Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

The Amino Acid Sequence of Ascorbate Peroxidase from Tea Has a High Degree of Homology to That of Cytochrome c Peroxidase from Yeast

The chloroplast isozyme of ascorbate peroxidase from tea leaveswas digested with lysyl endopeptidase, and the amino acid sequencesof the peptide fragments were determined. These sequences accountedfor 64% of the amino acids in the entire protein. The sequenceof one of the peptides can be aligned with the region whichincludes the proximal histidine that serves as the fifth ligandof the heme iron in guaiacol peroxidases and cytochrome c peroxidase.The sequences of the peptides from ascorbate peroxidase exhibita higher degree of homology to the sequence of cytochrome cperoxidase from yeast than to those of guaiacol peroxidasesfrom plants. In addition, three of the peptides from ascorbateperoxidase show a high degree of homology to triose-phosphateisomerase from maize. From the available amino acid sequencesand the enzymatic and molecular properties of ascorbate andcytochrome c peroxidases, we propose that these hydrogen peroxide-scavengingperoxidases that use either cytochrome c or ascorbate as theelectron donor originated from the same ancestral protein. (Received July 5, 1991; Accepted December 6, 1991)     

Functional Structure of the Oxygen-Evolving Unit of Photosystem II as Determined by Radiation Inactivation

The size of the complex that is essential for the electron-transferactivity from the oxygen-evolving center to the secondary electronacceptor, Q_B, is about 250 kDa, as determined by target-sizeanalysis after the radiation inactivation of functions of photosystemII (PS II). Inter-Chl tranfer of excitation energy was insensitiveto the radiation inactivation indicating that the masses ofCP47, CP43, and light-harvesting Chi a/b proteins are not includedin the functional size of the oxygen-evolving PS II complex.The transfer of electrons from the secondary electron donor,Z, to Q_B was catalyzed by a unit of only 65 kDa. The sizes ofthe complexes involved in these light-induced functions of PSII were dependent on the intensity of actinic light. Under saturatingintensities of light, the functional size of the complex fortransfer of electrons from Z to Q_B was 38 kDa, with a correspondingdecrease in the size of the oxygen-evolving PS II from 250 kDato 125 kDa [Takahashi, Mano and Asada (1985) Plant Cell Physiol.26: 383]. The protein of about 30 kDa functions in the photoreductionof the pheophytin molecule, as well as in the electron transferfrom Z to Q_A. Under low-intensity light, complexes having thesame sizes as those of the basal functional complexes undersaturating-intensity light are further required, probably tostabilize separated charges in the PS II reaction center andthe oxygen-evolving center. (Received June 20, 1990; Accepted September 18, 1990)     

Occurrence of Cu,Zn-Superoxide Dismutase in the Intrathylakoid Space of Spinach Chloroplasts

Thylakoids obtained from intact spinach chloroplasts showedno superoxide dismutase (SOD) activity, but Cu,Zn- and Mn-SODactivities were detected in the presence of Triton X-100. Thylakoidmembranes and the lumen fraction were separated by centrifugationafter treatment of the thylakoids with a Yeda pressure cell.Cu,Zn-SOD was found in the lumen fraction. Mn-SOD was detectedin the thylakoid fraction only after addition of 1% Triton X-100.Antibody against spinach Cu,Zn-SOD did not interact with thelatent Cu,Zn-SOD in the thylakoids unless Triton was added.These results indicate that Cu,Zn-SOD occurs in the lumen inaddition to the stroma of spinach chloroplasts, and Mn-SOD bindsto the thylakoid membranes. (Received February 29, 1984; Accepted May 28, 1984)     

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids

The permeability of phospholipid membranes to the superoxide anion (O₂^?) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O₂^? generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O₂^? was proportional to the intravesicular concentration of O₂^? over a range from 1 to 10 μm which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O₂^? was estimated to be 2.1 × 10^?6 cm s^?1 at pH 7.3 and 25 ° C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O₂^? unless Triton X-100 was added. O₂^? generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O₂^?.     

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)     

Intra- and Extra-Cellular Localization of "Cytosolic" CuZn-Superoxide Dismutase in Spinach Leaf and Hypocotyl

Immunogold-electron microscopic analysis of spinach leaves donewith the antibody specific for "cytosolic" CuZn-superoxide dismutase(SOD) indicates that SOD is localized in the apoplast, in thenucleus and in, or near, the tonoplast. The association of CuZn-SODwith the nucleus indicates it has a role in preventing fatalmutation caused by reactive species of oxygen. The localizingsite of CuZn-SOD in the apoplastic region of spinach leaf tissuescorresponds to that of the accumulation of lignin. In spinachhypocotyl "cytosolic" CuZn-SOD is localized in vascular tissueswhere lignification and the generation of superoxide respectivelywere shown by the phloroglucin-HCl reaction and formation offormazane from nitroblue tetrazolium. Because hydrogen peroxideis required for lignification via the peroxidase-catalyzed reaction,the CuZn-SOD in the apoplast appears to function in the biosynthesisof lignin by causing rapid disproportionation of the superoxideanion radical prior to its interaction with cellular componentsand peroxidase. (Received March 25, 1996; Accepted June 17, 1996)     

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)