Monodehydroascorbate Reductase in Spinach Chloroplasts and Its

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hydrogen peroxide in intact spinach chloroplasts (200-500 fimol mg"1 Chi h"1, ... Intact spinach chloroplasts were isolated from the fresh leaves and purified by ...

Plant & CellPhysiol. 25(3): 385-395 (1984) JSPP © 1984

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide M. Anwar Hossain, Yoshiyuki Nakano and Kozi Asada

The primary reaction product of chloroplast ascorbate peroxidase activity was shown to be monodehydroascorbate radical (MDA). MDA reductase (EC 1.6.5.4) was localized in spinach chloroplast stroma. The MDA reductase activity of spinach chloroplasts, using NAD(P)H as electron donor, could account for the regeneration of ascorbate from MDA produced by ascorbate peroxidase activity. In the absence of MDA reductase, MDA disproportionated to ascorbate (AsA) and dehydroascorbate (DHA). The DHA was reduced to AsA by DHA reductase (EC 1.8.5.1) in chloroplasts. Both NADH and NADPH served as the electron donor of partially purified MDA reductase from spinach leaves. Key words: Ascorbate peroxidase — Hydrogen peroxide — Monodehydroascorbate reductase (EC 1.6.5.4) — Regeneration (ascorbate) — Spinach chloroplast.

In illuminated chloroplasts superoxide anion radicals are produced in the thylakoid membranes through the autoxidation of the primary electron acceptor in PS I and diffuse out (Asada et al. 1983). The diffused superoxide anions are disproportionated to hydrogen peroxide and molecular oxygen by superoxide dismutase contained in the stroma and in the lumen of thylakoids (Asada et al. 1983). Recently it has been shown that the hydrogen peroxide is reduced to water, by an AsA specific peroxidase in spinach (Nakano and Asada 1981) and in pea (Jablonski and Anderson 1982) chloroplasts. Thus, AsA peroxidase plays an important role in scavenging hydrogen peroxide which inactivates CC>2-fixation cycle enzymes, such as fructose-1,6-bisphosphatase (Charles and Halliwell 1981), glyceraldehyde-3-phosphate dehydrogenase (Tanaka et al. 1982) and ribulose-5-phosphate kinase (Kaiser 1979). The concentration of AsA in chloroplasts is high (12 to 25 mM, Foyer et al. 1983), but to maintain the scavenging capacity for hydrogen peroxide produced in chloroplasts, the products of AsA oxidation by the chloroplast peroxidase should be recycled to AsA. Even if 10% of the photoreducing equivalents in PS I is used for the production of superoxide (15 /wnol ing"1 Chi h"1), hydrogen peroxide will be produced at a rate of 50 fiu s"1 in chloroplast assuming its Chi concentration of 25 mM (Asada et al. 1977). If the AsA regeneration system does not operate in chloroplasts, the AsA will be exhausted in only about 240 s by the hydrogen peroxide produced through AsA peroxidase reaction. It has been shown that the final oxidation product of the peroxidase reaction, DHA, is reduced by GSH in a reaction catalyzed by DHA reductase (EC 1.8.5.1) which is present in spinach (Nakano and Asada 1981) and in pea (Jablonski and Anderson 1981) chloroplasts. Spinach DHA reductase has been purified to near homogeneous Abbreviations: AsA, L-ascorbate; DHA, dehydroascorbate; MDA, monodehydroascorbate. 385

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The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

386

M. A. Hossain, Y. Nakano and K. Asada

state (Hossain and Asada 1984). The GSH is regenerated from GSSG by glutathione reductase using NADPH supplied by illuminated thylakoids and a scheme of PS I — NADPH^GSH has been proposed for the regeneration of AsA in chloroplasts. However, DHA reductase activity in spinach chloroplasts is lower (Nakano and Asada 1981) than the rate of photoreduction of hydrogen peroxide in intact spinach chloroplasts (200-500 fimol mg"1 Chi h"1, Nakano and Asada 1980). In this paper we report that the primary oxidation product of AsA peroxidase reaction is MDA radical. Several lines of evidence for the occurrence of MDA reductase (EC 1.6.5.4) in spinach chloroplasts and for its participation in the regeneration of AsA are presented. Purification and properties of spinach MDA reductase are also described.

Intact spinach chloroplasts were isolated from the fresh leaves and purified by Percoll density centrifugation (Nakano and Asada 1980). The intactness of the Percoll-purified chloroplasts was usually above 90% as estimated by the ferricyanide method (Heber and Santarius 1970). MDA reductase was assayed spectrophotometrically by following the decrease in absorbance at 340 nm due to NADH or NADPH oxidation using an absorbance coefficient of 6.2 mM"1 cm"1. For the standard assay MDA was generated by AsA oxidase (EC 1.10.3.3) (Yamazaki and Piette 1961). The reaction mixture contained 50 mM Tris-HCl, pH 7.6, 0.1 mM NADH, 2.5 mM AsA, AsA oxidase (0.14 unit, 1 /tmol ascorbate oxidized min"1 being 1 unit) and enzyme at 25°C. The reaction was initiated by the addition of AsA oxidase. AsA oxidase was used to give the near maximum oxidation rate of NADH and its amount corresponded to that which gave an oxidation rate of AsA of 0.14 mM min"1 under the following conditions: 50 mM Tris-HCl, pH 7.6, 1 mM AsA and AsA oxidase (0.14 units) in a final volume of 1 ml. Under these conditions, the steady state concentration of MDA was 2.1 jiu, as determined from the absorbance at 360 nm (Fig. 7). When necessary, corrections were made for the oxidation of NADH or NADPH by diaphorase before the addition of AsA oxidase. MDA was measured by following the increase in absorbance at 360 nm (Bielski et al. 1971) assuming an absorbance coefficient of 3.3 mM"1 cm"1 (Schuler 1977), using a Shimadzu multipurpose spectrophotometer MPS-2000, or a difference absorbance coefficient of 2.64 mM4 cm"1 between 360 and 400 nm using a Hitachi 356 dual wavelength spectrophotometer. AsA peroxidase and DHA reductase were assayed as previously described (Nakano and Asada 1981). Protein was determined according to Lowry et al. (1951) and Chi according to Arnon (1949). AsA peroxidase was purified from spinach leaves to near homogeneous state in polyacrylamide gel electrophoresis. Specific activity of the purified enzyme was 260 ,umol AsA oxidized min"1 mg"1 protein under the assay conditions of Nakano and Asada (1981) which is 520-fold over that of the leaf extract. Neither MDA reductase activity nor AsA oxidase activity was detectable in the purified enzyme. The purification procedure of the enzyme will be published elsewhere. MDA reductase was purified from spinach leaves. The fresh leaves (5 kg) were homogenized with 10 liters of 50 mM Tris-HCl buffer, pH 7.8, and the homogenate was squeezed through four layers of cheesecloth and centrifuged at 13,000 X^ for 20 min. The supernatant was brought to 40% saturation with solid ammonium sulfate and centrifuged at 13,000 X^ for 15 min. The collected precipitate was dissolved in 10 mM Tris-HCl buffer, pH 7.8, containing 5 mM 2-mercaptoethanol, and the enzyme was dialyzed against the same buffer. The dialyzed enzyme was applied to a DEAE-Sephacel column (10x6 cm) equilibrated with the same buffer. The column was washed with 4 liters of equlibrating buffer and the enzyme was eluted by

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Materials and Methods

Monodehydroascorbate reductase in chloroplasts

387

Results Monodehydroascorbate is the primary oxidation product in the chloroplast peroxidase reaction—AsA-

specific peroxidase found in chloroplasts (Kelly and Latzko 1979, Nakano and Asada 1981) are distinguished from "classical" plant peroxidase such as the enzyme from horseradish in the specificity of the electron donor. In the reaction mechanism proposed for the horseradish peroxidase, the peroxidase reaction intermediates with hydrogen peroxide, compounds I and II, univalently oxidize AsA producing MDA radical (Yamazaki et al. 1960). If the chloroplast peroxidase catalyzes in a similar way to horseradish peroxidase, MDA radical is expected as an intermediate. On addition of hydrogen peroxide to an AsA and AsA peroxidase mixture, we found an increase in absorbance at 360 nm and its level was kept for several minutes. Little increase in the absorbance was observed in the absence of either AsA, hydrogen peroxide or the enzyme. Its absorption spectrum (Fig. 1) was similar to that observed when AsA and AsA oxidase were mixed under the same conditions. The production of MDA in the reaction of AsA oxidase has been established (Yamazaki and Piette 1961). These observations indicate that the molecular species responsible for the absorbance at 360 nm was MDA, and MDA is produced in the AsA peroxidase reaction. Under the conditions where the steady state level of MDA was observed at 360 nm, the production rate of the radical (vp) is equal to the decay of the radical (v^). Thus a p =y d = 2£[MDA]2, where k is the second order reaction rate constant for the disproportionation of MDA to produce DHA and AsA. We determined the steady state concentrations of MDA using various concentrations of AsA peroxidase to change vp. vp was determined from the rate of disappearance of AsA at 290 nm which was multiplied by two to correct for the regeneration of AsA via the disproportionation of MDA. The steady concentration of MDA, [MDA], was proportional to the square root of vp and the slope of the curve, k, gave a value of 2.7 X 106 M"1 S -1 (Fig. 2). This value is about an order of magnitude higher than that determined by pulse radiolysis (2.5 X 105 M"1 S"1, at pH 7.0, Bielski et al. 1981). Under the same conditions as in Fig. 2, AsA peroxidase was replaced by AsA oxidase. The steady state concentration of MDA at various concentrations of AsA oxidase gave a curve similar to Fig. 2, as also observed by Yamazaki and Piette (1961). The k determined from the slope was 1.8 X 106 M -1 S -1 which is similar to the value obtained by AsA peroxidase. These observations confirm that MDA

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stepwise increase of KC1 concentration in the buffer. Most of the activity was eluted by 100 to 150 mM KC1. The active fraction was dialyzed against 10 mti Tris-HCl buffer, pH 7.8, containing 5 mM 2-mercaptoethanol and applied to a second DEAE-Sephacel column equilibrated with the dialysis buffer. The enzyme was eluted against a linear concentration gradient of KC1 (0-150 mni) in the same buffer. The enzyme was eluted by 85 to 110 mM KC1. The active fractions were concentrated and gel-filtered through a column of Sephadex G-100 (5x90 cm) equilibrated with 20 mM Tris-HCl buffer, pH 7.8, containing 5 mM 2-mercaptoethanol. MDA reductase purified in this way (880 mg; yield 10%) showed 10 fold increase in specific activity (0.25 fimol of NADH oxidized mg-1 protein min"1) over the leaf extract and showed no AsA peroxidase activity (Fig. 6, line a). AsA oxidase from cucumber was a product of Toyobo Co., Osaka. No increase of the oxidation of AsA was observed by the addition of 0.1 mM hydrogen peroxide, indicating the absence of AsA peroxidase in the cucumber AsA oxidase. Glucose oxidase (EC 1.1.3.4) was obtained from Sigma and DEAE-Sephacel from Pharmacia Fine Chemicals (Sweden). All other chemicals used were of analytical grade quality.

M. A. Hossain, Y. Nakano and K. Asada

388

• AsA peroxidase

X

(nm) Fig. 1

400

0-5 1 Ascorbate peroxidase (jug) Fig. 2

Fig. 1 Absorption spectrum of oxidation product of AsA peroxidase reaction. The reaction mixture (1 ml) contained 1 mM AsA, 180 /iu H2O2, 0.1 mM diethylenetriamine pentaacetic acid, and 50 mM Tris-HCl, pH 7.6. Where indicated, 0.4/

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