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: 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 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 (http://pymol.sourceforge.net/). 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. 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. 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An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation Sakihito Kitajima 1 *,. region of stromal APX is involved in its hydrogen peroxide-mediated inactivation. Discussion In this study, by using various chimeric APXs between hydrogen