The effect of azide on the spectral and catalytic properties of ascorbate oxidase

(1) 45% of the total copper of green zucchini ascorbate oxidase is EPR-detectable. At least two species of copper are present, one with a small A parallel (Type 1) and one with a large A parallel (Type 2). Computer simulated spectra indicated 50% contribution by each type of copper. (2) Azide inhibited ascorbate oxidase activity by an uncompetitive mechanism. EPR and optical spectra performed on titration of ascorbate oxidase with azide indicated the formation of a copper-azide complex. The Type 2 copper appears to be the binding site of azide. The involvement of the EPR non-detectable copper as an anion binding site with high affinity toward azide can not be excluded.     

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.     

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.     

Spectral study of ascorbate oxidase

The electronic, CD and EPR spectra of ascorbate oxidase isolated from the green zucchini squash (Cucurbita pepo medullosa) in 0.1 M phosphate buffer (pH 6.8) have been investigated. The visible absorption bands are clearly resolved in the CD spectrum, where the extrema occur at 735, 610, 550, 475 and 330 nm, while weak additional CD activity possibly occurs near 420 nm. The near-UV spectrum is dominated by the absorption of the aromatic amino acid residues centered at 280 nm, while resolved CD bands occur at 296, 291, 283, 265 and 240 nm. In the far-UV region the protein CD spectrum reflects its secondary structure: a single negative maximum at 218 nm suggests a predominant anti-parallel β conformation for ascorbate oxidase. The frozen solution EPR spectrum of the protein has been fitted according to a new computer simulation procedure. The following parameters were obtained: for the type 1 copper g_z = 2.222, g_x = 2.032, g_y = 2.056, A_z = 59 G, A_x = 11 G, and A_y = 5 G; for the type 2 copper g ? = 2.240, g_⊥ = 2.057, A? = 179 G and A_⊥ = 1 G. Of the eight copper atoms present in the protein four are EPR-detectable: three of type 1 and one of type 2, as shown by computer simulation of the EPR spectrum. Ascorbate oxidase is a rather unstable protein when purified and it is sensitive to a number of environmental factors. Aging of the protein leads to a decrease in the ratio between the type 1 and type 2 coppers. A new species formed at the early stages of the aging process, that has been spectrally characterized, suggests that the loss of the type 1 copper is preceded by a change in the symmetry of the original type 1 site from pseudotetrahedral to pseudotetragonal.     

Oxidation of reduced cucumber ascorbate oxidase

Reoxidation process of reduced cucumber ascorbate oxidase (1.10.3.3) with dioxygen was investigated in detail through absorption, circular dichroic (CD) and electron paramagnetic resonance (EPR) spectra. One of the three type I coppers and the type II copper were reoxidized more rapidly than other type I coppers. The principal active site of ascorbate oxidase was considered to be comprised of one type I, one type II and a pair of type III coppers similarly to the active sites in laccase and ceruloplasmin. Remaining two type I and a pair of type III coppers were also disclosed to contribute to the oxidation of L-ascorbate.     

An investigation on reduction process of cucumber ascorbate oxidase

Reduction process of cucumber ascorbate oxidase with L-ascorbate was investigated in detail through absorption and electron paramagnetic resonance (EPR) spectra under anaerobic condition. One of the three type I coppers (the type I copper which is oxidized rapidly (Sakurai, T. et al. (1985) Biochem. Biophys. Res. Commun. 131, 647-652)) and a pair of type III coppers only which contribute to the absorption at 330 nm were reduced faster than other two type I and the other pair of type III coppers, respectively. The principal active site of ascorbate oxidase was confirmed to be comprised of one type I, one type II and a pair of type III coppers. Type II copper seemed to be reduced after all type I and type III coppers have been reduced.     

Reassessment of copper stoichiometry in ascorbate oxidase

A very pure ascorbate oxidase solution was obtained by dissolving a crystalline sample of the enzyme. The ratio between 280 and 610 nm absorbancies was 22.5. It contained 8.0 +/- 0.2 Cu ions, 50% EPR detectable, per dimeric molecule (140,000 M.W.) with a molar extinction coefficient of 10,000 cm-1 at 610 nm. Two Cu ions were removed by treatment with N,N-diethyldithiocarbamate. The optical blue absorption band was unaffected, while two EPR detectable Cu ions were lost, with disappearance of the type 2 Cu signal. It is concluded that native ascorbate oxidase contains two type 1, two type 2, and four type 3 Cu ions.     

Spectral characteristics of the mechanism of oxidase activity of ceruloplasmin

The absorbance and EPR spectra of type 1 and 2 copper-binding centres which are present in ceruloplasmin (Cp) molecule were shown to disappear upon the reduction of the enzyme by ascorbate under anaerobic conditions. The fluorescence band attributed to type 3 Cu was altered concomitantly. The electron-accepting nitroxyl radical added to reduced Cp restored the absorbance, EPR and fluorescence spectra of the oxidase. Only type 1 and 3 copper ions, as judged by spectral changes, can be reduced by ascorbate and then reoxidized by the nitroxyl radical in the azide-treated Cp. The spectral properties of Cp provided by copper ions of different types change simultaneously and concordantly upon oxidation/reduction. This seems to be caused by cooperative interaction of these ions involved in the electron transfer from the donating substrate to the accepting molecule of the nitroxyl radical (in model studies of oxidase reaction) or oxygen (under natural conditions). The copper ions in the active centre of Cp constitute an intramolecular electron transport chain, which may, at least in vitro, function without one of its links.     

The effects of copper depletion on structural aspects of cytochrome c oxidase

The removal of copper from beef heart cytochrome c oxidase by either dialysis against potassium cyanide or by treatment with bathocuproine sulfonate produced changes in the enzyme which are indicative of a spin state transition. In the Soret region of the CD spectrum copper depletion of the enzyme caused a significant decrease in amplitude in combination with a red shift of the peak maximum for oxidized samples, while reduced copper-depleted samples exhibited decreased amplitude and a blue shift of the peak maximum. In the magnetic CD spectra of oxidized copper-depleted samples the peak at 420 nm was shifted to lower wave-length along with a significant increase in amplitude. In reduced samples the peak at 446 nm exhibited a slight red shift concomitant with a substantial decrease in amplitude. The conformational changes indicated by the CD and magnetic CD spectra when copper is removed from the enzyme were supported by the EPR spectra of the NO complex of the reduced copper-depleted enzyme. The removal of copper from cytochrome c oxidase caused the NO complex to exhibit a 3-line splitting pattern of gz in the EPR spectrum instead of the 9 lines seen in the NO complex of the native enzyme. When [15N]NO was used, a 2-line pattern was seen at gz when copper was removed from the enzyme. The changes in the CD and magnetic CD spectra and in the EPR spectra of the NO derivatives of cytochrome c oxidase can be explained by the rearrangement of the axial ligands to iron in cytochrome a3 as a result of copper depletion. These results emphasize the close structural interdependence of the metallic components of this enzyme.     

Studies on the origin of the near-infrared (800-900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 ('copper signal') of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (-170 to -190 degrees C). For some sets of samples spectra were recorded in the range 500-1100 nm. A particular efFort was made to study this correlation with what are called 'mixed valence' states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205-215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10-15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700-800 nm region did not appear to be more useful than the 800-900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

A tyrosine-derived free radical in apogalactose oxidase

Oxidation of apogalactose oxidase with ferricyanide leads to the formation of a stable free radical exhibiting distinctive optical absorption and EPR spectral features. The radical is associated with absorption in both near-UV and near-IR spectral regions, and its EPR spectrum is characteristic of an aromatic free radical with gav = 2.005. Reconstitution of both the apoenzyme and the free radical-containing form with copper substantially restores both the absorption spectra and the catalytic activity of the active enzyme, indicating that the preparation of the radical species does not significantly damage the protein. The absence of a free radical EPR signal in reconstituted and activated galactose oxidase containing nearly stoichiometric copper suggests the radical is an active site species relating to the free radical-coupled copper site previously proposed for this enzyme. Isotopic labeling experiments demonstrate that the radical derives from a tyrosine residue. The distinctive spectra associated with this radical indicate an environment which is different from that associated with the tyrosyl phenoxyl sites in other free radical enzymes.     

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of type-2-copper-depleted ascorbate oxidase.

The interaction of one-electron reduced metronidazole (ArNO2.-) with native and Type-2-copper-depleted ascorbate oxidase were studied in buffered aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. With ArNO2.-, reduction of Type 1 copper of the native enzyme and of the Type-2-copper-depleted ascorbate oxidase occurs via a bimolecular step and at the same rate. Whereas the native protein accepts, in the absence of O2, 6-7 reducing equivalents, Type-2-copper-depleted ascorbate oxidase accepts only 3 reducing equivalents with stoichiometric reduction of Type 1 copper. On reaction of O2.- with ascorbate oxidase under conditions of [O2.-] much greater than [ascorbate oxidase], removal of Type 2 copper results in reduction of all the Type 1 copper atoms, in contrast with reduction of the equivalent of only one Type 1 copper atom in the holoprotein. From observations at 610 nm, the rate of reduction of ascorbate oxidase by O2.- is not dependent on the presence of Type 2 copper. For the holoprotein, no significant optical-absorption changes were observed at 330 nm. It is proposed that electrons enter the protein via Type 1 copper in a rate-determining step followed by a fast intramolecular transfer of electrons within the protein. For the Type-2-copper-depleted protein, intramolecular transfer within the protein, however, is slow or does not occur. In the presence of O2, it is also suggested that re-oxidation of the partially reduced holoprotein occurs at steady state, as inferred from the observations at 330 nm and 610 nm. The role of Type 2 copper in ascorbate oxidase is discussed in terms of its involvement in redistribution of electrons within the protein or structural considerations.     

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.     

Oxidation of phenolate siderophores by the multicopper oxidase encoded by the Escherichia coli yacK gene

A gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned from Escherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron paramagnetic resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong optical A(610) (E(610) = 10,890 M(-1) cm(-1)) and copper stoichiometry were taken as evidence that, similar to ceruloplasmin, the enzyme likely contains multiple type 1 copper centers. The addition of copper led to immediate and reversible changes in the optical and EPR spectra of the protein, as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of the enzyme, but the other metals tested had no effect. In the presence of added copper, the enzyme displayed significant activity against two of the phenolate siderophores utilized by E. coli for iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as 3-hydroxyanthranilate, an iron siderophore utilized by Saccharomyces cerevisiae. Oxidation of enterobactin produced a colored precipitate suggestive of the polymerization reactions that characterize microbial melanization processes. As oxidation should render the phenolate siderophores incapable of binding iron, yacK MCO activity could influence levels of free iron in the periplasm in response to copper concentration. This mechanism may explain, in part, how yacK MCO moderates the sensitivity of E. coli to copper.     

The phenoloxidases of the ascomycete Podospora anserina. XI. The state of copper of laccases I, II and III.

1. Laccases I, II and III were (EC 1.14.18.1) prepared from the mycelium of the ascomycete Podospora anserina. The tetrameric laccase I(mol. wt 340 000, 16 copper atoms) and the monomeric laccases II and II (mol. wt 80 000, 4 copper atoms) have been studied by optical absorption-, circular dichroism-(CD)and electron paramagnetic resonance spectroscopy (EPR). 2. The visible and near ultraviolet difference absorption spectrum, which is apparently identical for all three laccases, shows two maxima at 330 and 610 nm and a shoulder at about 725 nm. The molar extinction coefficients of these bands are 4 times larger for the tetrameric laccase I compared to the monomeric laccases II and III which show values similar to other blue copper-containing oxidases. 3. CD spectra between 300 and 730 nm of the tree laccases are similar and contain at least 5-bands in the oxidized enzyme. If the enzyme is reduced, only a band at 307 nm remains. The molar ellipticity values of these bands are 4 times larger for laccase I than the corresponding bands of laccases II and III. It is inferred that the reducible bands are associated with the Type 1 Cu-2+. 4. In all three laccases the EPR-detectable copper accounts for only about 50% of the total copper content. The 9-GHz and 35-GHz spectra, which are identical for all three laccases, consist of two components of equal intensity. One component shows a rather small copper hyperfine coupling and a small deviation from axial symmetry. It is suggested that this copper is associated with the blue chromophore in analogy to Type 1 Cu-2+ in other blue copper proteins. The other component has a broader hyperfine coupling similar to Type 2 Cu-2+ as found in other copper proteins. The assumption that the experimental spectra result from a superposition of the spectra of equal amounts of Type 1 and Type 2 Cu-2+ has been verified by computer simulation. 5. It is suggested that the copper ions which are not detected by EPR are connected to the absorption band at 330 nm and that these ions are also essential for the function of these laccases.     

Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris

Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.     

Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation

The substrate specificity of catechol oxidase from Lycopus europaeus towards phenols is examined. The enzyme catalyzes the oxidation of o-diphenols to o-quinones without hydroxylating monophenols, the additional activity of tyrosinase. Substrates containing a -COOH group are inhibitors for catechol oxidase. The products of enzymic oxidation of caffeic acid were analyzed and isolated by HPLC with diode array detection. The neolignans of the 2,3-dihydro-1,4-benzodioxin type (3, 6-8), 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-2,3-dicarboxy-1,2-dihydro naphthaline (1) 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-3-carboxynaphthaline (5) and 2,6-bis-(3',4'-dihydroxyphenyl)-1-carboxy-3-oxacyclo-(3,0)-pent an-2-on-1-ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.     

Effect of nitrite on cytochrome oxidase

Nitrite causes changes in the optical and EPR spectra of cytochrome oxidase from heart and alters the spectral, redox and basic properties of cytochrome c. No utilization of nitrite by cytochrome oxidase was observed. However, nitrite inhibits the superoxide dismutase and oxidase activities of the enzyme. Changes in the properties of cytochrome oxidase were observed under effect of some products of nitrite reduction, e. g. nitric oxide, hydroxylamine, hydrazine; nitrate has no effect on the optical and EPR spectra or on the enzyme activity.     

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo).

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.