectrum of the most
abundant peak (38.0 min; Fig 3A, inset) had a
[M + H]+ ion at 1892.8 m⁄ z, and its MS ⁄ MS
spec-trum indicated that it was a peptide with the sequence
HDAGTYNK (Fig 3B) This matches a predicted
tryptic peptide corresponding to residues 33–43
(LGWHDAGTYNK) (Fig 4A), although Leu33,
Gly34, and Trp35 could not be assigned in the MS⁄ MS
spectrum Of these three residues, Trp35 is the only
reactive residue and therefore the most likely to
cross-link to the heme The 1892.8 m⁄ z value obtained was
15.7 higher than the sum of the calculated masses for
the protonated peptide LGWHDAGTYNK (1260.67)
plus heme (616.48), suggesting incorporation of an
oxy-gen atom We could not identify the two other minor
peaks (38.9 and 40.5 min) The side chain of Trp35
faces the distal cavity formed by the heme and
N-ter-minal half of the apoprotein of the catalytic site
(Fig 4B) Because the indole ring of Trp35 is 3.18 A˚
from the porphyrin ring in tsAPX (the distance
between nitrogen of the indole ring and C6 of the
por-phyrin ring) [10], the heme must move toward Trp35 to
form a covalent bond In active cytosolic APX,
ascor-bate binds to the c-meso edge of heme (a propionated
side of heme) [12] The loss of ascorbate-oxidizing
0 100 200 300 400 500 600 700 800
time (min)
0 100 200 300 400 500 600
A
38.0 min
38.9 min 40.5 min
50.9 min
0 100 200 300 400 500 600 700
240 280 320 360 400 440
nm
38.0 min
LGW(O-heme) (987.3)
Fig 3 Identification of the amino-acid
resi-due cross-linked to heme in H2O2-treated
tsAPX tsAPX was treated with 20 mol
H 2 O 2 at 25 C for 20 s and then digested
with trypsin (A) C18 RP-HPLC Peptide and
heme were detected at 220 nm (thin line)
and 400 nm (thick line), respectively Inset,
UV ⁄ Vis spectra of the product eluted at
38.0 min (B) MS ⁄ MS spectrum of the
product eluted at 38.0 min The peptide
sequences obtained from b and y fragment
ions are indicated mAU, Milliabsorbance
units.
Distal side
Proximal side
Trp35
Distal side
Proximal side Trp35
A
B
Fig 4 Deduced amino-acid sequence of tsAPX (A) and structure of its catalytic site [10] (B) The sequence of the tryptic peptide identi-fied from the MS ⁄ MS spectrum in Fig 3B is underlined.
Trang 4activity in the cross-linked form of tsAPX may
there-fore be due to the repositioning of heme, preventing it
from interacting with ascorbate
Effect of Trp35 mutation
To investigate the role of Trp35 in the cross-link and
the inactivation of APX by H2O2, we created a mutant
form of tsAPX in which Trp35 was changed to
phenyl-alanine (tsAPXW35F) We found that tsAPX in
O2-free 50 mm sodium phosphate, pH 7.0, had a
Soret band at 404 nm (e404¼ 105 mm)1Æcm)1) and
a shoulder around 380 nm (Fig 5A) This is similar
to the spectrum for yeast CCP, which has a
five-coordinated high-spin ferric heme [13] The spectrum
of tsAPXW35F, however, lacked the shoulder around
380 nm (Fig 5B) and had a more intense Soret band
(e405¼ 122 mm)1Æcm)1), which is typical of a
six-coordinated ferric heme, as found in the CCP mutant
[13,14] Spectra for these two APXs in the visible
region were similar, and both had two charge-transfer
bands (Fig 5A,B), which is characteristic of high-spin
heme species This is slightly different from a similar
mutant of cytosolic APX from soybean reported by
Badyal et al [15]; specifically, when the corresponding
tryptophan was changed to alanine in soybean
cytoso-lic APX, a peak appeared at 564 nm, which is
charac-teristic of low-spin heme species Furthermore, the spectra for the six-coordinated low-spin ferric forms, prepared by treatment with KCN, were similar for tsAPX and tsAPXW35F (Fig 5A,B) Finally, the Km and kcatvalues for tsAPXW35F were only slightly dif-ferent from those for tsAPX (Table 1) These results suggest that, except for a difference in the coordination
of the distal side of the heme ferric atom, the W35F mutation did not cause a significant change in the structure of tsAPX
Next, we examined the effects of the mutation on the interaction of tsAPX with excess H2O2 In con-trast with tsAPX, a cross-link was not observed in tsAPXW35F, even when it was treated with 20 mol
H2O2 for 120 s (Fig 2C,D) This agrees with the
MS⁄ MS results showing that Trp35 is the most likely site for cross-linking to heme This also indicates that the tryptic peptides of two unidentified minor peaks eluted at 38.9 and 40.5 m (Fig 3A) are probably parti-ally digested products containing Trp35 cross-linked to heme
In the presence of 20 mol H2O2, tsAPXW35F had a half-time of inactivation of 62 s, which is more than 6.2-fold longer than for tsAPX (< 10 s) (Fig 1) We obtained similar results when we included excess BSA
in the reaction to exclude the possible effect of con-taminating apoprotein (data not shown) These results strongly support the idea that the formation of the cross-link is at least part of the reason for the rapid inactivation of tsAPX
In these experiments, tsAPXW35F was inactivated
in 3 min by H2O2 Whether this reflects other aspects
of the inactivation mechanism for tsAPX is not clear;
it is possible that tsAPXW35F is inactivated by a distinct mechanism because the heme can no longer
be cross-linked to the enzyme The possibility of a distinct mechanism of inactivation is supported by the difference in the spectral changes for H2O2-treated tsAPXW35F and tsAPX (Fig 6A,B)
Discussion
In these studies, we showed that heme cross-links to the distal tryptophan in tsAPX within minutes when
Fig 5 Absorption spectra of tsAPX (A) and tsAPXW35F (B) treated
with or without 0.2 m M KCN in O 2 -free 50 m M sodium phosphate,
pH 7.0.
Table 1 Steady-state kinetic parameters of APXs.
K m(Asc)a
(l M )
KmðH2O 2 Þb
(l M )
k cat
(s)1Æheme)1)
a K m for ascorbate; b K m for H 2 O 2 ; c Kitajima et al.[6].
Trang 5treated with H2O2in the absence of ascorbate On the
basis of these results, we propose that the rapid
inacti-vation of tsAPX is at least partly due to repositioning
of heme caused by cross-linking between heme and the
distal tryptophan resulting from reaction with H2O2
Recently, Pipirou et al [16] reported that part of the
heme molecule is cross-linked to the distal tryptophan
in the cytosolic APX isoform when it reacts with
excess H2O2 in the absence of ascorbate They
pro-posed that a vinyl group of heme is bound to C1 of
the distal tryptophan and hydroxylated The cross-link
between heme and tryptophan may occur in a similar
way in tsAPX In a H2O2-tolerant APX isoform from
red algae [6,17], excess H2O2 also causes cross-linking
between heme and the apoprotein, but the ratio of
cross-linked heme to total heme was lower than in
tsAPX (data not shown) In CCP of yeast, the distal
tryptophan is also conserved, but it has not been
reported to cross-link to heme Thus, H2O2-mediated
cross-linking in perxoxidases other than tsAPX, if it
occurs, may be much slower
Why the heme rapidly forms a cross-link in tsAPX
is uncertain In CCP, a proximal tryptophan residue distant from the porphyrin is a major radical site in its reaction intermediate [18] In addition, when its reducing substrate, cytochrome c, is absent, the rad-ical is transferred to, oxidizes, and disrupts trypto-phan and tyrosine residues distant from the heme [19–21] Also, in cytosolic APX of pea [2] and in fungal lignin peroxidase [22], when the reducing sub-strate is absent, the radical is thought to transfer from porphyrin to tryptophans far from heme, resulting in their hydroxylation The relocation of the radical means that these amino-acid residues directly or indirectly donate electrons as endogenous reducing substrates to the porphyrin radical, protect-ing the enzyme from over-oxidation by excess H2O2 The cross-linking of Trp35 to heme of tsAPX thus suggests that the radical in the reaction intermediate
is located in porphyrin and Trp35, but that reloca-tion to residues distant from heme, if it occurs, is much slower than in other peroxidases As a result, the cross-linking of heme may occur readily in tsAPX
In bifunctional catalase–peroxidase, a bacterial homolog of APX, a radical is transferred from por-phyrin to another tryptophan residue that is connec-ted to a propionate side chain of porphyrin by a hydrogen-bonding network through two water mole-cules [23] The interaction of the propionate side chain with the protein is different for tsAPX and cytosolic APX, because of a unique 16-amino-acid stretch [10] that confers higher sensitivity to H2O2 [6]
A change in the interaction of the propionate with amino-acid residues may therefore influence transfer
of the radical in the reaction intermediate of tsAPX Theoretically, the propionate side chain is also involved in electron transfer from amino-acid residues
to the porphyrin [24]
The tendency of the radical to remain near the heme may allow a more rapid catalytic turnover, although at the expense of tolerance to H2O2 This might have been evolutionary pressure on the chloroplast APXs
In fact, the specific activities of chloroplast APXs reported to date are much higher than those of the cytosolic APXs [25–30]
In conclusion, we have shown that the rapid inacti-vation of tsAPX is at least partly due to cross-linking between heme and the distal tryptophan as a result of reaction with H2O2 Given the amino-acid sequence similarity between stromal and thylakoid-bound APXs (reviewed in [31]), the inactivation mechanism pro-posed here should also be relevant for thylakoid-bound APX
0.005 0.01 0.015 0.02 0.025 0.03
0.05
0.1
0.15
0.2
0.25
A
0
0.05
0.1
0.15
0.2
0 0.005 0.01 0.015 0.02
0 0
B
untreated
untreated
300 350 400 450 500 550 600 650 700
nm
untreated
untreated
Fig 6 Spectral change in tsAPX and tsAPXW35F treated with
20 mol H2O2 APXs were solubilized in O2-free 50 m M sodium
phosphate, pH 7.0, and then treated with H2O2 (A) tsAPX (2.1 l M )
before treatment and 3, 9, 18, 60, and 120 s after addition of
H 2 O 2 (B) tsAPXW35F (1.9 l M ) before treatment and 3, 60, 120,
180, and 300 s after the addition of H2O2 Spectral changes were
monitored at 22 C using a photodiode array spectrophotometer.
Trang 6Experimental procedures
Preparation of recombinant APXs
Expression plasmids for tsAPX were constructed as
des-cribed previously [6] Trp35 was mutated by PCR-mediated
site-directed mutagenesis The expression plasmids encoded
APXs corresponding to residues 92–386 of accession
num-ber AB022274, with methionine and glycine residues at the
N-terminus, which are not present in the native enzyme
Recombinant APXs produced in E coli BL21(DE3) were
purified by sequential steps of chromatography on HiPrep
16⁄ 10 DEAE FF (Amersham Bioscience, Piscataway, NJ,
USA), HiLoad 16⁄ 10 Phenyl Sepharose HP (Amersham
Bioscience), and HiLoad 16⁄ 60 Superdex 75 pg (Amersham
Bioscience) as previously described [6] Purified tsAPX and
tsAPXW35F appeared as single bands when separated by
SDS⁄ PAGE (data not shown)
O2-free APX solution was prepared by passing APX in
10 mm potassium phosphate, pH 7.0, 1 mm EDTA, 1 mm
ascorbate, and 0.15 m KCl through two Sephadex G25
col-umns (NAP5 and PD10 colcol-umns; Amersham Bioscience)
and elution with 50 mm sodium phosphate, pH 7.0, that
had been degassed by bubbling with N2gas Before
analy-sis, the concentration of APX was determined from the
absorption of heme
The absorption coefficients of the Soret peak for tsAPX
and tsAPXW35F were 105 [6] and 122 mm)1Æcm)2,
respect-ively The value for tsAPXW35 was determined according
to the heme content and UV⁄ Vis absorption spectra Heme
contents were determined by the pyridine hemochromogen
method [32] with horseradish peroxidase (Nacalai tesque,
Kyoto, Japan) as a standard (e¼ 100 mm)1Æcm)1 at 403
nm [33]) The heme contents per tsAPX and tsAPXW35F
molecule were 70% and 80%, respectively, indicating that
30% and 20% were the apoenzyme
Enzyme assay
APX activity was measured as described previously [6],
except that the reaction mixture was supplemented with
0.01 mgÆmL)1 BSA for the experiment in Fig 1E The Km
values for ascorbate and H2O2 and the kcat values were
determined as described previously [6]
HPLC and MS
HPLC analysis was performed using an LC-VP HPLC
system (Shimadzu, Kyoto, Japan) equipped with a
SPD-M10AVP photodiode array UV-Vis detector (Shimadzu)
The column was maintained at 40C For treatment of
APXs with H2O2, 20 mol H2O2 was manually added to
3.5 mL O2-free APX solution with stirring at 25C The
reaction was terminated by adding 0.5 mm ascorbic acid
H2O2 and ascorbate were removed by passing the sample
through an Econopack 10DG column (Bio-Rad, Hercules,
CA, USA) that had been equilibrated with 50 mm sodium phosphate, pH 7.0 For analysis of the heme–apoprotein cross-link, APX was denatured by adding a half volume of
8 : 3 HCl⁄ acetic acid before injection on to a C4 reversed-phase column (4.6· 250 mm; 5 lm; Vydac, Hesperia, CA, USA) Protein and heme were separated by delivery of 35% acetonitrile and 0.1% trifluoroacetic acid for 14 min, followed by a linear gradient of 35–45% acetonitrile over
15 min
For MS of undigested APX, acid-denatured APX sample was washed with a ZipTip C4 microcolumn (Millipore, Bedford, MA, USA), and MALDI-TOF analysis was per-formed on a Reflex III mass spectrometer (Bruker Dalto-nics, Bremen, Germany) in linear mode using sinapic acid (Fluka, Buchs, Switzerland) as the matrix MALDI spectra were externally calibrated using Protein Calibration Stand-ard II (Bruker Daltonics)
For MS of trypsin-digested samples, inactivated APX in
50 mm sodium phosphate, pH 7.0, was precipitated with acetone and dissolved in 4 m urea and 50 mm ammonium bicarbonate, pH 8.0 Sequencing-grade trypsin (Promega, Madison, WI, USA) was added to the solution at a molar ratio of 1 : 50 and incubated at 37C for 6 h The resulting reaction mixture was separated on a C18 reversed-phase column (4.6· 150 mm; TSK gel ODS-100S; Toso, Tokyo, Japan) Peptide and heme were separated by delivery of 0.1% trifluoroacetic acid for 10 min, followed by a linear gradient of 0–10% acetonitrile over 15 min, a linear ent of 10–40% acetonitrile over 30 min, and a linear gradi-ent of 40–100% acetonitrile over 5 min The peptide fraction showing both heme and peptide absorbance was isolated and concentrated by evaporation The sample was loaded on to a ZipTip C18 microcolumn (Millipore) and eluted with 60% acetonitrile and 0.1% formic acid for ana-lysis using a Q-TOF Ultima mass spectrometer (Waters Co., Milford, MA, USA) MS and MS⁄ MS data were acquired and processed automatically using MassLynx 4.0 software (Waters Co.)
Acknowledgements
We thank Ms Yuki Shinzaki for technical assistance This study was supported in part by the Research Association for Biotechnology, which is subsidized by the Ministry of Economy, Trade and Industry of Japan
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