Sulfite oxidase (EC 1.8.3.1) known to catalyse the aerobic oxidation of sulfite into sulfate, is found in the mitochondria of all eukaryotes. It was first isolated from bovine liver and subsequently purified from both animal and plant sources such as rat, dog, beef, humans, chicken and goat liver (animals) and plant Spinacia oleracea, Malva sylvestris, Nicotiana tabacum and Arabidopsis thaliana (plants). Various physiological properties of purified SO_x have been studied e.g. molecular weight, optimum pH, temperature, time, K_m & for sulfite metal ions activation and inhibition.

Sulfite oxidase (SO_x) (EC 1.8.3.1) is found in the mitochondria of all eukaryotes. It oxidizes sulfite to sulfate and via cytochrome c. It transfers the electrons produced to the electron transport chain (ETC), allowing generation of ATP by oxidative phosphorylation. This is the last step in the metabolism of sulfur-containing compounds and the sulfate is excreted. SO_x is a metallo-enzyme containing a molybdopterin cofactor and a heme group. It is one of the cytochrome b₅ and belongs to the enzyme super-family of molybdenum oxotransferases that also includes dimethylsulfoxide (DMSO) reductase, xanthine oxidase and nitrite reductase. In mammals, the expression level of SO_X is high in the liver, kidney and heart but very low in spleen, brain, skeletal muscle and blood^1–3 of functional SO_x causes sulfite deficiency. This causes neurological disorders, mental retardation, physical deformities, dislocated lenses, degradation of brain and early death thus sulfite level in body must normally be strictly maintained. SOx was first isolated from bovine liver and subsequently also purified from animal sources such as rat, dog, beef, humans, chicken and goat liver and plant sources such as Spinacia oleracea, Malva sylvestris, Nicotiana tabacum and Arabidopsis thaliana (Table 1).⁴ The present review deals with the distribution, purification, and characterization of SO_X from various sources. 

Purification and properties of sulfite oxidase from different sources

SO_X has been purified from different sources with varying molecular weight, opitimum pH, incubation temperature, K_m, metal ion requirement and inhibition.

Purification

 Goat SO_x was purified from liver by ammonium sulfate precipitation (50%), ion exchange chromatography (DEAE-cellulose, DE-52) and gel filtration by 92.56 fold purification with 7.30% yield,⁵ Sulfitobacter pontiacus SO_x was purified by cation- exchange absorber and ultrafiltration with 20 fold purification and a specific activity of 188 U/mg,⁶ Thermus thermophilus cell material SO_x was purified by ammonium sulfate precipitation (60% saturation), DEAE 52 column chromatography, superdex X26 gel filtration and Mono Q with 297 fold purification, 14% yield and specific activity of 56.67 U/mg,⁷ Arabidopsis thaliana SO_x was purified by Q30, superdex 200 FPLC with 12.84 fold purification and 10.7% yield,⁴ Nicotiana SO_x was purified from leaves by ion exchange chromatography followed by FPLC size exclusion with 100 fold purification,⁴ Malva sylvestris SO_X was purified from leaves by heat treatment, ion exchange chromatography and sephadex gel chromatography with 8.3 fold purification and 28% yield,⁸ Spinach SO_x was purified from thylakoid by ammonium sulfate acetone precipitation (50%), dialysis and DEAE- cellulose column chromatography,⁹ Thiobacillus novellus SO_x was purified by DEAE-cellulose, hydroxyapatite, affi-Gel blue with 206 fold purification and 3.4% yield,¹⁰ human and rat SO_X was purified from liver by heating, ammonium sulfate precipitate, acetone, DE-52 ion exchange chromatography with 6459 and 1113 folds purification and 9% and 6% yields respectively,¹¹ SO_x was purified from liver by heating (56°C), ammonium sulfate precipitation (50%), DEAE-cellulose and gel fractionation on Sephadex G-200 with 530 fold purification and 10% yield,¹² Hepatic SO_x was purified from chicken liver by heating (60°C), ammonium sulfate precipitation (40%) and DEAE cellulose ion exchange with 802 fold purification and 12% yield,¹³ Thiobacillus novellus SO_x was purified from 6 day old cells by sonication, ammonium sulfate precipitate (40%), heating and ammonium sulfate precipitation (40-70%), beef SO_X was purified from liver by ammonium sulfate precipitate (0-50%), heat (55^oC), first DEAE cellulose column, ammonium sulfate precipitation (0-50%), second DEAE cellulose column chromatography, ammonium sulfate precipitation (0-50%) and third DEAE cellulose column, ammonium sulfate precipitate (0-50%) with 233 fold purification, 7.8% yield and with a specific activity of 280 U/mg¹⁴ and dog sulfite oxidae was purified from liver by protamine sulfate supernatant, ammonium sulfate precipitation (0-50%), first DEAE cellulose column and second DEAE cellulose column with 385 fold purification, 16 % yield and specific activity of 27.10 U/mg.¹⁴ 

Molecular weights

The molecular weights of SO_x purified from different sources were in the range, 27 to 113 kDa. The molecular weights of purified enzyme from different sources has been summarized in Table 2. 

Optimum pH

The optimum pH of SO_x purified from different sources was in neutral to alkaline range i.e. pH 7-11. The functional groups in side chain of amino acid residues at the catalytic site as well as in the peptide backbone of enzyme are vital for catalytic action of enzyme. In alkaline pH range, the –NH₂ group of side chain of Lys, His, Arg controls the activity of SO_x. The optimum pH of SO_x from different sources is summarized in Table 3.

Optimum temperature

 Every enzyme works at its maximum rate at a specific temperature called as optimum temperature for that enzyme. SO_x isolated and purified from different sources had a wide range of optimum temperature 20-60°C. The optimum temperature of an enzyme can be explained on the basis of kinetic theory, which states that increased temperature increases the speed of molecular movement and thus the chances of molecular collisions which is directly proportional to incubation temp. The optimum temp. of SO_x from different sources is summarized in Table 4.

Michaelis kinetic constant (K_m)

 The measurement of Michaelis kinetic constant K_m for sulfite from various sources has been calculated from Lineweaver-Burk (LB) plot. K_m for SO_x was found in a wide range, 0.00092mM to 5.33mM. K_m value of SO_X from different sources is summarized in Table 5.

Inhibition studies

SO_x from various sources was inhibited by a number of compounds including metal ions and substrate analogues such as arsenite, EDTA, imidazole, Ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, diethylpyro-carbonate, N-ethyl-5-phenylisoxazolium-3'-sulfonate, 2,6-dichloroindophenol, cyanide, cyt c, ferricyanide, sodium tungstate, NaCl, potassium nitrate, potassium phosphate and Tris-HCl buffer. A list of various inhibitors is given in Table 6.

3D structure of SO_x

As a homodimer, SO_x contains two identical subunits, each with an N-terminal domain and a C-terminal domain. These two domains are connected by ten amino acids forming a loop. The N-terminal domain has a cofactor with three adjacent antiparallel beta sheets and five alpha helices. The C-terminal domain hosts a molybdopterin cofactor surrounded by thirteen beta sheets and three alpha helices. The molybdopterin cofactor has a Mo (VI) center, bonded to a sulfur from cysteine, an ene-dithiolate from pyranopterin and two terminal oxygens. It is at this molybdenum center that the catalytic oxidation of sulfite takes place (Figure 1).

Figure 1 3D structure of SOx.

Mechanism of action of SO_x

The active site of SO_x contains the molybdopterin cofactor and supports molybdenum in its highest oxidation state, +6 (Mo^VI). In the enzyme's oxidized state, molybdenum is coordinated by a cysteine thiolate, the dithiolene group of molybdopterin, and two terminal oxygen atoms. Upon reacting with sulfite, one oxygen atom is transferred to sulfite to produce sulfate and the molybdenum center is reduced by two electrons to Mo^IV. Water then displaces sulfate and the removal of two protons (H⁺) and two electrons returns the active site to its original state. A key feature of this oxygen atom transfer in enzyme is that the oxygen atom being transferred arises from water, not from dioxygen (O₂) (Figure 2).

Figure 2 A proposed mechanism of the oxidation of sulfite to sulfate by sulfite oxidase.

In reductive or oxidative sulfur metabolism

Assimilatory sulfate reduction in plants starts with the uptake of sulfate from soil into the roots and translocation via the xylem to green parts of the plant, where it is stored as the major anionic component of vacuolar sap.^15–28 Chloroplasts are the limiting organelles where sulfate reduction takes place, only cysteine synthesis enzymes are the ones localized not only in plastids but also in the cytosol and mitochondria. Sulfite is not only a key metabolite in assimilatory sulfate reduction, but also plays a central role in reductive or oxidative sulfur metabolism in both pro- and eukaryotes. In plants, sulfite is also the starting point for the formation of sulfolipids and an intermediate in the oxidation of reduced sulfur compounds to sulfate. Yet, it has to be considered that sulfite reduction and sulfolipid formation are chloroplast-based while sulfite oxidation is localized in peroxisomes.

Antimicrobial effects

Undissociated sulfonic acid and dissolved sulfur dioxide, respectively, are the most effective forms of S (IV) against microbial organisms, whereas sulfites (SO₂^–3) have only little or no effects. Hydrogen sulfite, the sulfite form most prevalent at normal food pH values, still has remarkable effects against microbial spoilage, but the effects are less strong than those of the undissociated acid. Furthermore, the intensity of the preservative effects is different for bacteria, yeast and mold fungus and even within these groups, there are also some significant variations. The minimum concentration for an inhibitory effect on bacteria at a pH value of 6.0 varies between 500mg/L and 2000mg/L of sodium sulfite. For an inhibitory effect on yeast and mold, generally lower pH values or higher concentrations of sodium sulfite are required.

Role of sulfite as preservative

Sulfite compounds are used as a preservative for food and drinks. Sulfites are a family of chemicals that includes sodium sulfite, sodium bisulfite, sodium metabisulfite, potassium sulfite and potassium metabisulfite. Sulfites are produced naturally during the fermentation of wine. However, the majority of sulfur compounds are produced through chemical synthesis. Sulfites are used to preserve the flavor and appearance of foods such as jam, gravy, shrimp, dried soup mixes, dehydrated potatoes and juices. Fresh or dried fruit and vegetables are sometimes dipped or sprayed with sulfites to not only to kill germs but also to preserve appearance and taste. Sulfites keep vegetables from turning brown. Sodium metabisulfite and potassium metabisulfite are sometimes added to fermenting beer and wine to inhibit microbes and increase shelf life. Sulfites are also added to meat products. Shrimp and lobster spoilage is greatly reduced when these foods are sprayed or dipped in sulfite solutions. Before the U.S. Department of Agriculture banned the practice, meat packagers used to add sulfites to raw meat in order to keep it looking fresh.

Use of sulfite as preservatives in medicine

Drug manufacturers take advantage of the antimicrobial and antioxidant properties of sulfites to preserve and increase the shelf life of medications. Sulfates are added to asthma inhalants, medicines given by injection, eye drops, oral tablets and liquids. The U.S. Food and Drug Administration (FDA) require that all sulfite containing food and drugs available in the U.S. list the amounts on their labels. However, there is no such rule in India.

Use of sulfite as antioxidants, antimicrobials and bleaching agents

Sulfites are antioxidants, antimicrobials and bleaching agents. As antioxidants prevent or reduce discoloration of fruits and vegetables, sulfites are commonly used in products such as dried apples, dehydrated potatoes, processed fruit and vegetable juices. It is used in wine-making, because it inhibits bacterial growth but does not interfere with the desired development of yeast. It is used in bleaching food starches and prevents rust and scale formation in boilers. Some sulfites are used in the production of cellophane for food packaging. However, it causes allergic reactions to asthmatics. It causes difficulty in breathing, stomach-ache, hives, bronchospasm and even anaphylactic shock. Sulfite sensitivity occurs most often in asthmatic adults, predominantly women and uncommonly reported in preschool children. The reactions of sulfite sensitive asthmatics to sulfites vary from person to person. It causes anaphylaxis (allergic reaction). Sulfites may also contribute to chronic skin and respiratory symptoms.

Food industry

Artificial ETC is composed of cyt c and SO_x formed by the layer-by-layer technique using a polyelectrolyte. The multilayer technology of sulfite oxidase-cyt c multilayer electrode may act as an anode in a bio-fuel cell and may be exploited as a biosensor for the detection of sulfite in wine and other foodstuffs.

Sulfite oxidases catalyze the physiologically vital oxidation of sulfite to sulfate in animals, plants and bacteria. Its enzymatic activity can be affected by various factors: pH, temperature and inhibitor. Each type of sulfite oxidase has a distinct optimum pH, pH stability, an optimum temperature and temperature stability. The molecular weights of SO_x purified from different sources were in the range, 27 to 113 kDa. The optimum pH of SO_x purified from different sources was in neutral to alkaline range i.e. pH 7–11. SOx isolated and purified from different sources had a wide range of optimum temperature 20-60°C K_m for SO_x was found in a wide range, 0.00092 mM to 5.33 mM. SO_x from various sources was inhibited by a number of compounds including metal ions and substrate analogues such as arsenite, EDTA, imidazole, Ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, diethylpyro-carbonate, N-ethyl-5-phenylisoxazolium-3'-sulfonate, 2,6-dichloroindophenol, cyanide, cyt c, ferricyanide, sodium tungstate, NaCl, potassium nitrate, potassium phosphate and Tris-HCl buffer. Sulfite oxidase is used in reductive or oxidative sulfur metabolism, antioxidants, antimicrobials and bleaching agents, preservatives in medicine and food industry. The conventional method is used for highly purified sulfite oxidase involves ammonium sulfate fractionation, gel filtration or anion exchange chromatography.. The characteristics of sulfite oxidas are assayed by enzyme activity and concentration, specific activity (SA), purification factor (PF), molecular weight, homogeneity and isoelectric point (pI). Sulfite oxidase is considered a promising enzyme with wide applications in both conventional and new industries and new efficient, cost effective techniques for its recovery and purification should be developed.

None.

The author declares there are no conflicts of interest.

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