Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)     

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

Intact spinach chloroplasts scavenge hydrogen peroxide witha peroxidase that uses a photoreductant as the electron donor,but the activity of ruptured chloroplasts is very low [Nakanoand Asada (1980) Plant & Cell Physiol. 21 : 1295]. Rupturedspinach chloroplasts recovered their ability to photoreducehydrogen peroxide with the concomitant evolution of oxygen afterthe addition of glutathione and dehydroascorbate (DHA). In rupturedchloroplasts, DHA was photoreduced to ascorbate and oxygen wasevolved in the process in the presence of glutathione. DHA reductase(EC 1.8.5.1 [EC] ) and a peroxidase whose electron donor is specificto L-ascorbate are localized in chloroplast stroma. These observationsconfirm that the electron donor for the scavenging of hydrogenperoxide in chloroplasts is L-ascorbate and that the L-ascorbateis regenerated from DHA by the system: photosystem IferredoxinNADPglutathione.A preliminary characterization of the chloroplast peroxidaseis given. (Received April 16, 1981; Accepted June 3, 1981)     

Purification of Ascorbate Peroxidase in Spinach Chloroplasts; Its Inactivation in Ascorbate-Depleted Medium and Reactivation by Monodehydroascorbate Radical

Ascorbate specific peroxidase in chloroplasts was purified fromspinach leaves. Spinach chloroplast peroxidase was a monomerwith a molecular weight of about 30,000 and showed an absorptionspectrum similar to a hemoprotein. The enzyme lost its activitywithin a minute in the absence of ascorbate under aerobic conditions.In addition to ascorbate, 20% sorbitol was necessary to stabilizethe enzyme. The inactivation of the enzyme in the ascorbate-depletedmedium was protected by other electron donors, pyrogallol, guaiacoland pyrocatechol, whose oxidation rates were very low comparedwith that of ascorbate. The inactivated enzyme recovered itsactivity with monodehydroascorbate radicals generated by theascorbate-ascorbate oxidase system. A mechanism of inactivationand reactivation of ascorbate peroxidase is proposed. (Received August 28, 1986; Accepted November 13, 1986)     

Inactivation Mechanism of Ascorbate Peroxidase at Low Concentrations of Ascorbate; Hydrogen Peroxide Decomposes Compound I of Ascorbate Peroxidase

One of the characteristic properties of ascorbate peroxidase(APX), which distinguishes it from guaiacol peroxidase, Cytc peroxidase and glutathione peroxidase, is the rapid inactivationof the enzyme under conditions where an electron donor is absent.When thylakoid-bound APX (tAPX) in 100 µM ascorbate wasdiluted 500-fold with an ascorbate-depleted medium, the enzymaticactivity was lost with half time of about 15 s. The inactivationof tAPX was suppressed under anaerobic conditions and also bythe addition of catalase, but it was unaffected by the additionof superoxide dismutase. These observations suggest that hydrogenperoxide at nanomolar levels, produced by autooxidation of ascorbateat lower than micromolar levels, might participate in the inactivationof tAPX. The participation of hydrogen peroxide was confirmedby the inactivation of tAPX upon incubation with hydrogen peroxideunder anaerobic conditions. In the absence of ascorbate, theheme of the two-electron-oxidized intermediate of tAPX (designatedCompound I) is decomposed by hydrogen peroxide. Thus, the instabilityof Compound I to hydrogen peroxide is responsible for the inactivationof APX when ascorbate is not available for Compound I and theenzyme cannot turnover. (Received October 16, 1995; Accepted February 21, 1996)     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Restoration by Glycerol of Cholate-Induced Inactivation of Oxygen Evolution in Spinach Chloroplasts

Electron transport in spinach chloroplasts treated with cholateor Tris in the presence and absence of 20% glycerol was measured.Glycerol suppressed the inhibitory action of cholate and Trison the donor side of photosystem II and also restored the Hillactivity previously lowered by cholate. This restoration requiredthe cholate-extract from the chloroplasts. (Received November 17, 1982; Accepted July 25, 1983)     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Scavenging of Hydrogen Peroxide in Prokaryotic and Eukaryotic Algae: Acquisition of Ascorbate Peroxidase during the Evolution of Cyanobacteria

Ascorbate (AsA) peroxidase was found in six species of cyanobacteriaamong ten species tested. Upon the addition of H₂¹⁸O₂ to thecells of AsA peroxidase-containing cyanobacteria, ¹⁶O₂ derivedfrom water and ¹⁸O₂ derived from H₂^I8O₂ were evolved in thelight. The evolution of ¹⁶O₂ was inhibited by DCMU and did notoccur in the dark, but ^I8O₂ was evolved even in the dark orin the presence of DCMU. Similar light-dependent evolution of¹⁶O₂ was observed in the cells of AsA peroxidase-containingEuglena and Chlamydomonas. However, the cells of AsA perox-idase-lackingcyanobacteria evolved only ¹⁸O₂ in either the light or dark.Furthermore, the quenching of chlorophyll fluorescence inducedby hydrogen peroxide was observed only in the cells of the AsAperoxidase-containing Synechocystis 6803, and not in the cellsof Anacystis nidulans which lacks AsA peroxidase. Thus, cyanobacteriacan be divided into two groups, those that has and those thatlacks AsA peroxidase. The first group scavenges hydrogen peroxidewith the peroxidase using a photoreductant as the electron donor,and the second group only scavenges hydrogen peroxide with catalase. (Received July 23, 1990; Accepted October 18, 1990)     

Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H₂O₂ could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H₂O₂-generating enzyme. This increase in PGA formation due to H₂O₂ was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O₂ and by the NAD(P)H-oxidizing system.     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13[EC]), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19[EC])and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11[EC]) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)     

Oxidation of Quercetin by Carotenoid Radical Generated in Illuminated Spinach Chloroplasts: The Effect of Ascorbate on Quercetin Oxidation

Carotenoid photobleaching in the presence of carbonylcyanidem-chlorophenylhydrazone (CCCP) was suppressed by quercetin,but not by ascorbate. When quercetin suppressed carotenoid photobleaching,quercetin was oxidized. The oxidation of quercetin was inhibitedby ascorbate with half-inhibition at about 10 µM. Ascorbatewas oxidized by CCCP-poisoned chloroplasts upon illumination.The rate of ascorbate oxidation in the presence of both ascorbateand quercetin was lower than that in the presence of ascorbatealone. Based on the present results, the physiological significanceof quercetin as an antioxidant and the redox reaction betweenascorbate and oxidized quercetin are discussed. (Received March 9, 1984; Accepted July 12, 1984)     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach

cDNAs encoding two cytosolic and two chloroplastic ascorbateperoxidase (AsAP) isozymes from spinach have been cloned recently[Ishikawa et al. (1995) FEBS Lett. 367: 28, (1996) FEBS Lett.384: 289]. We herein report the cloning of the fifth cDNA ofan AsAP isozyme which localizes in spinach glyoxysomes (gAsAP).The open reading frame of the 858-base pair cDNA encoded 286amino acid residues with a calculated molecular mass of 31,507Da. By determination of the latency of AsAP activity in intactglyoxysomes, the enzyme, as well as monodehydroascorbate (MDAsA)reductase, was found to be located on the external side of theorganelles. The cDNA was overexpressed in Escherichia coli (E.coli). The enzymatic properties of the partially purified recombinantgAsAP were consistent with those of the native enzyme from intactglyoxysomes. The recombinant enzyme utilized ascorbate (AsA)as its most effective natural electron donor; glutathione (GSH)and NAD(P)H could not substitute for AsA. The substrate-velocitycurves with the recombinant enzyme showed Michaelis-Menten typekinetics with AsA and hydrogen peroxide (H₂O₂); the apparentK_m values for AsA and H₂O₂were 1.89±0.05 mM and 74±4.0µM,respectively. When the recombinant enzyme was diluted with AsA-depletedmedium, the activity was stable over 180 min. We discuss theH₂O₂-scavenging system maintained by AsAP and the regenerationsystem of AsA in spinach glyoxysome. ¹Present address: Department of Biochemistry, Wakayama MedicalCollege, 27 Kyubancho, Wakayama, 640 Japan     

Internal Catalase Protects Herpes Simplex Virus from Inactivation by Hydrogen Peroxide

Herpes simplex virus 1 (HSV-1) was shown to contain catalase, an enzyme able to detoxify hydrogen peroxide by converting it to water and oxygen. Studies with a catalase inhibitor indicated that virus-associated catalase can have a role in protecting the virus from oxidative inactivation. HSV-1 was found to be more sensitive to killing by hydrogen peroxide in the presence of a catalase inhibitor than in its absence. The results suggest a protective role for catalase during the time HSV-1 spends in the oxidizing environment outside a host cell.     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Inactivation of Lipoxygenase by Hydrogen Peroxide,Cysteine and Some Other Reagents

The polarographic data show that H₂O₂ is not formed during the course of the coupled oxidation of antioxidants by lipoxygenase from defatted soybean meal. A lower concentration of H₂O₂ or autoxidizing cysteine has been found to induce an irreversible inactivation of the enzyme. Inactivation activity of cysteine is reduced either by the addition of catalase or under anaerobic condition. These facts are indicative of the oxidative function of autoxidizing cysteine for the enzyme. The inactivation by cysteine and H₂O₂ respectively is in additive and is impeded by the addition of competitive inhibitors such as linolelaidic and conjugated linoleic acids, indicating a possible reaction with a certain amino acid residue involved in the enzymic catalysis. The experimental evidences obtained with H₂O₂ and some other modifying reagents have been integrated to furnish a basis of later identification of the residue that is exerting the specific catalytic function.