The role of copper in the stability of ascorbate oxidase towards denaturing agents

The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.     

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.     

The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes

The reactivity with nitric oxide was investigated for a number of type-1, type-2 and type-3 copper proteins azurin from Pseudomonas aeruginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo medullosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77K. Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, photodissociation caused the reappearance of the original 625 nm absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 Cu1+-NO+ complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.     

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.     

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.     

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.     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

The role of cytochrome a in the proton pump of cytochrome-c oxidase

Cytochrome-c oxidase of bovine heart mitochondria was depleted of copper A by dialysis against 1 M KCN in the presence of dodecylmaltoside. There was no difference of the pH-dependence of the midpoint potential between the intact and the copper-depleted enzyme. Oxidation of reduced cytochrome a2+a3(3+).CN complex released about 1 proton/electron in the medium at pH 7.6. This release was inhibited by N,N'-dicyclohexylcarbodiimide. Again there was no difference between the intact and Cu-depleted enzyme. This limits the role of copper A in the mechanism of the proton pump. On the other hand, these experiments showed that cytochrome a could be a component of the proton pump.     

Characterization of cucumber ascorbate oxidase and its reaction with hexacyanoferrate (II)

The spectroscopic features of cucumber ascorbate oxidase (AOase) and its type-2 copper-depleted (T2D) derivative, and the electron pathway among the copper sites in the enzyme have been investigated. The electronic and CD spectra of native and T2D AOase in the visible region bear a striking resemblance to those of plastocyanin or azurin, which contain type-1 copper alone. The electronic absorption shoulder of the native enzyme at around 330 nm for the native enzyme which has been assigned to type-3 copper disappears with the depletion of the type-2 copper. The reduction of AOase with a large excess of hexacyanoferrate(II) results in a selective reduction of the type-2 Cu, giving rise to an additional EPR-detectable species which is considered to be originated from partly reduced type-3 copper. The type-1 copper is, however, not reduced even in the presence of excess hexacyanoferrate(II). The redox potential of type-1 Cu was determined to be +350 mV, which is distinctly lower than that of hexacyanoferrate(II-III). Type-2 copper was supposed to be a mediator of the electron transfer between type-1 and type-3 coppers in consideration of the extremely low activity of the T2D enzyme under the same condition. A comparison of the electron pathway in AOase with that in laccase is also argued.     

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.     

Reactions of nitric oxide with tree and fungal laccase

The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal and tree laccase, as well as with tree laccase depleted in type 2 copper, are reported. The products of the reactions were determined by NMR and mass spectroscopy, whereas the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All three copper sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site.     

The photoreactivity of the copper-NO complexes in cytochrome c oxidase and in other copper-containing proteins

The complexes of NO with CuB of cytochrome c oxidase in which cytochrome a3 may or may not be ligated to cyanide or fluoride are photodissociable. NO does not appear to react with CuB in complexes of cytochrome c oxidase in which sulphide or mercaptans are ligated to the haem iron of cytochrome a3. A comparison is made between the photoreactivity of the complexes of NO with cytochrome c oxidase and those with ceruloplasmin, ascorbate oxidase, and haemocyanin. It is shown that the photoreactivity of CuB 2+.NO in cytochrome c oxidase is not unique for this enzyme, but may also be observed in the complexes of NO with type-1 copper-containing enzymes. This would suggest that the ligation of CuB in cytochrome c oxidase shows some similarity to type-1 copper in blue oxidases.     

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)     

The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centres

The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1?Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.     

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.     

Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-A resolution containing a full complement of coppers

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.     

Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide

The combination of ascorbate, transition metal ions, and hydrogen peroxide (H(2)O(2)) is an efficient hydroxyl radical generating system called "the Udenfriend system." Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 microM-1 mM ascorbate. Subsequently, the plasma samples were incubated at 37 degrees C with 50 microM-1 mM iron (from ferrous ammonium sulfate), 60 or 100 microM copper (from cupric sulfate), and/or 200 microM or 1 mM H(2)O(2). Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H(2)O(2), no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H(2)O(2). Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H(2)O(2), as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous alpha-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H(2)O(2), ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.     

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.     

X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.     

Regulation of ascorbate oxidase expression in pumpkin by auxin and copper

Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.