Báo cáo khoa học: An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation pot

An inserted loop region of stromal ascorbate peroxidase is
involved in its hydrogen peroxide-mediated inactivation
Sakihito Kitajima
1
*, Ken-ichi Tomizawa
1
, Shigeru Shigeoka
2
and Akiho Yokota
3
1 Research Institute of Innovative Technology for the Earth (RITE), Soraku-gun, Kyoto, Japan
2 Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Japan
3 Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Ikoma, Japan
Ascorbate peroxidases (APXs) of plants are members
of the class I hydroperoxidase family, which includes
cytochrome c peroxidase of yeast and bifunctional cat-
alase–peroxidase of bacteria and archea [1]. In the cat-
alytic cycle, APX first reacts with hydrogen peroxide
and is converted to the two-electron-oxidized interme-
diate, compound I, where the ferric iron (Fe
III
) of the
heme moiety is oxidized to the oxyferryl (Fe
IV
¼ O)
species, and the porphyrin is oxidized to its free rad-
ical. In certain situations, the radical is transferred to
amino acid residues. Compound I, or the protein-
based radical, is then reduced back to the resting ferric
state in two successive one-electron transfer reactions
with ascorbate, generating two monodehydroascorbate

radicals.
There are two APX isoforms in chloroplasts, one of
which is soluble in the stroma and the other of which
is bound to the stromal side of thylakoid membranes.
Both isoforms are involved in the water–water cycle, a
system to scavenge reactive oxygen species in chloro-
plasts and dissipate excess excitation energy of photo-
systems [2]. The stromal and thylakoid-bound APXs,
however, are rapidly inactivated under oxidative stres-
ses, leading to photo-oxidative damage in leaves [3,4].
This inactivation is caused by interaction of hydrogen
peroxide with APX when reduction of compound I
Keywords
ascorbate peroxidase; chloroplast; Galdieria
partita; hydrogen peroxide; inactivation
Correspondence
A. Yokota, Graduate School of Biological
Science, Nara Institute of Science and
Technology (NAIST), Ikoma,
Nara 630-0192, Japan
Fax: +81 774 75 2320
Tel: +81 774 75 2307
E-mail:
*Present address
Graduate School of Science and
Technology, Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku, Kyoto, 606-8585,
Japan
(Received 18 February 2006, revised
13 April 2006, accepted 20 April 2006)

doi:10.1111/j.1742-4658.2006.05286.x
Ascorbate peroxidase isoforms localized in the stroma and thylakoid of
higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if
the second substrate, ascorbate, is depleted. However, cytosolic and micro-
body-localized isoforms from higher plants as well as ascorbate peroxidase
B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively
tolerant. We constructed various chimeric ascorbate peroxidases in which
regions of ascorbate peroxidase B, from sites internal to the C-terminal
end, were exchanged with corresponding regions of the stromal ascorbate
peroxidase of spinach. Analysis of these showed that a region between resi-
dues 245 and 287 was involved in the inactivation by hydrogen peroxide.
A 16-residue amino acid sequence (249–264) found in this region of the
stromal ascorbate peroxidase was not found in other ascorbate peroxidase
isoforms. A chimeric ascorbate peroxidase B with this sequence inserted
was inactivated by hydrogen peroxide within a few minutes. The sequence
forms a loop that binds noncovalently to heme in cytosolic ascorbate per-
oxidase of pea but does not bind to it in stromal ascorbate peroxidase of
tobacco, and binds to cations in both ascorbate peroxidases. The higher
susceptibility of the stromal ascorbate peroxidase may be due to a distorted
interaction of the loop with the cation and ⁄ or the heme.
Abbreviation
APX, ascorbate peroxidase.
2704 FEBS Journal 273 (2006) 2704–2710 ª 2006 The Authors Journal compilation ª 2006 FEBS
cannot proceed due to the absence of ascorbate [5]. In
contrast, the cytosolic [6] and microbody-localized [7]
isoforms are relatively tolerant to hydrogen peroxide.
It is not known why the stromal and thylakoid-bound
APXs are so susceptible to hydrogen peroxide when
others are not.
We have previously isolated a cDNA clone encoding

APX-B from the acidophilic and thermophilic red alga,
Galdieria partita [8]. This APX, like plant cytosolic
and microbody-localized APXs, was tolerant to hydro-
gen peroxide [8]. The amino acid sequence of its N-ter-
minal half was similar to those of the chloroplastic
APXs, whereas the C-terminal half showed a gapped
pattern similar to cytosolic and microbody-localized
APXs of higher plants [8]. This finding raised the
hypothesis that a region within the C-terminal half of
the chloroplastic APXs is involved in their susceptibil-
ity to hydrogen peroxide.
Results
Preparation of chimeric APXs
To test the hypothesis, we prepared a set of chimeric
APX proteins. For Gal
70)208
⁄ Spi
209)365
, Gal
70)244
⁄
Spi
245)365
, Gal
70)287
⁄ Spi
288)365
and Gal
70)298
⁄

Spi
299)365
, N-terminal regions of APX-B (70–208,
70–244, 70–287, and 70–298, respectively) were fused
to the C-terminal region of stromal APX at sites
downstream of residues 208, 244, 287 and 298,
respectively (Fig. 1). All residue numbers used in this
study correspond to stromal APX of spinach. The
first Met of APX-B corresponds to Met70 of the
stromal APX.
With hydrophobic interaction and gel filtration chro-
matography, APX-B was purified to give a single band
in SDS ⁄ PAGE, but the four chimeric APXs and
Fig. 1. Alignment of amino acid sequences of C-terminal half-regions of ascorbate peroxidase (APX)-B and stromal APX of spinach. Recombi-
nation sites for creating chimeric APXs are indicated by vertical bars. Helices were assigned according to the structure of cytosolic APX of
pea [14]. Identical and similar amino acid residues are marked by asterisks and dots, respectively.
Table 1. Enzyme properties of ascorbate peroxidases (APXs). Asc, ascorbate.
Soret peak
a
(nm)
Soret peak
b
K
m
(Asc)
c
(lM) K
m
(H
2

O
2
)
d
(lM) k
cat
(s
)1
Æheme
)1
)
nm m
M
)1
Æcm
)1
Galdieria APX-B 407
e
406 102 117 ± 3
e
42 ± 2
e
2190 ± 41
e
Spinach stromal APX 404 307 ± 29 39 ± 3
Tobacco stromal APX 405 404 105 395 ± 27 22 ± 1 2510 ± 90
Gal
70)208
⁄ Spi
209)365

406 259 ± 36 35 ± 2
Gal
70)244
⁄ Spi
245)365
407 160 ± 28 31 ± 4
Gal
70)287
⁄ Spi
288)365
405 169 ± 10 56 ± 4
Gal
70)298
⁄ Spi
299)365
405 165 ± 7 50 ± 4
Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
407 403 98 256 ± 37 41 ± 6 946 ± 43
a
Measured in the elution buffer (see Experimental procedures).
b
Measured in oxygen-free 50 mM potassium phosphate, pH 7.0.
c
K
m

value
was determined with various concentrations of ascorbate (0–0.5 m
M) and a fixed concentration of hydrogen peroxide (0.1 mM).
d
K
m
value
was determined with various concentrations of hydrogen peroxide (0–0.1 m
M) and a fixed concentration of ascorbate (0.5 mM).
e
See [8].
S. Kitajima et al. Inactivation of stromal ascorbate peroxidase
FEBS Journal 273 (2006) 2704–2710 ª 2006 The Authors Journal compilation ª 2006 FEBS 2705
stromal APX of spinach were still contaminated by
other proteins (data not shown). Specific activities of
the chimeric APX samples per absorbance of Soret
peak (roughly corresponding to heme amount) were
60–80% of that of APX-B, and that of the spinach
stromal APX sample was 140% of that of APX-B. By
comparing specific activities per protein and per
absorbance of Soret peak, the purity of these five
APXs was roughly estimated at 5–30%.
K
m
values for ascorbate and hydrogen peroxide
and wavelengths of the Soret peak of chimeric APXs
are listed in Table 1. The values for the stromal
APX of spinach determined in this study were sim-
ilar to those reported previously [9]. K
m

values
of the chimeric APXs for ascorbate ranged from 160
to 259 lm, and those for hydrogen peroxide from 31
to 56 lm. These values were similar to those of
APX-B and the stromal APX of spinach. For chi-
meric APXs in the elution buffer, the wavelength of
the Soret peak ranged from 405 to 407 nm, lying in
the range between those of the APX-B and the stro-
mal APX of spinach. On the basis of these results,
we judged that these APX samples could be used for
the following experiments.
Susceptibility of Gal
70)208
⁄ Spi
209)365
,
Gal
70)244
⁄ Spi
245)365
, Gal
70)287
⁄ Spi
288)365
and
Gal
70)298
⁄ Spi
299)365
to depletion of ascorbate

To examine the susceptibility of the chimeric APXs
to hydrogen peroxide inactivation, APX solutions
were diluted 100-fold with 50 mm Mes ⁄ KOH buffer,
pH 7.0, and incubated at 25 °C. Bovine serum albu-
min at 10 lgÆml
)1
was also included in the buffer to
eliminate the possibility that small amounts of con-
taminating proteins could interact nonspecifically
with hydrogen peroxide and influence APX inactiva-
tion. This dilution lowered the ascorbate concentra-
tion to 10 lm. Under these conditions, a small
amount of hydrogen peroxide, produced from oxygen
by auto-oxidation of the remaining ascorbate, reacts
with APX [5] and leads to inactivation of the stro-
mal APX. This property has the advantage of allow-
ing a precise comparison of the susceptibility of
various chimeric APXs to hydrogen peroxide; the
concentration of generated hydrogen peroxide and
the rate of inactivation are relatively lower than
when an excess amount of hydrogen peroxide is
exogenously added, and we could determine the half-
time of the inactivation quantitatively.
Whereas recombinant APX-B retained its initial
activity for up to 3 h, the half-inactivation time (t
1 ⁄ 2
)
of the recombinant stromal APX was 13 min. This
value is higher than those for the native enzymes
[5,10], but is similar to that of the recombinant enzyme

reported previously [8,9]. The t
1 ⁄ 2
values for Gal
70-208
⁄
Spi
209-365
, Gal
70)244
⁄ Spi
245-365
and Gal
70-287
⁄ Spi
288-365
were 49, 57 and 289 min, respectively. There was very
little inactivation of Gal
70-298
⁄ Spi
299-365
(Fig. 2A). No
inactivation of any APX was observed in medium sup-
plemented with 0.5 mm ascorbate (Fig. 2B), indicating
that the inactivation is due to depletion of ascorbate
and subsequent generation of hydrogen peroxide. The
chimeric APXs containing amino acid residues 209–
365 and 245–365 of the stromal APX showed more
20
40
60

80
100
120
Remaining activities (%)
A
B
0
20
40
60
80
100
Remaining activities (%)
0
0 306090120150180
Incubation time at 25ºC (min)
Fig. 2. Effect of ascorbate depletion on the activity of ascorbate
peroxidases (APXs). Each protein solution was diluted with 50 m
M
Mes ⁄ KOH buffer, pH 7.0, supplemented with 10 lgÆml
)1
of bovine
serum albumin without or with 0.5 m
M ascorbate, to give final con-
centrations of 10 l
M ascorbate (A) or 0.5 mM ascorbate (B),
respectively. After incubation at 25 °C for the indicated times, the
remaining activities were determined. Closed square, APX-B; open
square, Gal
70)208

⁄ Spi
209)365
; closed circle, Gal
70)244
⁄ Spi
245)365
;
open circle, Gal
70)287
⁄ Spi
288)365
; closed triangle, Gal
70)298
⁄
Spi
298)365
; open triangle, stromal APX of spinach. The initial activit-
ies of APX-B (36 n
M), Gal
70)208
⁄ Spi
209)365
,Gal
70)244
⁄ Spi
245)365
,
Gal
70)287
⁄ Spi

288)365
,Gal
70)298
⁄ Spi
298)365
and stromal APX of spin-
ach were 0.17, 0.019, 0.054, 0.097, 0.079, 0.10 lmol ascor-
bateÆmin
)1
Æml
)1
, respectively. The standard deviations of five
measurements are indicated (bars).
Inactivation of stromal ascorbate peroxidase S. Kitajima et al.
2706 FEBS Journal 273 (2006) 2704–2710 ª 2006 The Authors Journal compilation ª 2006 FEBS
rapid inactivation than that containing the sequence
from 288 to 365. This suggests that the amino acid res-
idues from 245 to 287 of the stromal APX are import-
ant in determining susceptibility to hydrogen peroxide.
Susceptibility of Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
to hydrogen peroxide
A region from 245 to 287 of the stromal APX is a part
of a loop (231–274) located near the heme molecule
[11] and contains a unique 16 amino acid sequence
between residues 248 and 265 which is not found in

cytosolic APX of higher plants and APX-B (Fig. 1)
[8,11,12]. To examine the function of this insertion in
determining hydrogen peroxide susceptibility, we cre-
ated another chimeric APX (Gal
70)244
⁄ Spi
245)273
⁄
Gal
274)337
). Here, the sequence from amino acid resi-
dues 245–273 of APX-B was substituted by the corres-
ponding region of the stromal APX with the 16 amino
acid insertion (Fig. 1). Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
could be purified to give a single band in SDS ⁄ PAGE
(data not shown).
The wavelengths of the Soret peaks of APX-B
and Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
were 406 nm
(102 mm

)1
Æcm
)1
) and 403 nm (98 mm
)1
Æcm
)1
), respect-
ively, in oxygen-free 50 mm potassium phosphate buf-
fer, pH 7.0 (Table 1). Upon reduction by dithionite,
the Soret peaks shifted to 435 nm (108 mm
)1
Æcm
)1
)
and 434 nm (102 mm
)1
Æcm
)1
) with the b-peak at 555
and 556 nm, respectively. A cyanide complex of the
oxidized form gave peaks at 420 nm (112 mm
)1
Æcm
)1
)
and 420 nm (109 mm
)1
Æcm
)1

) with the b-peak at 542
and 542 nm, respectively (Fig. 3A,B). The K
m
value
for ascorbate is in the range of values for the parental
APXs. The K
m
value for hydrogen peroxide was sim-
ilar to those of both parental APXs. The k
cat
value cal-
culated from the maximum activity and heme contents
was decreased but remained at no less than 43%
(946 s
)1
Æheme
)1
) of that of APX-B (Table 1). These
facts suggested that interaction of the heme molecule
with neighboring amino acid residues and water mole-
cules of the active site was not significantly changed,
except for interaction with the loop (described below).
The susceptibility of Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
to hydrogen peroxide was compared with that of
APX-B and stromal APX. In this experiment, we used

recombinant stromal APX of tobacco for comparison
instead of that of spinach, because the tobacco stromal
APX could be purified to give a single band in
SDS ⁄ PAGE (data not shown). The absorption coeffi-
cients at the Soret peak of the tobacco stromal APX
was 105 mm
)1
Æcm
)1
(404 nm) in oxygen-free 50 mm
potassium phosphate buffer, pH 7.0 (Table 1).
Even when ascorbate was removed, the enzymes
were not inactivated if the enzyme solution was kept
oxygen-free (Fig. 4A–C). When 20 equivalents of
hydrogen peroxide relative to APX was added, APX-B
retained approximately 40% of the initial activity even
after incubation for 10 min (Fig. 4A). Gal
70)244
⁄
Spi
245)273
⁄ Gal
274)337
lost enzyme activity within a few
minutes (Fig. 4B), as did tobacco stromal APX
(Fig. 4C). Rapid inactivation was also observed in
A
0
2
4

6
8
10
12
14
16
0
84
0
8
60
3
6
0
8
50
3
5
m
n
m
M
-1
•cm
-1
er
tnu
deta
NCK
et

in
o
ih
tid
0
20
40
60
80
100
120
300 700600500400
nm
m
M
-1
•cm
-1
B
0
2
4
6
8
10
12
14
16
0
8408

60
3608
5
0
35
m
n
m
M
-1
•cm
-1
ertnudeta
etinoihtid
NCK
ertnudeta
N
C
K
etinoihtid
300 700600500400
nm
0
20
40
60
80
100
120
m

M
-1
•cm
-1
ertnudeta
etinoih
t
id
N
C
K
Fig. 3. Absorption spectra of ascorbate peroxidase (APX)-B and Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
. APX-B (A) and Gal
70)244
⁄ Spi
245)273
⁄
Gal
274)337
(B) in oxygen-free 50 mM potassium phosphate buffer, pH 7.0, were analyzed. —, untreated; - - -, treated with dithionite; – Æ –,
treated with 0.2 m
M potassium cyanide.
S. Kitajima et al. Inactivation of stromal ascorbate peroxidase
FEBS Journal 273 (2006) 2704–2710 ª 2006 The Authors Journal compilation ª 2006 FEBS 2707
similar experiments using crude soluble extract of

Escherichia coli containing recombinant stromal APX
of spinach [13] and thylakoid-bound APX purified
from spinach [5]. These results indicated that an inser-
ted loop region of stromal APX is involved in its
hydrogen peroxide-mediated inactivation.
Discussion
In this study, by using various chimeric APXs
between hydrogen peroxide-tolerant APX-B and sen-
sitive stromal APX, we have proved the hypothesis
that a region in the C-terminal half of stromal APX
is involved in its susceptibility to hydrogen peroxide
and indicated that this region is in a loop unique to
chloroplastic APXs. However, it is not presently
known why this region accelerates the hydrogen perox-
ide-mediated inactivation. In cytosolic APX of pea [14]
and stromal APX of tobacco [11], the loop binds to a
cation located near a Trp residue (Trp265 in Fig. 5) at
the proximal side of the heme. In the usual catalytic
cycle, the increase in electrostatic potential caused by
the cation is thought to prevent radical transfer from
the pophyrin of compound I to the proximal Trp
[14,15]. However, the radical is suggested to be trans-
ferred to the Trp when ascorbate is absent [16]. The
loop structure may therefore influence the location of
the radical through interaction with the cation and
thus affect the reaction of radicals to excess hydrogen
peroxide.
Alternatively, higher susceptibility may be due to
lack of binding of the loop to the heme. In contrast to
cytosolic APX, whose loop binds noncovalently to a

propionate side chain of porphyrin at His239 (Fig. 5A)
[14], Arg239 of stromal APX binds to Ala259 and
Pro260, and thus the loop cannot bind to it (Fig. 5B)
[11]. Consequently, the heme in stromal APX is more
loosely associated with the apoprotein than in cytosolic
APX, and the structure of the catalytic site may be
easily disordered when it reacts with hydrogen per-
oxide.
Considering the sequence similarity of the thylakoid-
bound APX isoform to stromal APX [17], the long
loop is probably involved in inactivation of thylakoid-
bound APX in the same way as in stromal APX.
One might expect that the stromal APX would
lose susceptibility to hydrogen peroxide through
removal of the inserted sequence of the loop region.
We created a gene for such a chimeric APX by
inserting the loop region of APX-B (lacking insert)
into the stromal APX of spinach. Unfortunately, we
could not test this idea, because crude extracts of
E. coli harboring the chimeric APX gene exhibited
neither APX activity nor Soret absorption, for rea-
sons that are unknown.
In conclusion, we have shown that a unique loop
structure is involved in susceptibility of stromal APX
to hydrogen peroxide. However, the molecular mech-
anism of this inactivation is still unknown. To assess
this question, structural changes to the heme and sur-
rounding amino acid residues of the inactivated APX
should be clarified. It would also be interesting to
investigate why such a feature was conserved during

the evolution of chloroplastic APXs, despite high sus-
ceptibility to hydrogen peroxide being a disadvantage
for plant adaptation to the land environment.
0
20
40
60
80
100
120
0
20
40
60
80
100
A
B
0
20
40
60
80
100
0 120 240 360 480 600
C
Incubation time at 25ºC (s)
Remaining activities (%) Remaining activities (%) Remaining activities (%)
Fig. 4. Effect of excess amounts of hydrogen peroxide on the activ-
ity of ascorbate peroxidases (APXs). APXs in oxygen-free 50 m

M
potassium phosphate buffer, pH 7.0, were mixed with (open circle)
or without (closed circle) 20 equivalents of hydrogen peroxide relat-
ive to APX. After incubation at 25 °C for the indicated times, the
remaining activities were determined. (A) APX-B. (B) Gal
70)244
⁄
Spi
245)273
⁄ Gal
274)337
. (C) Stromal APX of tobacco. The concentrat-
ions of APXs in the solution were 7.0, 3.6 and 4.4 l
M for APX-B,
Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
and stromal APX, respectively. The
standard deviations of five measurements are indicated (bars).
Inactivation of stromal ascorbate peroxidase S. Kitajima et al.
2708 FEBS Journal 273 (2006) 2704–2710 ª 2006 The Authors Journal compilation ª 2006 FEBS
Experimental procedures
Construction of plasmids for expression
of chimeric APXs in Escherichia coli
Sequences for chimeric APXs between stromal APX and
APX-B were amplified by two successive rounds of PCR.
PCR amplification was performed with a pfu turbo DNA

polymerase (Stratagene, La Jolla, CA). A pET16b (Nov-
agen, Madison, WI) vector that contained the DNA
sequence for APX-B [8] and a pET3a (Novagen) vector that
contained the truncated DNA sequence for stromal APX of
spinach [9] were used as the templates for PCR. Suitable
DNA fragments produced by PCR were ligated using T4
DNA ligase (Takara Bio, Ohtsu, Shiga, Japan). The liga-
tion products were amplified with two of the 5¢- and 3¢-end
primers and digested with NcoI and XhoI for cloning into
pET16b downstream of the T7 promoter. A cDNA of
tobacco stromal APX was cloned by RT-PCR from total
RNA extracted from leaves of Nicotiana tabacum cv.
‘Xanthi’. The 5¢- and 3¢-end primers (5¢-AGATATCCA
TGGGCGCCGCGTCTGATTCTGATCAGTTG-3¢ and
5¢-CCCCCTCGAGGGCAAATTAAAACAAACGGCAGA
AC-3¢, respectively) for PCR were designed according to
the nucleotide sequence of tobacco stromal APX (accession
number AB022274), with some modifications to create the
NcoI site (CCATGG) at the putative cleavage site of the
transit peptide and the XhoI site (CTCGAG) downstream
of the stop codon. The cDNA fragment thus amplified was
inserted into the NcoI ⁄ XhoI site downstream of the T7 pro-
moter of pET16b. Amplified fragments were confirmed for
accuracy by sequencing.
Purification of the proteins
Recombinant APXs, except for Gal
70)244
⁄ Spi
245)273
⁄

Gal
274)337
and the stromal APX of tobacco, were produced
in E. coli strain BL21 (DE3). The proteins were purified
using a HiLoad 16 ⁄ 10 Phenylsepharose HP column (Amer-
sham Bioscience, Piscataway, NJ) and a HiLoad 26 ⁄ 60
Superdex 75 prep grade column (Amersham Biosciences) as
previously described for purification of recombinant stro-
mal APX of spinach [8]. To purify Gal
70)244
⁄ Spi
245)273
⁄
Gal
274)337
and the stromal APX of tobacco, the crude pro-
teins extracted from E. coli were fractionated using a Hi-
Prep 16 ⁄ 10 DEAE FF column (Amersham Biosciences) as
described by Yoshimura et al. [9], prior to loading onto the
HiLoad 16 ⁄ 10 Phenylsepharose HP and HiLoad 26 ⁄ 60 Su-
perdex 75 prep grade columns. Purified APXs in the elution
buffer (10 mm potassium phosphate buffer, pH 7.0, 1 mm
EDTA, 1 mm ascorbate, 0.15 m KCl) were concentrated by
Amicon Centriprep YM-10 (Millipore, Bedford, MA) and
stored at ) 80 °C. Protein concentration was determined by
the procedure of Bradford [18] with bovine serum albumin
as the standard. APX samples other than APX-B, tobacco
stromal APX and Gal
70)244
⁄ Spi

245)273
⁄ Gal
274)337
were
shown by SDS ⁄ PAGE to be contaminated by other pro-
teins (data not shown).
Absorption coefficients of purified APX-B, tobacco stro-
mal APX and Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
were deter-
mined according to their heme contents and absorption
spectra. Heme content was determined by the pyridine
hemochromogen method [19], using dog heart myoglobin
(Sigma-Aldrich, Tokyo, Japan) as the standard. Heme con-
tents per polypeptide of APX-B, tobacco stromal APX or
Gal
70)244
⁄ Spi
245)273
⁄ Gal
274)337
were 55–65%, indicating
that 45–35% of the polypeptide was the apoenzyme.
Enzyme assay
APX activity was measured at 25 °C in a reaction mixture
that contained 50 mm sodium phosphate, pH 7.0, and

0.5 mm ascorbic acid. The reactions were initiated by addi-
tion of hydrogen peroxide to a final concentration
of 0.1 mm. The hydrogen peroxide-dependent oxidation of
ascorbate was monitored by the decrease in absorbance of
Trp-265
His-239
Trp-265
His-239
A
Trp-265
Arg-239
Ala-259
Pro-260
B
Trp-265Trp-265
Arg-239
Ala-259
Pro-260
Fig. 5. The structure of the active site in cytosolic ascorbate peroxidase (APX) of pea (A) and stromal APX of tobacco (B). The loop structure
between helices F and G is shown as a blue line. The 16-residue insert is shown as a green line. Hydrogen bonds are indicated by a broken
line. Fe, nitrogen, oxygen and the cation near Trp265 are shown in cyan, blue, red and magenta, respectively. Residue numbers refer to stro-
mal APX of spinach; see Fig. 1. These structures were drawn using the program
PYMOL ( />S. Kitajima et al. Inactivation of stromal ascorbate peroxidase
FEBS Journal 273 (2006) 2704–2710 ª 2006 The Authors Journal compilation ª 2006 FEBS 2709
ascorbate at 290 nm (e ¼ 2.8 mm
)1
Æcm
)1
). The concentra-
tion of hydrogen peroxide was determined from the absorp-

tion at 240 nm (e ¼ 0.0394 mm
)1
Æcm
)1
).
Preparation of ascorbate- and oxygen-free APX
solution
APX in the elution buffer was passed twice through Sepha-
dex G25 columns (NAP5 and PD10 columns; Amersham
Biosciences) equilibrated with 50 mm potassium phosphate,
pH 7.0. The equilibration buffer and solutions of eluted
APX were thoroughly degassed by flushing with N
2
gas.
Prior to analysis, APX concentration was determined from
the absorption of heme.
Acknowledgements
We thank Ms Yuki Shinzaki, Mr Yukihisa Yamauchi
and Ms Satoko Sugahara for their technical assistance.
We thank Dr Shigeharu Harada for helpful advice on
drawing three-dimensional structures of APXs. This
study was partly supported by the Petroleum Energy
Center and the Research Association for Biotechno-
logy, subsidized by the Ministry of Economy, Trade
and Industry of Japan.
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