Spectroscopic Evidence That Salicylic Acid ... - Semantic Scholar

Biosci. Biotechnol. Biochem., 66 (3), 646–650, 2002

Note

Spectroscopic Evidence That Salicylic Acid Converts a Temporally Inactivated Form of Horseradish Peroxidase (Compound III) to the Irreversibly Inactivated Verdohemoprotein (P-670) Tomonori KAWANO,*,**,† Shoshi MUTO,*** Masaru ADACHI,**** Hiroshi HOSOYA,***** and Fr áed áeric LAPEYRIE* *UMR INRA-UHP Interactions Arbre W Micro-Organismes, Centre Institut National de la Recherche Agronomique de Nancy, F-54280 Champenoux, France **Present address: Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan ***Nagoya University Bioscience Center, Chikusa-ku, Nagoya 464-8601, Japan ****Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-2192, Japan *****Department of Biological Science, Graduate School of Science and PRESTO-JST, Hiroshima University, Higashi-Hiroshima 739-8526, Japan Received August 13, 2001; Accepted November 16, 2001

We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidasecatalyzed reactions are discussed. Key words:

Compound III; horseradish peroxidase; P-670; salicylic acid; verdohemoprotein

Salicylic acid (SA) is well recognized as a plantdefense-inducing chemical produced by most higher plants in response to pathogen attacks.1,2) In our previous study on the mechanism for production of reactive oxygen species during the defense-induction processes in plants, especially in suspension-cultured tobacco cells3) and epidermal cells of Vicia fava4) used as model systems, we observed that SA plays a key role in extracellular superoxide-anion (O2„)-generating reaction catalyzed by extracellularly localized peroxidases secreted from the cells.3) The peroxidase's catalytic cycle coupled to consumption of H2 O2 is well known for oxidation of various substrates. In the presence of H2 O2, the native form of enzyme (FeIII) is converted to Compound I (heme with FeIV＝O and porphyrin radicals) that catalyzes the oxidation of substrates by converting itself into Compound II (FeIV＝O). Compound II also catalyzes the oxidation of substrates while reconverting itself into the native form, and thus the

peroxidase cycle is completed. Based on the results from chemiluminescence studies3) and electron spin resonance spectroscopic experiments,6) we have proposed a hypothesis that Compounds I and II of tobacco enzymes catalyze the monovalent oxidation of SA, yielding SA free radicals, and SA free radicals may be involved in a subsequent series of reactions producing O2„. Similarly to SA, other peroxidase substrates rich in tobacco plants7,8) and pathogenic fungi9) are also monovalently oxidized into free radical species. These free radical species may be the mediators for the peroxidase-dependent O2„ generating reactions in the presence of dissolved oxygen. It has been proposed that not only guaiacol-utilizing plant peroxidases but also ascorbate peroxidase can utilize SA as a substrate by a similar mechanism.5) In our previous studies, we have employed a major horseradish peroxidase isoenzyme belonging to the C group (HRP-C) as a model guaiacol-utilizing peroxidase for studying the mechanism of SA-oxidizing reactions commonly observed for many plant peroxidases, and conˆrmed that HRP-C also catalyzes the SA-dependent O2„ generating reaction.6) In addition, we obtained the electron spin resonance spectroscopic evidence in support of HRP-C-catalyzed formation of SA free-radical species from SA6). In addition to SA's role as a substrate in the peroxidase cycle-dependent O2„ generation, here we report a novel behavior of SA as an irreversible inactivator of HRP-C in the presence of excess H2 O2. It is known that the native form of HRP-C is readily converted to the temporal inactive state known as Com-

To whom corresponding should be addressed. Fax: +81-824-24-0734; E-mail: kawanotom＠sci.hiroshima-u.ac.jp Abbreviations : HRP-C, horseradish peroxidase C; P-670, horseradish peroxidase-derived verdohemoprotein; SA, salicylic acid

†

Irreversible Inactivation of Peroxidase by Salicylate

pound III (FeII＝O2, FeIII-O ), and also in the presence of excess of H2 O2 (probably releasing a cerComtain amount of O2„ required for HRP-C W pound III conversion), the enzyme is arrested at Compound III.11) We observed that the irreversibly inactivated protein known as the verdohemoprotein (named after its green color) or P-670 pigment (named after its absorption maximum at 670 nm) was produced from Compound III in the presence of SA. SA, HRP-C, and other chemicals were purchased from Sigma (St. Louis, MO., USA). HRP-C was used without further puriˆcation. The concentration of HRP-C was measureed spectroscopically (e403 nm＝ 102 mM„1・cm„1 ).10) Absorption spectra of HRP-C dissolved in 1 ml of 20 mM K-phosphate (pH 6.0) were recorded on a Beckman DU-70 spectrophotometer at room temperature with a spectral bandwidth of 1.0 nm in a cuvette with a 1-cm light path. Native enzyme has absorption maxima at 404, 500, and 639 nm (Fig. 1A, absorption maxima at 500 and 639 nm are shown). Compound I which has characteristic absorption maxima at 404, 550, and 650 nm, is reportedly formed after addition of 0.5–1.5 molar equivalents of H2 O2 to HRP-C.12) However, Compound I was not stable and was easily converted to Compound II that has characteristic absorption maxima at 420, 527, and 556 nm. By addition of 2–15 molar equivalents of H2 O2, accumulation of Compound II was stably observed (Fig. 1B, absorption maxima at 527 and 556 nm are shown). In the presence of 40–100 molar equivalents of H2 O2, absorption spectra of HRP-C showed absorption maxima at 415, 545, and 578 nm (Fig. 1C, absorption maxima at 545 and 578 nm are shown) indicating that the majority of HRP-C molecules has been converted to Compound III, a temporal inactive form of HRPC.12,13) As previously reported by Adediran,14) Compound III of HRP-C was very stable at neutral and high pH, and also at pH 6.0 no decay of the absorption maxima (at 545 and 578 nm) was observed within 15 min (data not shown). Addition of SA to native HRP-C induced no changes in the absorption spectrum, and the enzymes arrested at Compounds I and II are readily reduced back to the native form by SA, conˆrming that SA is a good substrate for the H2 O2-requiring catalytic cycle of HRP-C (data not shown). Here in this study, the eŠects of SA on Compound III were examined. SA (3–30 molar equivalents to HRP-C) was added to Compound III generated by addition of H2 O2 (100 molar equivalents) to HRP-C (Fig. 2). Absorption spectra were recorded immediately after addition of various concentrations of SA to the HRP-C reaction mixture rich in Compound III. We observed the spontaneous decay in the characteristic absorption doublet (545 and 578 nm) of Compound III, indicating that SA spontaneously

647

„ 2

Fig. 1. Spectral Features of HRP-C in the Presence and Absence of H2 O2. Typical absorption spectra collected for 10 mM HRP-C dissolved in 20 mM K-phosphate buŠer (pH 6.0) are shown. A, Native enzyme. Arrowheads indicate the absorption maxima at 500 and 639 nm. B, Compound II, generated from HRP-C in the presence of 100 mM H 2 O2 (10 molar equivalent to HRP-C). Arrowheads indicate absorption maxima at 527 and 556 nm. C, Compound III, generated from HRP-C in the presence of 1 m M H2 O2 (100 molar equivalents to HRP-C). Arrowheads indicate absorption maxima at 545 and 578 nm. For (B) and (C), spectra were obtained immediately after addition of H 2 O2 to native HRP-C.

converted the Compound III to other forms of HRPC. On the other hand, an increase in absorption maximum at 670 nm was observed. It is known that an increase in the absorption maximum at 670 nm in any heme-containing protein indicates the formation of P-670, the irreversibly inactivated from of hemoproteins, so-called verdohemoproteins.15,16) Even in the absence of SA, Compound III is slowly converted to P-670, taking several hours. In contrast, the SAenhanced conversion of Compound III to P-670 was shown to be very rapid. This indicates that SA e‹ciently accelerates the conversion of Compound

648

T. KAWANO et al.

Fig. 3. Interaction between SA and HRP-C. Interrelationship among ˆve redox states of HRP-C and P-670 are illustrated. Numbers (2–6) indicate the formal oxidation state of heme. Reaction paths 3ª5ª4ª3 indicate one catalytic cycle of the enzyme. O2„, superoxide; SA+, an intermediate product of SA oxidation; SA, SA free radicals.

Fig. 2. EŠects of SA on Formation of P-670 from Compound III. Typical data from 4 sets of experiments are shown. A, Changes in absorption spectra after addition of SA to Compound III (derived from 10 mM HRP-C). Arrows indicate the increase and decrease in absorption. The spectrum recorded in the absence of SA shows the highest absorbency for Compound III and the lowest for P-670. The spectrum recorded after addition of 300 mM SA (30 molar equivalent) shows the lowest Compound III and the highest P-670. The spectrum with 150 mM SA (15 molar equivalent) shows the intermediate level of absorbance. B, eŠects of SA concentration on the formation of P-670.

III to P-670, thus irreversibly inactivating the enzyme. Since the conversion of HRP-C to Compound III is reversible, Compound III is often considered as a source of O2„. Especially in the reaction mixtures containing indole-3-acetic acid (IAA), plant peroxidases (but not non-plant peroxidases such as myeloperoxidase) are readily converted to Compound III by a complex mechanism.10) When Compound III is re-converted to native enzyme, bound oxygen is released as O2„.10) These IAA-dependent reactions catalyzed by HRP-C can be followed by the O2„speciˆc chemiluminescence of a Cypridina luciferin analog.17) However, in the SA-dependent O2„ generating peroxidase reactions, O2„ is supplied from the conventional peroxidase cycle other than the Com-

pound III forming reaction.6) Instead of release of O2„, formation of Compound III may lower the level of SA-dependent generation of O2„, since Compound III is not capable of SA oxidation. Conversion of Compound III to verdohemoproteins (P-670) may further lower the concentration of catalytic active enzyme. In our previous reports, we summarized that SA plays multiple roles in the plants.3,6) First, SA inhibits the decomposition of H2 O2 by catalase by binding to it (i), and that by a Fenton-type reaction by chelating the catalytic metal ions such as Fe 2+ and Cu+ (ii). As a consequence, SA lowers the production of hydroxy radicals (iii) by blocking the Fenton-type reaction. In addition, SA behaves as an eŠective scavenger of hydroxy radicals (iv). Thus, SA protects the cellular components from highly reactive hydroxy radicals (v). On the other hand, SA promotes the production of less reactive oxygen radical species, O2„ via peroxidase reaction (vi). In the P-670 forming reaction occurring in plant tissues, SA may contribute to the maintenance of the H2 O2 concentration required for total reactions, and Compound III-conversion enhancement of HRP-C W by generation of O2„. In this study, we initiated the conversion of HRP-C to Compound III by addition of excess of H2 O2, so that eŠects of SA on H2 O2 concentration and generation of O2„ can be ignored. Due to SA's reactivity against hydroxy radicals and

Irreversible Inactivation of Peroxidase by Salicylate

inhibitory action against the hydroxy radical-generating reactions, we can eliminate the possibility that hydroxy radicals are involved in the SA-induced inactivation of HRP-C. However, at present, we cannot propose the mechanism involved in SA-induced P-670 formation. In conclusion, roles for SA in HRP-C-catalyzed reactions are summarized in Fig. 3. Compound III is a complex (FeII-O2 ) formed from heme and O2„, thus production of 1 molar equivalent O2„ (rather than 40–100 molar equivalents of H2 O2 ) eŠectively converts the native form of HRP-C to Compound III.12) As we have previously reported,6) addition of SA to HRP-C produces O2„ via a catalytic cycle requiring H2 O2, thus the SA-induced O2„ may contribute to the conversion of the native enzyme to Compound III. Furthermore, the resultant Compound III may react with SA, ˆnally yielding P-670. As a consequence, SA may accelerate the irreversible conversion of Compound III to P-670. This may partially explain why the SA-dependent O2„ production in HRP-C reaction mixture is only transient.6) Peroxidases play multiple roles in plant defense mechanisms. As noted in our previous studies3,7–9) and other plant systems,18,19) peroxidases may contribute to the production of reactive oxygen species such as O2„. On the other hand, plant peroxidases largely contribute to the expense of reactive oxygen species by consuming H2 O2. Since P-670 no longer participates in either the production or removal of reactive oxygen species, SA action on conversion of active enzyme to P-670 may wave a great impact on the plant's ability to control the level of reactive oxygen species. Our ˆnding may induce discussion over the action of SA and peroxidases during plant defense mechanism.

5)

6)

7)

8)

9)

10)

11)

12)

References 1)

2)

3)

4)

Delaney, T. P., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., GaŠney, T., GulRella, M., Kessmann, H., Ward, E., and Ryals, J., Central role of salicylic acid in plant defense resistance. Science, 266, 1247–1250 (1994). Conrath, U., Chen, Z., Ricigliano, J. R., and Klessig, D. F., Two inducers of plant defense responses, 2,6dichloroisonicotinic acid and salicylic acid, inhibit catalase activity in tobacco. Proc. Natl. Acad. Sci. USA, 92, 7143–7147 (1995). Kawano, T., Sahashi, N., Takahashi, K., Uozumi, N., and Muto, S., Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: The earliest events in salicylic acid signal transduction. Plant Cell Physiol., 39, 721–730 (1998). Mori, I. C., Pinontoan, R., Kawano, T., and Muto, S., Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba. Plant Cell Physiol., 42, 1383–1388 (2001).

13)

14)

15)

16)

17)

649

Kvaratskhelia, M., George, S. J., and Thorneley, R. N., Salicylic acid is a reducing substrate and not an eŠective inhibitor of ascorbate peroxidase. J. Biol. Chem., 272, 20998–21001 (1997). Kawano, T. and Muto, S., Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture. J. Exper. Bot., 51, 685–693 (2000). Kawano, T., Pinontoan, R., Uozumi, N., Miyake, C., Asada, K., Kolattukudy, P. E., and Muto, S., Aromatic monoamine-induced immediate oxidative burst leading to an increase in cytosolic Ca 2+ concentration in tobacco suspension culture. Plant Cell Physiol., 41, 1251–1258 (2000a). Kawano, T., Pinontoan, R., Uozumi, N., Morimitsu, Y., Miyake, C., Asada, K., and Muto, S., Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: Mechanism for oxidative burst mediating Ca 2+ in‰ux. Plant Cell Physiol., 41, 1259–1266 (2000b). Kawano, T., Sahashi, N., Uozumi, N., and Muto, S., Involvement of apoplastic peroxidase in the chitosaccharide-induced immediate oxidative burst and a cytosolic Ca 2+ increase in tobacco suspension culture. Plant Peroxidase Newslett., 14, 117–124 (2000c). Gazaryan, I. G., Lagrimini, L. M., Ashby, G. A., and Thorneley, R. N. F., Mechanism of indole-3acetic acid oxidation by plant peroxidases: anaerobic stopped-‰ow spectrophotometric studies on horseradish and tobacco peroxidases. Biochem. J. 313, 841–847 (1996). Rutter, R., Valentine, M., Hendrich, M. P., Hager, L. P., and Debrunner, P. G., Chemical nature of the porphyrin pi cation radical in horseradish peroxidase compound I. Biochemistry, 22, 4769–4774 (1983). Renganathan, V. and Gold, M. H., Spectral characterization of the oxidized states of lignin peroxidase, an extracellular heme enzyme from the white rot basidiomycete Phanerochaete chrysosporium. Biochemistry, 25, 1626–1631 (1986). Miyazawa, J., Kano, A., and Hasegawa, H., Allosteric regulation of cell wall-bound peroxidase and induction of acquired resistance to tomato wilt disease by 4-hydroxybenzoic hydrazide. Ann. Phytopathol. Soc. Japan, 64, 16–23 (1998). Adediran, S. A., Kinetics of the formation of p-670 and of the decay of Compound III of horseradish peroxidase. Arch. Biochem. Biophys., 327, 279–284 (1996). Goodwin, D. C., Grover, T. A., and Aust, S. T., Roles of e‹cient substrates in enhancement of peroxidase-catalyzed oxidations. Biochemistry, 36, 139–147 (1997). Krylov, S. N. and Dunford, H. B., Evidence for a free radical chain mechanism in the reaction between peroxidase and indole-3-acetic acid at neutral pH. Biophys. Chem., 58, 325–338 (1996). Kawano, T., Kawano, N., Hosoya, H., and Lapeyrie, F., A fungal auxin antagonist, hypaphorine competitively inhibits indole-3-acetic acid-dependent su-

650

18)

T. KAWANO et al.

peroxide generation by horseradish peroxidase. Biochem. Biophys. Res. Commun. 288, 546–551 (2001). Kiba, A., Toyota, K., Ichinose, Y., Yamada, T., and Shiraishi, T., Species-speciˆc suppression of superoxide-anion generation of surfaces of pea leaves by the suppressor from Mycosphaella pinodes. Ann.

19)

Phytopathol. Soc. Jpn., 62, 508–512 (1996). Vera-Estrella, R., Blumwald, E., and Higgins, V. J., EŠect of speciˆc elicitors of Cladosporium fulvum on tomato suspension cells: Evidence for involvement of active oxygen species. Plant Physiol., 99, 1208–1215 (1992).