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 Kitajima1*, Ken-ichi Tomizawa1, Shigeru Shigeoka2and Akiho Yok

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation Sakihito Kitajima1*, Ken-ichi Tomizawa1, Shigeru Shigeoka2and Akiho Yokota3

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

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 (FeIII) of the

heme moiety is oxidized to the oxyferryl (FeIV¼ 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: yokota@bs.naist.ac.jp

*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 perper-oxidase 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.

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 Gal70)208⁄ Spi209)365, Gal70)244⁄ Spi245)365, Gal70)287⁄ Spi288)365 and Gal70)298⁄ Spi299)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

Km(Asc) c (l M ) Km(H2O2) d (l M ) kcat(s)1Æheme)1)

nm m M )1Æcm)1

a

Measured in the elution buffer (see Experimental procedures).bMeasured in oxygen-free 50 m M potassium phosphate, pH 7.0.cK m value was determined with various concentrations of ascorbate (0–0.5 m M ) and a fixed concentration of hydrogen peroxide (0.1 m M ) d Kmvalue was determined with various concentrations of hydrogen peroxide (0–0.1 m M ) and a fixed concentration of ascorbate (0.5 m M ) e See [8].

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%

Km 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] Km 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 Gal70)208⁄ Spi209)365,

Gal70)244⁄ Spi245)365, Gal70)287⁄ Spi288)365and

Gal70)298⁄ Spi299)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 condiconcentra-tions, 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 (t1⁄ 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 t1⁄ 2 values for Gal70-208⁄ Spi209-365, Gal70)244⁄ Spi245-365 and Gal70-287⁄ Spi288-365 were 49, 57 and 289 min, respectively There was very little inactivation of Gal70-298⁄ Spi299-365 (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

A

B

0

20 40 60 80 100

0

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)1of 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 m M ascorbate (B), respectively After incubation at 25
C for the indicated times, the remaining activities were determined Closed square, APX-B; open square, Gal70)208⁄ Spi 209 )365; closed circle, Gal70 )244 ⁄ Spi 245 )365; open circle, Gal70)287⁄ Spi288)365; closed triangle, Gal70)298⁄ Spi298)365; open triangle, stromal APX of spinach The initial activit-ies of APX-B (36 n M ), Gal70)208⁄ Spi 209 )365, Gal70 )244 ⁄ Spi 245 )365, Gal70)287⁄ Spi288)365, Gal70)298⁄ Spi298)365and stromal APX of spin-ach were 0.17, 0.019, 0.054, 0.097, 0.079, 0.10 lmol ascor-bateÆmin)1Æml)1, respectively The standard deviations of five measurements are indicated (bars).

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 Gal70)244⁄ Spi245)273⁄ Gal274)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 (Gal70)244⁄ Spi245)273⁄

Gal274)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) Gal70)244⁄ Spi245)273⁄ Gal274)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 Gal70)244⁄ Spi245)273⁄ Gal274)337 were 406 nm

(102 mm)1Æcm)1) and 403 nm (98 mm)1Æcm)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)1Æcm)1)

and 434 nm (102 mm)1Æcm)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)1Æcm)1)

and 420 nm (109 mm)1Æcm)1) with the b-peak at 542 and 542 nm, respectively (Fig 3A,B) The Km value for ascorbate is in the range of values for the parental APXs The Km value for hydrogen peroxide was sim-ilar to those of both parental APXs The kcatvalue cal-culated from the maximum activity and heme contents was decreased but remained at no less than 43% (946 s)1Æheme)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 Gal70)244⁄ Spi245)273⁄ Gal274)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)1Æcm)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) Gal70)244⁄ Spi245)273⁄ Gal274)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

m n

e r t

n a t d

N K

e t n i h t d

0

20

40

60

80

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120

nm

-1•c

B

0 2 4 6 8 10 12 14 16

0

m n

e r t

n a t d

e t n i h t d

N K

e r t

n a t d

N K

e t n i h t d

nm 0

20 40 60 80 100 120

-1•c

e

r

t

n a t d

e t n

i

h

t

d

N K

Fig 3 Absorption spectra of ascorbate peroxidase (APX)-B and Gal70)244⁄ Spi 245 )273 ⁄ Gal 274 )337 APX-B (A) and Gal70 )244 ⁄ Spi 245 )273 ⁄ Gal274)337(B) in oxygen-free 50 m M potassium phosphate buffer, pH 7.0, were analyzed —, untreated; - - -, treated with dithionite; – Æ –, treated with 0.2 m M potassium cyanide.

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

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120

0

20

40

60

80

100

A

B

0

20

40

60

80

100

C

Incubation time at 25ºC (s)

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) Gal70)244⁄

Spi245)273⁄ Gal274)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,

Gal70)244⁄ Spi245)273⁄ Gal274)337and stromal APX, respectively The

standard deviations of five measurements are indicated (bars).

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

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 Gal70)244⁄ Spi245)273⁄

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 Gal70)244⁄ Spi245)273⁄

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 Gal70)244⁄ Spi245)273⁄ Gal274)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 Gal70)244⁄ Spi245)273⁄ Gal274)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

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-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 (http://pymol.sourceforge.net/).

ascorbate at 290 nm (e¼ 2.8 mm)1Æcm)1) The

concentra-tion of hydrogen peroxide was determined from the

absorp-tion at 240 nm (e¼ 0.0394 mm)1Æcm)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 N2 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

References

1 Welinder KG (1992) Superfamily of plant, fungal and

bacterial peroxidases Curr Opin Struct Biol 2, 388–393

2 Asada K (1999) The water–water cycle in chloroplasts:

scavenging of active oxygens and dissipation of excess

photons Annu Rev Plant Physiol Plant Mol Biol 50, 601–

639

3 Yoshimura K, Yabuta Y, Ishikawa T & Shigeoka S

(2000) Expression of spinach ascorbate peroxidase

iso-enzymes in response to oxidative stresses Plant Physiol

123, 223–233

4 Mano J, Ohno C, Domae Y & Asada K (2001)

Chloro-plastic ascorbate peroxidase is the primary target of

methyl viologen-induced photooxidative stress in

spi-nach leaves: its relevance to monodehydroascorbate

radical detected with in vivo ESR Biochim Biophys Acta

1504, 275–287

5 Miyake C & Asada K (1996) Inactivation mechanism

of ascorbate peroxidase at low concentrations of

ascorbate; hydrogen peroxide decomposes Compound

I of ascorbate peroxidase Plant Cell Physiol 37, 423–

430

6 Asada K (1992) Ascorbate peroxidase: a hydrogen

peroxi-dase scavenging system in plants Physiol Plant 85, 235–

241

7 Ishikawa T, Yoshimura K, Sakai K, Tamoi M, Takeda

T & Shigeoka S (1998) Molecular characterization and

physiological role of a glyoxysome-bound ascorbate per-oxidase from spinach Plant Cell Physiol 39, 23–34

8 Kitajima S, Ueda M, Sano S, Miyake C, Kohchi T, Tom-izawa K, Shigeoka S & Yokota A (2002) Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants Biosci Biotechnol Biochem 66, 2367–2375

9 Yoshimura K, Ishikawa T, Nakamura Y, Tamoi M, Takeda T, Tada T, Nishimura K & Shigeoka S (1998) Comparative study on recombinant chloroplastic and cytosolic ascorbate peroxidase isozymes of spinach Arch Biochem Biophys 353, 55–63

10 Nakano Y & Asada K (1987) Purification of ascorbate peroxidase in spinach chloroplast; its inactivation in ascorbate-depleted medium and reactivation by mono-ascorbate radical Plant Cell Physiol 28, 131–140

11 Wada K, Tada T, Nakamura Y, Ishikawa T, Yabuta Y, Yoshimura K, Shigeoka S & Nishimura K (2003) Crys-tal structure of chloroplastic ascorbate peroxidase from tobacco plants and structural insights into its instability

J Biochem 134, 239–244

12 Jespersen HM, Kjaersgard IV, Ostergaard L & Welinder

KG (1997) From sequence analysis of three novel ascor-bate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate per-oxidase Biochem J 326, 305–310

13 Sano S, Ueda M, Kitajima S, Takeda T, Shigeoka S, Kur-ano N, Miyachi S, Miyake C & Yokota A (2001) Charac-terization of ascorbate peroxidases from unicellular red alga Galdieria partita Plant Cell Physiol 42, 433–440

14 Patterson WR & Poulos TL (1995) Crystal structure

of recombinant pea cytosolic ascorbate peroxidase Biochemistry 34, 4331–4341

15 Cheek J, Mandelman D, Poulos TL & Dawson JH (1999) A study of the K+-site mutant of ascorbate per-oxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side J Biol Inorg Chem 4, 64–72

16 Hiner AN, Martinez JI, Arnao MB, Acosta M, Turner

DD, Lloyd Raven E & Rodriguez-Lopez JN (2001) Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide Eur J Biochem 268, 3091–3098

17 Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y & Yoshimura K (2002) Regulation and function of ascorbate peroxidase isoenzymes J Exp Bot 53, 1305–1319

18 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein util-izing the principle of protein-dye binding Anal Biochem

72, 248–254

19 Tomita T, Tsuyama S, Imai Y & Kitagawa T (1997) Purification of bovine soluble guanylate cyclase and ADP-ribosylation on its small subunit by bacterial tox-ins J Biochem 199, 531–536