Plant Sulfite Oxidase Research Articles

Sulfite Oxidase Activity Level Determines the Sulfite Toxicity Effect in Leaves and Fruits of Tomato Plants

Increasing plant tolerance to sulfites/SO2 can lead to the development of tolerant crops to biotic and abiotic stresses. Plant sulfite oxidase (SO) is a molybdo-enzyme that oxidizes excess SO2/sulfite into non-toxic sulfate. The effect of toxic sulfite on leaves and fruits was studied in tomato plants with different SO expression: wild-type, SO overexpression (OE) and SO RNA interference (Ri). Sulfite-dipped ripe-fruits and sulfite treated leaf discs of Ri plants impaired in SO activity were more susceptible, whereas OE plants were more resistant, as revealed by remaining chlorophyll and tissue damage levels. Application of molybdenum further enhanced the tolerance of leaf discs to sulfite by enhancing SO activity in OE lines, but not in wild-type or Ri plants. Notably, incubation with tungsten, the molybdenum antagonist, overturned the effect of molybdenum spray in OE plants, revealed by remaining chlorophyll content and SO activity. The results indicate that SO in tomato leaves and ripe fruits determines the resistance to sulfite and the application of molybdenum enhances sulfite resistance in OE plants by increasing SO activity. Overall, the results suggest that SO overexpression can be employed, with or without molybdenum application, for developing fruit and vegetable crops tolerant to sulfite/SO2 containing pre- and postharvest treatments.

Sulfite Oxidase Activity Is Essential for Normal Sulfur, Nitrogen and Carbon Metabolism in Tomato Leaves.

Plant sulfite oxidase [SO; E.C.1.8.3.1] has been shown to be a key player in protecting plants against exogenous toxic sulfite. Recently we showed that SO activity is essential to cope with rising dark-induced endogenous sulfite levels in tomato plants (Lycopersicon esculentum/Solanum lycopersicum Mill. cv. Rheinlands Ruhm). Here we uncover the ramifications of SO impairment on carbon, nitrogen and sulfur (S) metabolites. Current analysis of the wild-type and SO-impaired plants revealed that under controlled conditions, the imbalanced sulfite level resulting from SO impairment conferred a metabolic shift towards elevated reduced S-compounds, namely sulfide, S-amino acids (S-AA), Co-A and acetyl-CoA, followed by non-S-AA, nitrogen and carbon metabolite enhancement, including polar lipids. Exposing plants to dark-induced carbon starvation resulted in a higher degradation of S-compounds, total AA, carbohydrates, polar lipids and total RNA in the mutant plants. Significantly, a failure to balance the carbon backbones was evident in the mutants, indicated by an increase in tricarboxylic acid cycle (TCA) cycle intermediates, whereas a decrease was shown in stressed wild-type plants. These results indicate that the role of SO is not limited to a rescue reaction under elevated sulfite, but SO is a key player in maintaining optimal carbon, nitrogen and sulfur metabolism in tomato plants.

Oxygen reactivity of mammalian sulfite oxidase provides a concept for the treatment of sulfite oxidase deficiency.

Mammalian sulfite oxidase (SO) is a dimeric enzyme consisting of a molybdenum cofactor- (Moco) and haem-containing domain and catalyses the oxidation of toxic sulfite to sulfate. Following sulfite oxidation, electrons are passed from Moco via the haem cofactor to cytochrome c, the terminal electron acceptor. In contrast, plant SO (PSO) lacks the haem domain and electrons shuttle from Moco to molecular oxygen. Given the high similarity between plant and mammalian SO Moco domains, factors that determine the reactivity of PSO towards oxygen, remained unknown. In the present study, we generated mammalian haem-deficient and truncated SO variants and demonstrated their oxygen reactivity by hydrogen peroxide formation and oxygen-consumption studies. We found that intramolecular electron transfer between Moco and haem showed an inverse correlation to SO oxygen reactivity. Haem-deficient SO variants exhibited oxygen-dependent sulfite oxidation similar to PSO, which was confirmed further using haem-deficient human SO in a cell-based assay. This finding suggests the possibility to use oxygen-reactive SO variants in sulfite detoxification, as the loss of SO activity is causing severe neurodegeneration. Therefore we evaluated the potential use of PEG attachment (PEGylation) as a modification method for future enzyme substitution therapies using oxygen-reactive SO variants, which might use blood-dissolved oxygen as the electron acceptor. PEGylation has been shown to increase the half-life of other therapeutic proteins. PEGylation resulted in the modification of up to eight surface-exposed lysine residues of SO, an increased conformational stability and similar kinetic properties compared with wild-type SO.

Elucidating the Catalytic Mechanism of Sulfite Oxidizing Enzymes Using Structural, Spectroscopic, and Kinetic Analyses

Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor-dependent enzymes that are found in plants, animals, and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification and in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates, the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid kinetic studies of PSO are also discussed.

A fast and sensitive HPLC method for sulfite analysis in food based on a plant sulfite oxidase biosensor

A reliable and sensitive analysis of sulfites in food is essential in food monitoring. However, the established methods exhibit deficiencies in the very low concentration ranges (below 10 mg/L SO 2 ), especially with more complex food matrices. With a focus on these challenges, an HPLC method with immobilized enzyme reactor (HPLC–IMER) for the analysis of sulfites in food was optimized and compared to a standard method. A modulated sample preparation procedure and the use of a novel sulfite oxidase from Arabidopsis thaliana were explored to make the method applicable for most food samples. The plant sulfite oxidase turned out to be superior to the commercially available animal sulfite oxidase in terms of detection limit (0.01 mg/L SO 2 ), linear range (0.04–20 mg/L SO 2 ) and stability. In a small scale comparison within our laboratory, as well as in a standardized proficiency testing, the HPLC–IMER was compared to an established distillative method. The enzyme-based method is not only more sensitive and specific, it also yields higher sulfite recoveries in almost all samples while exhibiting better statistic method parameters.

Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase

Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O(2) and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O(2)] with a second-order rate constant of k(ox) = 8.7 x 10(4) +/- 0.5 x 10(4) m(-1)s(-1). When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed.

Spectroscopic Characterization of YedY: The Role of Sulfur Coordination in a Mo(V) Sulfite Oxidase Family Enzyme Form

Electronic paramagnetic resonance (EPR), electronic absorption, and magnetic circular dichroism spectroscopies have been performed on YedY, a SUOX fold protein with a Mo domain that is remarkably similar to that found in chicken sulfite oxidase, Arabidopsis thaliana plant sulfite oxidase, and the bacterial sulfite dehydrogenase from Starkeya novella. Low-energy dithiolene --> Mo and cysteine thiolate --> Mo charge-transfer bands have been assigned for the first time in a Mo(V) form of a SUOX fold protein, and the spectroscopic data have been used to interpret the results of bonding calculations. The analysis shows that second coordination sphere effects modulate dithiolene and cysteine sulfur covalency contributions to the Mo bonding scheme. In particular, a more acute O(oxo)-Mo-S(Cys)-C dihedral angle results in increased cysteine thiolate S --> Mo charge transfer and a large g(1) in the EPR spectrum. The spectrosocopic results, coupled with the available structural data, indicate that these second coordination sphere effects may play key roles in modulating the active-site redox potential, facilitating hole superexchange pathways for electron transfer regeneration, and affecting the type of reactions catalyzed by sulfite oxidase family enzymes.

Hibiscus chlorotic ringspot virus upregulates plant sulfite oxidase transcripts and increases sulfate levels in kenaf (Hibiscus cannabinus L.)

Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is required for encapsidation and virus systemic movement. To better understand the roles of HCRSV CP in virus infection and its interactions with host proteins, a cDNA library of kenaf (Hibiscus cannabinus L.) was constructed and screened by using a yeast two-hybrid system (YTHS) to identify CP-interacting proteins. One protein identified was sulfite oxidase (SO) and the interaction was confirmed in vitro and in vivo. The interaction was found to be associated with peroxisomes by immunofluorescent labelling of peroxisomes by an anti-SKL signal peptide antibody. Our YTHS results showed that only the P and S domains of CP interacted with SO from kenaf. This is probably due to the exposure of these two domains on the outer surface of the capsid. Peroxisomes were observed to aggregate in HCRSV-infected cells, and biochemical assays of total protein from kenaf leaf extracts showed that SO activity and SO-dependent H(2)O(2)-generating activity in the HCRSV-infected leaves increased compared with that in mock-inoculated kenaf plants.

Significance of Plant Sulfite Oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.

Sulfur K-edge Spectroscopic Investigation of Second Coordination Sphere Effects in Oxomolybdenum-Thiolates: Relationship to Molybdenum−Cysteine Covalency and Electron Transfer in Sulfite Oxidase

Second-coordination sphere effects such as hydrogen bonding and steric constraints that provide for specific geometric configurations play a critical role in tuning the electronic structure of metalloenzyme active sites and thus have a significant effect on their catalytic efficiency. Crystallographic characterization of vertebrate and plant sulfite oxidase (SO) suggests that an average O(oxo)-Mo-S(Cys)-C dihedral angle of approximately 77 degrees exists at the active site of these enzymes. This angle is slightly more acute (approximately 72 degrees) in the bacterial sulfite dehydrogenase (SDH) from Starkeya novella. Here we report the synthesis, crystallographic, and electronic structural characterization of Tp*MoO(mba) (where Tp* = (3,5-dimethyltrispyrazol-1-yl)borate; mba = 2-mercaptobenzyl alcohol), the first oxomolybdenum monothiolate to possess an O(ax)-Mo-S(thiolate)-C dihedral angle of approximately 90 degrees . Sulfur X-ray absorption spectroscopy clearly shows that O(ax)-Mo-S(thiolate)-C dihedral angles near 90 degrees effectively eliminate covalency contributions to the Mo(xy) redox orbital from the thiolate sulfur. Sulfur K-pre-edge X-ray absorption spectroscopy intensity ratios for the spin-allowed S(1s) --> Sv(p) + Mo(xy) and S(1s) --> Sv(p) + Mo(xz,yz) transitions have been calibrated by a direct comparison of theory with experiment to yield thiolate Sv(p) orbital contributions, c(j)(2), to the Mo(xy) redox orbital and the Mo(xz,yz) orbital set. Furthermore, these intensity ratios are related to a second coordination sphere structural parameter, the O(oxo)-Mo-S(thiolate)-C dihedral angle. The relationship between Mo-S(thiolate) and Mo-S(dithiolene) covalency in oxomolydenum systems is discussed, particularly with respect to electron-transfer regeneration in SO.

Plant Sulfite Oxidase as Novel Producer of H2O2: COMBINATION OF ENZYME CATALYSIS WITH A SUBSEQUENT NON-ENZYMATIC REACTION STEP

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.

Spectroscopic and Kinetic Studies of Arabidopsis thaliana Sulfite Oxidase: Nature of the Redox-Active Orbital and Electronic Structure Contributions to Catalysis

Plant sulfite oxidase from Arabidopsis thaliana has been characterized both spectroscopically and kinetically. The enzyme is unusual in lacking the heme domain that is present in the otherwise highly homologous enzyme from vertebrate sources. In steady-state assays, the enzyme exhibits a pH maximum of 8.5 and is also found to function as a selenite oxidase. Sulfite at the lowest experimentally feasible concentrations reduces the enzyme within the dead-time of a stopped-flow instrument at 5 degrees C, indicating that the A. thaliana enzyme has a limiting rate constant for reduction, k(red), at least 10 times greater than that of the chicken enzyme (190 s(-1)). The EPR parameters for the high- and low-pH forms of the A. thaliana enzyme have been determined, and the g-values are found to resemble those previously reported for the vertebrate enzymes. Finally, the A. thaliana enzyme has been probed by resonance Raman spectroscopy. A detailed analysis of the vibrational spectrum in the region where Mo=O stretching modes are anticipated to occur has been performed with the help of density functional theory calculations, evaluated in the context of the Raman data. Calculated frequencies obtained for two model systems have been compared to experimental resonance Raman spectra of oxidized A. thaliana sulfite oxidase catalytically cycled in both H2(16)O and H2(18)O. The vibrational frequency shifts observed upon (18)O-labeling of the enzyme are consistent with theoretical models in which either the equatorial oxygen or both equatorial and axial atoms of the dioxomolybdenum center are labeled. Importantly, the vibrational mode description is consistent with the active site possessing geometrically inequivalent oxo ligands and a Mo d(xy) redox-active molecular orbital oriented in the equatorial plane forming a pi-bonding interaction solely with the equatorial oxo, O(eq). Electron occupancy of this Mo=O(eq) pi* redox orbital upon interaction with substrates would effectively labilize the Mo=O(eq) bond, providing the dominant contribution to lowering the activation energy for oxygen atom transfer.

Structures of the Mo(V) Forms of Sulfite Oxidase from Arabidopsis thaliana by Pulsed EPR Spectroscopy

The Mo(V) center of plant sulfite oxidase from Arabidopsis thaliana (At-SO) has been studied by continuous wave and pulsed EPR methods. Three different Mo(V) EPR signals have been observed, depending on pH and the technique used to generate the Mo(V) oxidation state. At pH 6, reduction by sulfite followed by partial reoxidation with ferricyanide generates an EPR spectrum with g-values similar to the low-pH (lpH) form of vertebrate SOs, but no nearby exchangeable protons can be detected. On the other hand, reduction of At-SO with Ti(III) citrate at pH 6 generates a Mo(V) signal with large hyperfine splittings from a single exchangeable proton, as is typically observed for lpH SO from vertebrates. Reduction of At-SO with sulfite at high pH generates the well-known high-pH (hpH) signal common to all sulfite oxidizing enzymes. It is proposed that, depending on the conformation of Arg374, the active site of At-SO may be in "closed" or "open" forms that differ in the degree of accessibility of the Mo center to substrate and water molecules. It is suggested that at low pH the sulfite-reduced At-SO has coordinated sulfate and is in the "closed form". Reoxidation to Mo(V) by ferricyanide leaves bound sulfate trapped at the active site, and consequently, there are no ligands with exchangeable protons. Reduction with Ti(III) citrate injects an electron directly into the active site to generate the [Mo(V)[triple bond]O(OH)]2+ unit that is well-known from model chemistry and which has a single exchangeable proton with a large isotropic hyperfine interaction. At high pH, the active site is in the "open form", and water can readily exchange into the site to generate the hpH SO.

Molecular Basis of Intramolecular Electron Transfer in Sulfite-oxidizing Enzymes Is Revealed by High Resolution Structure of a Heterodimeric Complex of the Catalytic Molybdopterin Subunit and a c-Type Cytochrome Subunit

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 A) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.

The Crystal Structure of Plant Sulfite Oxidase Provides Insights into Sulfite Oxidation in Plants and Animals

The molybdenum cofactor (Moco) containing sulfite oxidase (SO) from Arabidopsis thaliana has recently been identified and biochemically characterized. The enzyme is found in peroxisomes and believed to detoxify excess sulfite that is produced during sulfur assimilation, or due to air pollution. Plant SO (PSO) is homodimeric and homologous to animal SO, but contains only a single Moco domain without an additional redox center. Here, we present the first crystal structure of a plant Moco enzyme, the apo-state of Arabidopsis SO at 2.6 Å resolution. The overall fold and coordination of the Moco are similar to chicken SO (CSO). Comparisons of conserved surface residues and the charge distribution in PSO and CSO reveal major differences near the entrance to both active sites reflecting different electron acceptors. Arg374 has been identified as an important substrate binding residue due to its conformational change when compared to the sulfate bound structure of CSO.

Identification and Biochemical Characterization ofArabidopsis thaliana Sulfite Oxidase: A NEW PLAYER IN PLANT SULFUR METABOLISM

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 ). 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.