Existence of aa 3-Type Ubiquinol Oxidase as a Terminal Oxidase in Sulfite Oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an α-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa ₃-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 °C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A₁ and myxothiazol, which are inhibitors of mitochondrial bc ₁ complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.     

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.     

Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

One of two copper atoms is not necessary for the cytochrome c oxidase activity of Pseudomonas AM 1 cytochrome aa3

From Pseudomonas AM 1 grown in a medium deficient in Cu, aa3-type cytochrome c oxidase was purified which contained 2 molecules of haem a and one atom of Cu per molecule. The enzyme showed absorption peaks at 428 and 595 nm in the oxidized form and at 442 and 604 nm in the reduced form, and its CO complex showed peaks at 432 and 602 nm. The enzyme in the oxidized state showed an obscure absorption peak around 800 nm instead of a peak at 820 nm. One mol of the enzyme oxidized maximally 76, 75, and 98 mol of the ferrocytochromes c of Candida krusei, horse and Pseudomonas AM 1 per sec, respectively. These reactions were 50% inhibited by 7 microM KCN. The product of reduction of O2 catalyzed by the enzyme was concluded to be H2O on the basis of the ratio of ferrocytochrome c oxidized to O2 consumed.     

Molybdenum Oxidation by Thiobacillus ferrooxidans

Thiobacillus ferrooxidans AP19-3 oxidized molybdenum blue (Mo⁵⁺) enzymatically. Molybdenum oxidase in the plasma membrane of this bacterium was purified ca. 77-fold compared with molybdenum oxidase in cell extract. A purified molybdenum oxidase showed characteristic absorption maxima due to reduced-type cytochrome oxidase at 438 and 595 nm but did not show absorption peaks specific for c-type cytochrome. The optimum pH of molybdenum oxidase was 5.5. The activity of molybdenum oxidase was completely inhibited by sodium cyanide (5 mM) or carbon monoxide, and an oxidized type of cytochrome oxidase in a purified molybdenum oxidase was reduced by molybdenum blue, indicating that cytochrome oxidase in the enzyme plays a crucial role in molybdenum blue oxidation.     

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).     

Purification and characterization of glycolate oxidase from pumpkin cotyledons

Glycolate oxidase was purified and crystallized from cotyledons of germinating pumpkin seedlings. The molecular weight of the enzyme was determined to be 280,000-320,000, consisting of 8 identical subunits with molecular weight of 38,000. There are two absorption peaks at 340 and 450 nm, indicating the glycolate oxidase is a flavin protein. Several kinetic parameters were determined, Km (glycolate) 0.33 mM and Km (O2) 76.2 microM at pH 8.0. Oxalate and oxalacetate were found to be potent competitive inhibitors against glycolate; the Ki values for oxalate and oxalacetate were 4.5 and 7.8 mM, respectively. Fatty acids such as linoleic acid inhibited the enzyme noncompetitively; the Km for linoleic acid was 0.63 mM. The regulation of glycolate oxidase in the glycolate pathway occurring in leaf peroxisomes is discussed.     

Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.

Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome-c oxidase

The oxidase cho of Methylobacillus flagellatus KT was purified to homogeneity by nondenaturing gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cbo with a pH optimum of 8.3. When TMPD served as electron donor for the oxidase cho, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and of only ascorbate. The kinetic constants, determined at pH 7.0, were as follows: oxidation by the enzyme of reduced TMPD at pH 7.0 was characterized by KM = 0.86 mM and Vmax = 1.1 mumol O2/(min mg protein), and oxidation of reduced cytochrome c from horse heart was characterized by KM = 0.09 mM and Vmax = 0.9 mumol O2/(min mg protein) Cyanide inhibited ascorbate/TMPD oxidase activity (Ki = 4.5-5.0 microM). The soluble cytochrome cH (12 kDa) partially purified from M. flagellatus KT was found to serve as the natural electron donor for the oxidase cbo.     

Menaquinol oxidase activity and primary structure of cytochrome bd from the amino-acid fermenting bacterium Corynebacterium glutamicum

Cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-G+C gram-positive bacterium Corynebacterium glutamicum. Inhibition of NADH oxidase activity in the membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. Cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. Its reduced form showed absorption peaks at 627, 595, and 560 nm, which were due to haem d, high-spin protohaem, and low-spin protohaem, respectively. The air-oxidised form showed a peak at 645 nm, which might be due to oxygenated ferrous haem d. The spectral features and the size of subunit I are more similar to the properties of cytochromes bd from Proteobacteria, such as Escherichia coli, than to those of cytochrome bd from low-G+C gram-positive bacteria, such as Bacillus stearothermophilus. The menaquinol oxidase activity of the purified cytochrome bd was low, but was enhanced about fivefold by pre-incubating the enzyme with menaquinones. The order of effectiveness of quinols as oxidase substrates was clearly different from that of quinones as the activators of enzyme activity. Furthermore, activation was destroyed by ultraviolet irradiation of the pre-incubated enzyme and then restored by a second incubation with menaquinone. These results indicate that the enzymatic properties of this new oxidase are more similar to the properties of cytochromes bd from low-G+C gram-positive bacterial than to those of proteobacterial counterparts. They also suggest that the enzyme has a second quinone-binding site essential for full activity, in addition to the active centre for substrate oxidation. By using probes based on partial peptide sequences of the subunits, the genes for the two subunits of C. glutamicum cytochrome bd were cloned. The deduced amino acid sequence demonstrated that subunit I lacks the C-terminal half of the Q loop and that the primary structure of C. glutamicum cytochrome bd is more similar to that of other gram-positive bacteria than to proteobacterial cytochromes bd.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

Purification and characterization of <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase with thermal,organic solvent,and detergent tolerance

A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.     

Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.     

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.     

Cytochrome c oxidase of Pseudomonas AM 1: purification, and molecular and enzymatic properties

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Pseudomonas AM 1 to an electrophoretically homogeneous state and some of its properties were studied. The oxidase showed absorption peaks at 428 and 598 nm in the oxidized form, and at 442 and 604 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 602 nm. The enzyme molecule was composed of two kinds of subunits with molecular weights of 50,000 and 30,000 and it contained equimolar amounts of heme a and copper atom. The enzyme rapidly oxidized Candida krusei and horse ferrocytochromes c as well as Pseudomonas AM 1 ferrocytochrome c. The reactions catalyzed by the enzyme were strongly inhibited by KCN.     

Purification and characterization of veratryl alcohol oxidase from Comamonas sp. UVS and its role in decolorization of textile dyes

In the present work, we have purified veratryl alcohol oxidase (VAO) enzyme from Comamonas UVS to evaluate its potential to decolorize textile dyes. VAO was purified (13.9 fold) by an ion exchange followed by the size exclusion chromatography. Molecular weight of the VAO was estimated to be about 66 kDa by SDS-PAGE. The optimum pH and temperature of oxidase were 30°C and 65°C, respectively. VAO showed maximum activity with n-propanol among the various substrates (n-propanol, veratryl alcohol, L-dopa, tryptophan, etc.). Under standard assay conditions, K_m value of the enzyme was 2.5 mM towards veratrole. The enzyme activity was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had a slight inhibitory effect. The purified enzyme was able to decolorize textile dyes, Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h at 40 μg/mL concentration. GC-MS analysis of the metabolites suggested oxidative cleavage and desulphonation of these dyes.     

Purification and characterization of ACC oxidase from Artocarpus altilis

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase was purified to homogeneity from breadfruit (Artocarpus altilis) by a six-step chromatography procedure to yield an 87-fold increase in purification with a recovery of 9.5%. SDS-PAGE revealed that the purified enzyme had a molecular mass of 42.3 kDa and a Km of 28.2 μM. The enzyme showed marked inhibition by cobalt sulphate, sodium metabisulphite, sodium dithionite, n-propyl gallate, zinc sulphate and hydrogen peroxide. Dithiothreitol (DTT) enhanced enzyme activity when it was included in the assay medium. Optimum activity of ACC oxidase was obtained at a pH of 7.2 at 28 °C.     

Purification and properties of the membranal thiol oxidase from porcine kidney

A thiol oxidase was purified from porcine kidney cortex by chromatography of detergent-solubilized plasma membranes on cysteinylsuccinamidopropyl-glass beads, hydroxyapatite, and Sephacryl S-200. The oxidase was purified 2600-fold; 28% recovery of activity was obtained. With glutathione as substrate, the apparent Km was 0.73 mM and the V max was a 4.4 U/mg protein. The reaction catalyzed was 2 RSH + O2----RSSR + H2O2, and superoxide production was not detected during the reaction. Other low molecular weight thiols, including cysteine, dithiothreitol, N-acetylcysteine, and cysteamine, were substrates for the oxidase; 2-mercaptoethanol, reductively denatured ribonuclease A, and chymotrypsinogen A were not substrates. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band corresponding to 70 kDa; gel filtration on a Sephacryl column produced a single elution of activity with a protein corresponding to 120 kDa, indicating that the functional form is a dimer. On a high-pressure gel permeation column the protein eluted at 70 kDa under dilute conditions but at greater than 200 kDa at high concentrations, indicating that the protein also aggregates into larger multimers. Activity was inhibited by copper chelators, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), H2O2, and N-ethylmaleimide, suggesting the presence of copper and a sulfhydryl group at the active site. Following treatment with metal chelators, enzyme activity was reconstituted with CuSO4, but not with FeSO4. The purified enzyme contained 1 mol copper per subunit which was undetectable by electron paramagnetic resonance, suggesting that the copper is in a binuclear complex.     

Oxalate oxidase from Ceriporiopsis subvermispora: biochemical and cytochemical studies.

The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pI of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has Km and kcat values of 0.1 mM and 88 s-1, respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed.