Identification and Biochemical Characterization ofArabidopsis thaliana Sulfite Oxidase

Abstract

In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC 1.8.3.1). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K m values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants. In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC 1.8.3.1). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K m values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants. molybdenum cofactor molybdopterin nitrate reductase sulfite oxidase A. thaliana SO recombinant A. thaliana SO N. tabacum SO high pressure liquid chromatography fast protein liquid chromatography Molybdenum plays an important role as the active center in molybdenum enzymes catalyzing essential redox reactions in the global C, N, and S cycles (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1480) Google Scholar). Molybdenum enzymes are important for such diverse metabolic processes as sulfur detoxification and purine catabolism in mammals (2Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (444) Google Scholar), nitrate assimilation in autotrophs, and phytohormone synthesis in plants (3Mendel R.R. Schwarz G. Crit. Rev. Plant Sci. 1999; 18: 33-69Crossref Google Scholar). With the exception of nitrogenase, in all molybdenum enzymes studied so far, molybdenum is part of a so-called molybdenum cofactor (Moco).1 Sulfite oxidase (SO) from vertebrate sources is among the best studied molybdenum enzymes. Mammalian and avian SO (EC 1.8.3.1) is a homodimeric protein consisting of an N-terminal heme domain and a C-terminal Moco domain (4Cohen H.J. Fridovich I. J. Biol. Chem. 1971; 246: 359-366Abstract Full Text PDF PubMed Google Scholar, 5Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1977; 252: 2017-2025Abstract Full Text PDF PubMed Google Scholar), and its crystal structure has been solved (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 (438) Google Scholar). SO catalyzes the reaction SO32− + H2O → SO42− + 2H+ + 2e−, which is the important terminal step in the oxidative degradation of cysteine, methionine, and membrane components such as the sulfatides. Deficiency of this enzyme in humans leads to major neurological abnormalities and early death (7Garrett R.M. Johnson J.L. Graf T.N. Feigenbaum A. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6394-6398Crossref PubMed Scopus (123) Google Scholar). In mammals, SO is localized in the intermembrane space of mitochondria (8Cohen 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 on to cytochromec, the physiological electron acceptor. SO has been extensively studied by EPR (9Raitsimring A.M. Pacheco A. Enemark J.H. J. Am. Chem. Soc. 1998; 120: 11263-11278Crossref Scopus (65) Google Scholar) and resonance Raman spectroscopy (10Johnson M.K. Garton S.D. Oku H. J. Biol. Inorg. Chem. 1997; 2: 797-803Crossref Scopus (29) Google Scholar) as well as rapid kinetic methods, and a detailed model for the reaction mechanism has been published (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1480) Google Scholar). Together with eukaryotic assimilatory nitrate reductase (NR), SO forms a family of molybdenum enzymes that contain a dioxomolybdenum center (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1480) Google Scholar, 2Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (444) Google Scholar). Like SO, NR is a homodimeric enzyme with a Moco domain, a heme domain, and a third FAD-binding domain that does not occur in SO (2Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (444) Google Scholar). In plants, during primary sulfate assimilation in the chloroplast, sulfate is reduced via sulfite to organic sulfide, which is essential for cysteine biosynthesis (11Leustek T. Saito K. Plant Physiol. 1999; 120: 637-644Crossref PubMed Scopus (389) Google Scholar). However, there are reports that sulfite can be reoxidized to sulfate (e.g. when plants are subjected to SO2 gas (as reviewed in Ref. 12Heber U. Hüve K. Int. Rev. Cytol. 1998; 177: 255-286Crossref Google Scholar) or when isolated chloroplasts are fed with radioactively labeled sulfite) (13Dittrich A.P.M. Pfanz H. Heber U. Plant Physiol. 1992; 98: 738-744Crossref PubMed Scopus (29) Google Scholar). Sulfite oxidation in intact chloroplasts is enhanced by light and sensitive to inhibitors of photosynthetic electron transport (13Dittrich A.P.M. Pfanz H. Heber U. Plant Physiol. 1992; 98: 738-744Crossref PubMed Scopus (29) Google Scholar, 14Jolivet P. Bergeron E. Zimierski A. Meunier J.-C. Phytochemistry. 1995; 38: 9-14Crossref Scopus (17) Google Scholar) and thus was interpreted to be primarily due to nonenzymatic reactions during electron transport. Another sulfite-oxidizing activity has been found to be associated with thylakoid membranes (15Jolivet P. Bergeron E. Meunier J.-C. Phytochemistry. 1995; 40: 667-672Crossref Scopus (19) Google Scholar), although this enriched enzyme fraction exhibited properties that were different from the hepatic enzyme. Another report describes the purification of a SO activity from the plant Malva sylvestris (16Ganai B.A. Masood A. Baig M.A. Phytochemistry. 1997; 45: 879-880Crossref Scopus (16) Google Scholar); however, the enzyme had a molecular mass of only 27 kDa. Here we report the cloning of the plant SO gene from Arabidopsis thaliana (At-SO) and the biochemical characterization of the encoded protein. Additionally, native SO was purified fromNicotiana tabacum (Nt-SO) leaves, and its properties were compared with At-SO. At-SO is a molybdenum enzyme with molybdenum-MPT as molybdenum cofactor. In contrast to animal SO, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only known molybdenum enzyme lacking other redox-active centers. Another difference from animal SO is that At-SO is localized mostly in the peroxisomal fraction. Finally, using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species belonging to herbacious and woody plants. The expressed sequence tag (GenBankTM accession number AV 540461) was used to screen an A. thaliana cDNA library (λ YES), resulting in the isolation and identification of a cDNA encoding for A. thaliana SO (At-sox) which was subcloned into pBluescript SK+ (Stratagene), yielding pBS-At-sox. Hybridizations and Southern blotting were performed according to standard molecular biology techniques. The full-length At-sox cDNA (1443 base pairs) showed a single open reading frame of 1182. For overexpression and purification of recombinantA. thaliana SO (rAt-SO), the QIAexpress kit (Qiagen) for metal ion chelating chromatography of His-tagged proteins was used. The His6 tag was fused to the C-terminal end of the protein separated by two additional amino acids. At-sox was PCR-cloned into NcoI- and BglII-digested pQE-60, resulting in pQE60-At-sox. Initial experiments were performed by using pQE60-At-sox to express recombinant At-SO (rAt-SO) in Escherichia coli M15 cells. However, in order to express large amounts of protein with a high saturation of Moco, the expression cassette of pQE60-At-sox(EcoRI-HindIII fragment) was subcloned into pQE80, yielding pQE80-At-sox, allowing a pREP4-independent expression. Recombinant At-SO (rAt-SO) was expressed from pQE80-At-sox in the E. coli strain TP1000 (genotype ΔmobAB), which was previously used for expression of recombinant human SO with a saturation of Moco (17Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). TP1000/pQE80-At-sox was grown aerobically at 37 °C overnight in LB supplemented with antibiotics. This culture was diluted 1:50 into 2× YT medium containing 1 mm Na2MoO4 and 0.1 mmisopropyl-β-thiogalactoside. Aerobic growth was continued for 24 h at 22 °C. Purification of soluble rAt-SO was performed at 4 °C by using Ni2+-nitrilotriacetic acid Superflow matrix (Quiagen). Purified protein was analyzed by SDS-PAGE on 12.5% polyacrylamide gels, and pure fractions were dialyzed against 20 mm Tris/HCl, pH 8.0, 1 mm EDTA, concentrated by ultrafiltration using a YM10 membrane (Amicon), sterile filtrated through 0.2-μm membranes, and stored at 4 °C or in aliquots at −70 °C. The protein preparation was stable at 4 °C for several weeks after dialyzing and concentrating to 2–10 mg/ml. Concentration of rAt-SO was determined by UV spectroscopy using the calculated extinction coefficient of ε = 69.820m−1 cm−1. Polyclonal antibodies were generated against purified rAt-SO protein (Eurogentec). Size exclusion chromatography was performed with an FPLC system (Amersham Pharmacia Biotech) using the (Amersham Pharmacia The was in 20 mm Tris/HCl, mm 1 mm EDTA, pH 8.0, and of purified rAt-SO was separated at a of The molecular was determined using a from the of standard proteins N. tabacum were in liquid in of mm 1 mm EDTA, 1 1 mm pH of cell and the was at for 20 The as crude was to a that was previously and with of the enzyme was with a of m in of were analyzed by SDS-PAGE and with the generated in this fractions were concentrated and subjected to size exclusion chromatography in aliquots using a (Amersham Pharmacia Biotech) in 20 mm 1 mm EDTA, pH Finally, the fractions were in 20 mm Tris/HCl, 1 mm EDTA, pH 8.0, and to a The enzyme was with a of m in 20 of SO was at °C in 20 mm to pH with containing 0.1 mm The reaction was in a single of = at of SO activity was as the of 1 of reductase activity was as described (4Cohen H.J. Fridovich I. J. Biol. Chem. 1971; 246: 359-366Abstract Full Text PDF PubMed Google Scholar). in crude protein extracts was determined as described Biochem. PubMed Scopus Google Scholar). kinetic were performed aerobically at °C using a light and a of experiments were performed with mm pH experiments were performed using in which the pH was with in than mm acid was used. kinetic was at pH with using a of than K and the of sulfite and The UV-visible spectra of and reduced rAt-SO were using a and compared with the of and reduced sulfite oxidase from R. Biophys. 1995; PubMed Scopus Google Scholar) SO was reduced with sulfite or and a was and The electron resonance spectra were with a with an m and a at K was using a liquid was and by to the stable oxidation according to Johnson and Rajagopalan J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and analysis were performed as described in previously G. R.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). was by with a standard isolated from oxidase for which the was = J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). active Moco was by the in the of additional as described Schwarz G. J. A. J. J. R.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). were and as described R. R. Arch. Biochem. Biophys. PubMed Scopus Google Scholar) and separated in a For the was against purified rAt-SO as described T. T. K. K. T. K. Plant Physiol. 1992; PubMed Scopus Google Scholar) and diluted to Using the amino acid sequence of human or SO a in an A. thaliana expressed sequence tag (GenBankTM AV 540461) that was and showed an open reading frame of amino with to the primary sequence of the Moco domain of SO 1 However, the sequence for the heme domain known from animal SO was lacking in this the that the cDNA was at the the structure of this gene encoding for a SO was analyzed (GenBankTM The sequence showed a single open reading frame with and was on to the at however, no sequence encoding for a heme domain was found or of this of Arabidopsis with the isolated cDNA as that the gene encoding for the Arabidopsis SO (At-sox) was a single gene not The cDNA sequence was in number as plant SO The of the protein of Moco from animal with from plant SO and plant NR 1 that the At-SO has to SO than SO and NR to the of molybdenum enzymes, a the SO and Arabidopsis NR can be 1 However, there are several the of the SO and of are in the plant as well 1 For the domain of animal and plant SO and of NR are 1 In order to that the isolated cDNA for a SO, the properties of the gene been For recombinant the isolated was into a expression allowing the expression and purification as His-tagged protein from E. rAt-SO was purified to than and N-terminal of the purified protein the The protein exhibited a sulfite SO activity when using as electron acceptor. activity was found with as electron not as the heme domain is known to electron the Moco domain and in hepatic SO J.L. Rajagopalan K.V. J. Biol. Chem. 1977; 252: 2017-2025Abstract Full Text PDF PubMed Google Scholar). The K m for sulfite in the was determined to be which is in the range as found for SO R. Biophys. 1995; PubMed Scopus Google Like hepatic SO D.L. Rajagopalan K.V. Biophys. PubMed Scopus Google Scholar), rAt-SO be by The and the nitrate a at the at at in 20 mm at and nitrate at 1 rAt-SO was to nitrate when using reduced as electron not FPLC size exclusion chromatography of rAt-SO in two The major at which to a molecular mass of while the at which to on the size of At-SO from the amino acid sequence can be that the of the protein is In eukaryotic molybdenum enzymes, molybdenum is by an cofactor. analysis of the oxidation the of the Moco to rAt-SO The of to rAt-SO was determined to be rAt-SO was in the rAt-SO Moco to the NR in crude extracts of the NR activity not the of the cofactor into be the properties of rAt-SO that plant SO the Moco as all other of the SO The UV-visible spectra of rAt-SO showed the of a heme The of the with an at and a at is similar to the for the Moco domain of SO J.L. Rajagopalan K.V. J. Biol. Chem. 1977; 252: 2017-2025Abstract Full Text PDF PubMed Google Scholar) and of the recombinant Moco domain of human SO R.M. Rajagopalan K.V. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). to and S.D. Garrett R.M. Rajagopalan K.V. Johnson M.K. J. Am. Chem. Soc. 1997; Scopus Google Scholar), are in the reduced enzyme. The EPR spectra of rAt-SO at pH and pH with sulfite are in high the EPR with = is to the exhibited by the enzyme the = S. Biochem. J. PubMed Scopus Google Scholar). the enzyme a in = and the of due to strongly = 8.0, S. Biochem. J. PubMed Scopus Google Scholar). With however, the EPR was a due to at two species, although the to that at pH with the enzyme. a of not a pure to the pH with the vertebrate forms of the due at in part to the of the plant enzyme pH not no EPR with the at pH with the enzyme S. Biochem. J. PubMed Scopus Google Scholar) is with the Arabidopsis protein not in 0.1 m the of the UV-visible and EPR at high however, is evident that the molybdenum center of the At-SO is similar to that of the vertebrate The of a or pure pH EPR is due to in and a values of active Using a against a single at was in of crude protein extracts of A. thaliana leaves, the size of which is to the size of the protein as from the primary sequence 4 similar was when crude extracts of from N. and 4 Thus, in species of and plants and of herbacious and woody plants, a protein is expressed with a size similar to rAt-SO and with that are a wide that the generated against rAt-SO SO proteins in all other plants can that plant contain only a single Moco domain and that are In plants, showed that SO were using as electron (15Jolivet P. Bergeron E. Meunier J.-C. Phytochemistry. 1995; 40: 667-672Crossref Scopus (19) Google Scholar) or radioactively labeled sulfur P. Bergeron E. Zimierski A. Meunier J.-C. Phytochemistry. 1995; 38: 9-14Crossref Scopus (17) Google Scholar). SO as a plant molybdenum enzyme is the only sulfite-oxidizing activity in plants, no SO activity be in Plant Moco are by the of activity of all molybdenum enzymes and R.R. 1997; PubMed Scopus Google Scholar). In A. Moco derived from are however, all of are and high of molybdenum enzymes PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). we the N. where Moco are J. A. I. R. Scopus Google Scholar, R.R. J.L. and of Scholar) that were used previously for Moco biosynthesis in plants Schwarz G. J. A. J. J. R.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, G. J. T. J. G. A. H. R.R. Plant Cell. 2000; PubMed Scopus Google Scholar). of SO activity in cell of in the Moco and showed that had SO of of activity in the standard 4 and oxidase were in J. A. I. R. Scopus Google and G. J. T. J. G. A. H. R.R. Plant Cell. 2000; PubMed Scopus Google not in the are in the of molybdenum into and can be by in the of high G. J. T. J. G. A. H. R.R. Plant Cell. 2000; PubMed Scopus Google Scholar, R.R. Scopus Google Scholar). SO activity in grown in a medium containing 1 mm was found to be to of activity 4 which is a the of Moco and the SO activity in plants. that in N. of the to sulfite is due to the molybdenum enzyme The of the sulfite-oxidizing In plants, the enzymes for primary sulfate assimilation are localized in the where sulfate is reduced via sulfite to organic (11Leustek T. Saito K. Plant Physiol. 1999; 120: 637-644Crossref PubMed Scopus (389) Google Scholar). SO the of sulfite we SO is localized in the extracts of N. were separated into fractions and SO was after SDS-PAGE of that SO be found in the peroxisomal fraction not in which with the of the at the of the enzyme 1 with animal SO, additional amino are an of the that was found to be in the crystal structure of 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 (438) Google Scholar). The experiments were with from different and with from Arabidopsis not and the as in were we that SO is not localized in the and not with primary sulfate that in plant crude extracts SO activity can be that is on the biosynthesis of Moco, we to that this activity is to the that was and native was purified to than by ion chromatography by FPLC size exclusion chromatography resulting in an of the protein. N-terminal of the purified protein that of amino were to the of A. thaliana SO The K m for sulfite was determined to be which is in the range as found for rAt-SO molecular of molybdenum in plants we isolated a cDNA from A. thaliana encoding for a protein with high to animal The plant sequence for a Moco domain, the heme domain was of the structure of this gene the of sequence for a domain, which is with the of SO activity of both expressed and native SO, the of a UV-visible and the molecular mass of the cross-reacting protein in different plant that such a single domain SO protein is in The crystal structure of 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 (438) Google Scholar) that the heme domain and the Moco domain not be The structure of the the molybdenum domain and the separated heme domain a high for this part of the protein A. G. Enemark J.H. J. Biol. Inorg. Chem. 1999; PubMed Scopus Google Scholar). Together with the the molybdenum center and the electron of the heme domain to the Moco domain be a of the heme is that in plants the of the SO electron be by a expressed protein. on the of the native electron for plant SO, well be that a protein electron transport SO and the native protein is This from the purification where native SO purified from N. only the single Moco was found for the of while in a expressed heme domain was with the Moco domain of SO from U. J. Schwarz G. R. C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). For however, is that plant SO an electron protein of a redox similar to a into that experiments a peroxisomal for plant SO, the described for plant E. Biochem. J. 1999; PubMed Scopus Google Scholar) are a to the role of physiological electron for plant eukaryotes, plant SO is the molybdenum enzyme known to and the only lacking other redox-active centers. The of Moco of from different sources SO and NR enzymes as of a the so-called SO family (2Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (444) Google Scholar). Arabidopsis is the where both and of this family are which a system for of a molybdenum domain that can be part of an oxidase or of a The and spectral properties of At-SO are so similar to the Moco domain of the animal that is to that this enzyme in as an This is by SO activity in N. In mutants, SO activity is strongly as compared with the that the molybdenum enzyme SO in as This enzyme is among plants as by the that antibodies against rAt-SO a cross-reacting protein of in a wide range of species belonging to a of both herbacious and woody plants. SO catalyzes than of the sulfite there is a activity in N. This activity be nonenzymatic as described by (13Dittrich A.P.M. Pfanz H. Heber U. Plant Physiol. 1992; 98: 738-744Crossref PubMed Scopus (29) Google Scholar) for electron transport in chloroplasts or due to the fraction that was described in chloroplasts P. Bergeron E. Zimierski A. Meunier J.-C. Phytochemistry. 1995; 38: 9-14Crossref Scopus (17) Google Scholar, P. Bergeron E. Meunier J.-C. Phytochemistry. 1995; 40: 667-672Crossref Scopus (19) Google Scholar). However, sulfite oxidation in has to be in order to with the sulfate assimilation that is for the of the plant found At-SO in the peroxisomal fraction than in which that the of SO is not to the sulfur assimilation be that plant SO has a For has been that peroxisomal is by of sulfite S. T. U. Plant Physiol. 1998; Scopus Google Scholar). are to the physiological role of SO in plants, as well as and properties. T. for and R. for N-terminal protein