Plant Sulfite Oxidase as Novel Producer of H2O2

Abstract

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome. Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome. Sulfite oxidases (SO) 3The abbreviations used are: SO, sulfite oxidase; rAt-SO, recombinant A. thaliana SO, cyt c, cytochrome c. from vertebrates (published as EC 1.8.3.1) play an essential role in sulfur detoxification by catalyzing the reaction SO32-+H2O+2(cytc)ox→SO42-+2H++2(cytc)red (1Wilson H.L. Rajagopalan K.V. J. Biol. Chem. 2004; 279: 15105-15113Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), which is the terminal step in the oxidative degradation of cysteine and methionine. Different electron acceptors were reported to interact with vertebrate SO including cytochrome c, ferricyanide, and oxygen (2MacLeod R.M. Farkas W. Fridovich I. Handler P. J. Biol. Chem. 1961; 236: 1841-1846Abstract Full Text PDF PubMed Google Scholar, 3Cohen H.J. Fridovich I. J. Biol. Chem. 1971; 246: 359-366Abstract Full Text PDF PubMed Google Scholar, 4Cohen H.J. Fridovich I. J. Biol. Chem. 1971; 246: 367-373Abstract Full Text PDF PubMed Google Scholar). In mammals, SO is localized in the intermembrane space of mitochondria (5Cohen H.J. Betcher-Lange S. Kessler D.L. Rajagopalan K.V. J. Biol. Chem. 1972; 247: 7759-7766Abstract Full Text PDF PubMed Google Scholar) where electrons derived from sulfite are passed to cytochrome c, the physiological electron acceptor. Vertebrate SO is a homodimeric protein with monomers subdivided into a Moco domain and a heme domain, as verified by the atomic structure of chicken SO (6Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Recently we have described plant SO (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) from Arabidopsis thaliana, which is the fourth molybdenum enzyme present in plants in addition to nitrate reductase, xanthine dehydrogenase, and aldehyde oxidase. Cloning and characterization of plant SO was possible by using sequence homologies to the mammalian counterpart. However, in contrast to the animal enzyme plant SO lacks the heme domain, which is evident from the amino acid sequence, its enzymological and spectral properties (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), and the atomic structure (8Schrader N. Fischer K. Theis K. Mendel R.-R. Schwarz G. Kisker C. Structure (Camb.). 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Also its subcellular localization differs from that of animals, in plants we showed SO to be localized in peroxisomes (9Nowak K. Luniak N. Witt C. Wüstefeld Y. Wachter A. Mendel R.-R. Hänsch R. Plant Cell Physiol. 2004; 45: 1889-1894Crossref PubMed Scopus (62) Google Scholar). SO is wide spread and highly conserved within the plant kingdom; the SO gene is present in higher and lower plants, and the protein encoded seems to be highly conserved because antibodies directed against Arabidopsis SO detect proteins of the correct size in a wide range of herbaceous and also woody plants (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Obviously, SO has an important function if it was conserved during evolution. The major function of plant SO seems to be different from the role it plays in animals where it oxidizes sulfite derived from the decomposition of sulfur-containing amino acids. In plants, sulfur metabolism is different because plants assimilate sulfur by reducing sulfate via sulfite to sulfide to form cysteine (for recent reviews, see Leustek (10Leustek T. Somerville C.R. Meyerowitz E.M. The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD2002: 1-16Google Scholar) and Saito (11Saito K. Plant Physiol. 2004; 136: 2443-2450Crossref PubMed Scopus (312) Google Scholar)). This sulfur assimilation pathway is localized in chloroplasts. How do we unravel the role of plant SO? Previous data showed that plants subjected to sulfur-labeled SO2 gas readily converted the radio-active label into sulfate (12Garsed S.G. Read D.J. New Phytol. 1977; 78: 111-119Crossref Scopus (32) Google Scholar, 13Van der Kooij T.A.W. de Kok L.J. Haneklaus S. Schnug E. New Phytol. 1997; 135: 101-107Crossref Scopus (63) Google Scholar), yet the corresponding enzyme catalyzing this reaction was not characterized at that time. Most importantly, this reaction was interpreted as the key step (“safety valve”) in protecting plants from acid rain that has been one major cause of forest damage (14Heber U. Hüve K. Int. Rev. Cytol. 1998; 177: 255-286Crossref Google Scholar). Excess of sulfite derived from SO2 gas is detrimental for the cell because sulfite is toxic. Now that plant SO is isolated and characterized (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 8Schrader N. Fischer K. Theis K. Mendel R.-R. Schwarz G. Kisker C. Structure (Camb.). 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 9Nowak K. Luniak N. Witt C. Wüstefeld Y. Wachter A. Mendel R.-R. Hänsch R. Plant Cell Physiol. 2004; 45: 1889-1894Crossref PubMed Scopus (62) Google Scholar), it is important to study the special properties that make SO a unique catalyst and that were responsible for conserving SO during evolution among plants. The clue to understand the role of SO seems to lie in its peroxisomal localization, either because only there the substrate is available or because in this organelle the reaction product could be further metabolized. SO2 gas can readily penetrate through biomembranes (15Rennenberg H. Herschbach C. Yunus M. Iqbal M. John Wiley and Sons, Chichester, UK1996: 285-294Google Scholar), and sulfite is generated during sulfate assimilation in the chloroplasts. Hence substrate availability is no convincing argument for explaining peroxisomal localization of SO. However, peroxisomes are known as the compartment in which reactive oxygen is produced, and therefore, identifying both the electron acceptor and the reaction products could be the clue for describing the special properties that make plant SO unique. Here we identified molecular oxygen as the terminal electron acceptor for plant SO and showed that it converts molecular oxygen into hydrogen peroxide. Furthermore we provide evidence that this hydrogen peroxide is able to non-enzymatically oxidize sulfite. Thus, under in vitro conditions plant SO combines both an enzymatic and a non-enzymatic reaction. Protein Expression and Purification—For overexpression and purification of recombinant A. thaliana SO the QIAexpress kit (Qiagen) for metal ion chelating chromatography of His-tagged proteins was used. The protein was expressed and purified from pQE80-At-sox in Escherichia coli strain TP1000 (genotype ΔmobAB) as described earlier (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Purification of soluble rAt-SO was performed at 4 °C by using nickel-nitrilotriacetic acid superflow matrix (Qiagen) followed by anion exchange chromatography on a 10 ml SourceQ15 column (Amersham Biosciences). Moco-containing SO did not bind to the column, whereas the inactive Moco-free form of SO bound to the column and eluted between 250 and 300 mm NaCl (8Schrader N. Fischer K. Theis K. Mendel R.-R. Schwarz G. Kisker C. Structure (Camb.). 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The protein preparation was stable at 4 °C for several weeks after dialyzing and concentrating to 2-10 mg/ml. Concentration of recombinant plant SO was determined by UV spectroscopy using the calculated extinction coefficient of ϵ = 69.820 m-1 cm-1. Detection of Plant SO Protein—Protein of leaves and roots were extracted and analyzed as described earlier (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). For immuno-detection, protein was separated on a 10% SDS-PAGE, and the polyclonal anti-rAt-SO antibody (Eurogentec) was diluted 1:500 prior to use. SO Assays—SO was routinely assayed for the activity to reduce ferricyanide as described previously (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In parallel, oxygen measurements were performed with an inoLab OxiLevel2 electrode (Wissenschaftlich-Technische Werkstätten GmbH) in 2.2-ml volume by using 2 μg of recombinant plant SO, oxygen-saturated 0.1 m Tris acetate buffer (pH 7.25) over a time of 2 min and expressed as oxygen consumption (mg/liter) per second. In the mixed SO + catalase assay, 2 μg of recombinant plant SO and 94 units of bovine catalase (Sigma) were incubated in 0.1 m Tris acetate buffer. For quantification of sulfite and sulfate used and produced by recombinant plant SO, respectively, the reaction was stopped by heating at 95 °C for 5 min, and sulfite and sulfate were determined as described below. All data presented are replicates from at least three independent experiments and expressed as mean ± S.D. Detection of Hydrogen Peroxide—Determination of H2O2 formation was performed according to Nag S. K. Plant PubMed Scopus Google Scholar) with SO reaction was stopped at different time by of the of in 5 ml of and diluted to a volume of ml with of the was at H2O2 formation was using a H2O2 formation was also determined using as described by M. E. W. J. 1998; PubMed Google Scholar). of of into sulfite mm to sulfate by recombinant plant SO was determined either by using or by using gas 18O according to the described by R. A. M. M. T. J. 1997; PubMed Scopus Google Scholar). min of 2 of per ml of reaction was and sulfate was with (Sigma) in a of was by min, of were into were into a for by a to an are expressed in and Sulfite of were into an ion exchange chromatography and sulfite were separated on an column 250 4 and eluted with a mm mm at a of ml were by a with an 4 The of this is for both Expression of SO in and of SO on plant was SO protein was in plant of Arabidopsis including and The protein of SO in leaves was to be in a range of of the This quantification was performed by a of recombinant SO with the extracted SO as with the purified antibody against recombinant Arabidopsis SO not this was to the of SO μg of proteins of Arabidopsis leaves and respectively, only were over the to after SO was during the and in the the protein was as SO is an Different electron acceptors were described to interact with purified animal SO including cytochrome c, ferricyanide, and (2MacLeod R.M. Farkas W. Fridovich I. Handler P. J. Biol. Chem. 1961; 236: 1841-1846Abstract Full Text PDF PubMed Google Scholar, 3Cohen H.J. Fridovich I. J. Biol. Chem. 1971; 246: 359-366Abstract Full Text PDF PubMed Google Scholar). derived from sulfite are passed via the heme domain of the enzyme cytochrome c, the physiological electron acceptor R.M. A. Rajagopalan K.V. U. S. A. 1998; PubMed Scopus (123) Google Scholar). Plant SO, is to interact with cytochrome c because it lacks a heme plant SO is not localized in mitochondria animal SO but in an electron acceptor cytochrome c has to be animal SO was described to function with with lower we the consumption of molecular oxygen by plant SO. The experiments showed that plant SO is able to use molecular oxygen as terminal electron acceptor The of was in the range as the with the acceptor ferricyanide determined in experiments (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). H2O2 during Sulfite plant SO molecular oxygen as terminal electron acceptor, the could be the products of this sulfate is the reaction and as plant SO is localized in peroxisomes (9Nowak K. Luniak N. Witt C. Wüstefeld Y. Wachter A. Mendel R.-R. Hänsch R. Plant Cell Physiol. 2004; 45: 1889-1894Crossref PubMed Scopus (62) Google Scholar) it is to hydrogen peroxide as the product of sulfite oxidation by plant SO. H2O2 can be in the SO by different Nag S. K. Plant PubMed Scopus Google Scholar) described the formation of a from the only in the of H2O2 and its at This was The is known to react with H2O2 but not with reactive oxygen M. E. W. J. 1998; PubMed Google Scholar). Also H2O2 formation during sulfite oxidation by plant SO. of catalase units per to both the and the H2O2 H2O2 Sulfite in a and rain H2O2 is known as one of the non-enzymatic for Chem. 2001; Scopus Google Scholar). we under the conditions of sulfite could be non-enzymatically by we the of sulfite in and it oxidation in the of sulfite a of in sulfate were in an sulfite and this did not with for to not conditions and reaction as described under sulfite did not oxidize to sulfate. However, the addition of physiological of H2O2 in the range to the of sulfite into sulfate and this oxidation at different Thus, under conditions H2O2 is able to oxidize sulfite non-enzymatically to sulfate. Plant Sulfite an with a sulfite oxidation by plant SO we showed that H2O2 is we determined the of non-enzymatic sulfite oxidation to the reaction by plant SO. both we use of catalase that converts H2O2 into and molecular oxygen according to the 2 H2O2 2 + we the reaction of plant SO in the of of consumption of plant SO is with catalase in the reaction For each molecule of oxygen by plant SO one molecule of H2O2 is and two molecules of H2O2 one molecule of oxygen in the catalase addition of catalase the oxygen consumption of plant SO by this can be not This is another for H2O2 as a reaction product of plant SO. plant SO combines with a subsequent non-enzymatic reaction of H2O2 with the of sulfate in with the enzymatic step This can be with catalase in the reaction to sulfite data from experiments with plant SO where the of sulfite and the of sulfate are over time. In the of plant SO sulfate at a time as with the reaction in the of or on of the substrate was to sulfate within in the mixed with 250 units of this time was after the of we the reaction for plant SO. enzymatic reaction with oxygen as electron acceptor to the of In a non-enzymatic this H2O2 is used to oxidize a molecule of sulfite to into we 18O measurements using and the mixed SO + catalase reaction with SO The of catalase non-enzymatic sulfite as the oxygen during sulfite oxidation R. Chem. Rev. PubMed Scopus Google and results not it is known that at and sulfite does not readily exchange its with J. Chem. Scopus Google Scholar, Chem. 2003; PubMed Scopus Google Scholar). sulfate be under the conditions of H2O2 sulfate labeling be This was performed with two different The results in that in the of catalase only of the label is in that plant SO combines an enzymatic with a subsequent non-enzymatic sulfite of sulfite oxidation with 18O in the of 18O ± ± 18O ± ± in a The of plant SO was calculated on the of determined in the of catalase to only the enzymatic reaction catalase is higher because of the of the subsequent non-enzymatic The of plant SO was calculated to be and that for catalase is one of the J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This that at the catalase used in the experiments H2O2 generated by SO will be the of sulfite oxidation by plant SO in the and in the of of ± and ± respectively, were the non-enzymatic reaction of sulfite with H2O2 has an of ± Most plant SO and catalase are in the cell in peroxisomes (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Nowak K. Luniak N. Witt C. Wüstefeld Y. Wachter A. Mendel R.-R. Hänsch R. Plant Cell Physiol. 2004; 45: 1889-1894Crossref PubMed Scopus (62) Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). that and catalase is one of the excess is by sulfite with sulfite S. T. U. Plant Cell Physiol. 1998; Scopus Google Scholar). we to catalase is by sulfite under that of sulfite catalase at In the SO + catalase the of sulfite is by an in oxygen consumption because catalase is and does not to the oxygen This is only because at higher sulfite of plant SO is also by its the data presented it is evident that plant SO is a expressed in plant at time with only Different from vertebrate SO that of a heme domain and a molybdenum domain, the heme domain is in the plant enzyme (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 8Schrader N. Fischer K. Theis K. Mendel R.-R. Schwarz G. Kisker C. Structure (Camb.). 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). While vertebrate SO is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor R.M. A. Rajagopalan K.V. U. S. A. 1998; PubMed Scopus (123) Google Scholar), plant SO is localized in peroxisomes and does not react with cytochrome c (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Nowak K. Luniak N. Witt C. Wüstefeld Y. Wachter A. Mendel R.-R. Hänsch R. Plant Cell Physiol. 2004; 45: 1889-1894Crossref PubMed Scopus (62) Google Scholar). plant SO an electron acceptor with a to a but no evidence was for the of plant SO with a protein (7Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Hänsch R. Mendel R.-R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), C. and R. R. in a heme domain was with the Moco domain of SO from U. B. J. Schwarz G. R. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results identified oxygen as the terminal electron acceptor for plant SO that converts oxygen into Thus, plant SO is the eukaryotic the EC for (EC the animal with cytochrome c as the electron acceptor into the of c (EC that in vitro the non-enzymatic oxidation of sulfite by H2O2 to the reaction of plant SO. and H. Scopus Google Scholar) that oxidation of sulfite in plants might be by on the of the electron in or by or by However, no was it was that in and rain H2O2 is one of the for This reaction is the key step during the of acid H2O2 is highly soluble in with about of higher The reaction at on the of Chem. 2001; Scopus Google Scholar). The of oxidation of SO2 gas by H2O2 with between 2 and and to oxidative at J.H. and to John Wiley and Sons, New Scholar). The via with a acid ion where the of the step as J.H. and to John Wiley and Sons, New Scholar, J. Chem. Scopus Google Scholar). labeling experiments and the use of catalase provide evidence that under in vitro conditions plant SO combines its enzymatic sulfite oxidation with a subsequent non-enzymatic step using its reaction product H2O2 as for another molecule of sulfite. in vitro the non-enzymatic step the of sulfate and the consumption of oxygen. In known to in peroxisomes be in sulfite oxidation as could be the of this Sulfite is a toxic that can which is sulfite and it can to which are metabolic (14Heber U. Hüve K. Int. Rev. Cytol. 1998; 177: 255-286Crossref Google Scholar). its by oxidation to sulfate is a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere or during the decomposition of sulfur-containing amino acids. to characterization of plant SO, this reaction was interpreted as the key step in protecting plants from acid rain that has been a major cause of forest damage (14Heber U. Hüve K. Int. Rev. Cytol. 1998; 177: 255-286Crossref Google Scholar, and and Scholar). only sulfite but also H2O2 is as a toxic in recent it that at lower H2O2 also plays an important role as to different (for see R. Plant Biol. PubMed Scopus Google Scholar) and C. K. A. Plant Biol. 2004; PubMed Scopus Google Scholar)). The of H2O2 for Arabidopsis has been reported to be in the range between and mm S.D. C. G. Plant Physiol. 2001; PubMed Scopus Google Scholar, S. H. B. G. G. P. PubMed Scopus Google Scholar). in H2O2 the of H2O2 during sulfite oxidation also the peroxisomal localization (9Nowak K. Luniak N. Witt C. Wüstefeld Y. Wachter A. Mendel R.-R. Hänsch R. Plant Cell Physiol. 2004; 45: 1889-1894Crossref PubMed Scopus (62) Google Scholar) of plant SO because this organelle catalase as one of the excess this we have no for the of non-enzymatic sulfite oxidation in the For we to a model the possible interaction between plant SO and catalase in the peroxisome. sulfite H2O2 as a reaction product of plant SO is by higher sulfite catalase and H2O2 derived from the SO reaction oxidizes non-enzymatically a sulfite it has to be into that under conditions H2O2 derived from peroxisomal could to the non-enzymatic detoxification of sulfite as The of this is not and further are to and for