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1.
1H-NMR relaxation measurements of Rhus laccase showed that a portion of the relaxivity was specifically abolished by less than stoichiometric EDTA. Another portion of relaxivity was removed by addition of N3(-) to the EDTA saturated enzyme. This treatment or selective removal of the Type 2 Cu left a large residual paramagnetic relaxivity (1700 M-1s-1) which was assigned to the Type 1 Cu. It is concluded that only a portion of the laccase relaxivity can be assigned to the Type 1 Cu and that this copper type does not behave homogeneously: the two fractions have different relaxivity, 5200 and less than or equal to 2400 M-1s-1 respectively.  相似文献   

2.
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.  相似文献   

3.
New epr features consistent with a novel type of Cu(II) are observed in partially reduced Type 2 copper depleted laccase molecules. Cu(II) hyperfine lines appear near 2590 G and 2770 G, and a rhombic g1 feature is also observed. These reflect a Cu(II) emergent on reductive disruption of the binuclear Type 3 site in T2D laccase. Additionally, much of the new, magnetically isolated Cu(II) is retained on full reoxidation of partly reduced Type 2 copper depleted laccase. The proportion of disrupted Type 3 Cu(II) sites remaining after reoxidation appears to depend on the prior distribution of electrons within T2D laccase.  相似文献   

4.
On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 206, 513-529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three-domain structure for laccase closely related to ascorbate oxidase and a six-domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.  相似文献   

5.
Isolation of mannose-binding proteins from human and rat liver   总被引:7,自引:1,他引:7       下载免费PDF全文
The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.  相似文献   

6.
A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora hermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7–0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging from 10 to 120 and from 1 to 45 μM; and kcat values ranging from 50 to 16000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase ≈ Myceliophthora laccase ≈ Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a ‘blue’, type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.  相似文献   

7.
A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.  相似文献   

8.
A new procedure is described for preparing tree laccase that is missing the type 2 copper. The derivative has only about 5% of the activity of the native enzyme, and some, or all, of the residual activity could be due to traces of holoprotein. The type 1 copper is fully oxidized in the purified type-2-depleted protein, while the type 3 site is reduced to the extent of at least 85%. However, the type 3 coppers can be reoxidized by treatment with excess H2O2. Reconstitution is achieved by incubation with Cu(I), and the remetalated protein exhibits the activity and the spectral properties of the native enzyme. The type 2 copper is removed by dialysis against a redox buffer containing ferri- and ferrocyanide ions as well as EDTA. More than 25% of the total copper is removed from laccase during the procedure, but the type-2-depleted fraction is readily isolated by means of an ion-exchange column. The practical advantages of this procedure are described. Finally, the simplicity of the method raises hopes that the mechanism of depletion can be defined.  相似文献   

9.
Due to conflicting reports on the properties of Rhus laccase depleted in type 2 copper a further investigation of this protein derivative has been undertaken. In contrast to most other reports it is shown that the type 3 copper site retains its absorbance at 330 nm when type 2 copper is removed. The type 3 copper ions are oxidized in the resting protein and part of the type 3 Cu(II) can be made electron paramagnetic resonance (epr) detectable on reduction by ascorbate. This new epr signal is highly rhombic and the epr parameters are comparable to those found in other metalloproteins containing Cu(II) in binuclear sites. Certain preparations of type 2 deficient protein exhibit lower extinction coefficients at 330 nm. Since these protein derivatives have lost some type 3 copper, it is inferred that the absorbance at 330 nm is dependent on a native type 3 copper site. Also in contrast to other reports, it is found that the extinction coefficient at 614 nm of the type 1 Cu(II) decreases from 5700 to 4700 M?1cm?1 when type 2 copper is removed. The oxidized-reduced difference spectrum also shows a substantial decrease in the absorbance between 700 and 800 nm. The changes in absorbance above 600 nm are probably due to a modification of the type 1 Cu(II) site on removal of type 2 copper. The present results also suggest some explanations to the apparent discrepancies among the earlier reports.  相似文献   

10.
We have studied the removal of the type-2 copper from tree laccase (Rhus vernicifera) by treatment with EDTA at pH 5.2 in the presence of a redox buffer containing ferri- and ferrocyanide. The efficiency with which the copper is removed depends on the Fe(CN) 6(4-)/Fe(CN) 6(3-) ratio. We have varied this ratio from approx. 2:1 to about 50:1 and the best results were obtained with the highest ratio, i.e., the most cathodic solution potential. Nevertheless, the presence of Fe(CN) 6(3-) is required for the procedure to be effective. Although we cannot exclude the possibility that a mixed-valence form of laccase is the reactive species, we believe the results are better explained by a model which assumes that the removal of the type-2 copper depends upon an ordered sequence of oxidation-reduction reactions. Specifically, we propose that the copper is released as the monovalent ion from previously reduced laccase and then reoxidized in solution and sequestered with EDTA. The reoxidation step drives the reaction because recombination with the protein is inhibited when copper is in the divalent form. In testing this model, we have also shown that the type-2 copper can be removed under strictly reducing conditions when 4,4'-dicarboxy-2,2'-biquinoline (BCA) is present to complex the copper(I) ion. Although the BCA method is effective, the reaction takes longer, perhaps because of the limited solubility of BCA at the pH values of interest. Finally, we have found that the best results are obtained with either method when a cyanometalate ion such as Fe(CN) 6(3-) or Co(CN) 6(3-) is present in the medium. The exact role of this factor has yet to be established, but there is no indication that free cyanide has a role in the process. The most likely interpretation is that some type of binding interaction with the protein facilitates copper release.  相似文献   

11.
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.  相似文献   

12.
(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.  相似文献   

13.
The interactions of one-electron reduced metronidazole (ArNO2.-) and O2.- with native and Type-2-copper-depleted Vietnamese- and Japanese-lacquer-tree laccases were studied in aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. On reaction with ArNO2.-, in the absence of O2, the holo- and the Type-2-copper-depleted proteins accept, with reduction of Type 1 copper, 2 and 1 reducing equivalents respectively. On reaction with O2.- of both holo- and Type-2-copper-depleted Vietnamese-lacquer-tree laccase, almost complete reduction of Type 1 copper was observed and, after completion of the reaction, some (less than 20%) reoxidation of Type 1 copper occurs. Reduction of Type 1 copper of the laccases by these one-electron donors occurs via a bimolecular step; however, the rate of reduction of Vietnamese-lacquer-tree laccase is over 10 times that of Japanese-lacquer-tree laccase. It is inferred that electrons enter the protein via Type 1 copper with, in the case of the holoprotein, subsequent rapid intramolecular transfer of 1 reducing equivalent within the protein. Furthermore it is suggested that intra-molecular electron transfer to Type 3 copper atoms is slow and, in the case of Type-2-copper-depleted protein, may not occur. This slow process may partially account for the variation of the catalytic activities of 'blue' oxidases.  相似文献   

14.
Tree laccase, a multi-copper oxidase, has been studied as a copper donor in conjunction with the demetalated forms of three blue copper proteins. Copper transfer could be observed under reducing conditions in the absence of air. Only about 10% of the total copper in laccase could be transferred regardless of the amount of acceptor present in solution, hence, the laccase is heterogeneous as isolated. Potential sources of the heterogeneity are considered. After transfer, laccase could be partially resolved into copper-deficient and nearly holoprotein fractions that would not donate copper when recombined with acceptor protein. EPR results in conjunction with thiol titrations indicate that there is no net loss of type 1 copper from laccase but that there is loss of type 2 copper as well as a small amount of type 3 copper. Very little transfer is observed when type 2-depleted laccase is used as the donor. Finally, the implications that these results could have in the elucidation of possibly more physiologically relevant processes are briefly summarized.  相似文献   

15.
A. Seelig  B. Ludwig  J. Seelig  G. Schatz 《BBA》1981,636(2):162-167
The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at ?40°C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichiometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.  相似文献   

16.
A new type of Cu(II) epr signals have been produced in native and type 2 copper depleted Rhus vernicifera laccase. They are shown to originate from one of the type 3 copper ions that are epr silent in the resting enzyme. The new epr signals show high rhombicity in g tensor and are similar to those observed in other proteins, such as superoxide dismutase and half-met hemocyanin. The half-reduced type 3 copper pair is formed by reduction with an electron from type 1 Cu(I) but only after a reoxidation of the copper pair, either by peroxide or dioxygen. It is suggested that the half-reduction of the type 3 copper pair only occurs in molecules where type 2 copper ion is either reduced or absent.  相似文献   

17.
A new rhombic EPR signal was recently discovered in the partially reduced type 2 copper-depleted Rhus vernicifera laccase (Reinhammar, B. (1983) J. Inorg. Biochem., in press). The signal originates from one of the type 3 Cu(II) ions that becomes EPR-detectable as a result of the selective reduction of the other copper ion in the exchange-coupled Cu(II)-Cu(II) pair. The 14N and 1H and 63,65Cu electron nuclear double resonance (ENDOR) of this uncoupled Cu(II) now have been collected and represent the first ENDOR measurements of a type 3 copper site. The data indicate that the copper is coordinated by at least three nitrogenous ligands, at least one of which is an imidazole. H/D exchange suggests a nearby H2O or OH-, perhaps as a fourth ligand. A similar EPR signal is seen for CuB of reduced cytochrome c oxidase under turnover conditions. The 14N ENDOR, and, therefore, the structure, of this site corresponds extremely closely to that of the laccase type 3 (Cu(II).  相似文献   

18.
Intramolecular electron transfer within zucchini squash ascorbate oxidase is inhibited in a novel manner in the presence of an equimolar concentration of Ag(+). At pH 5.5 in acetate buffer reduction of the enzyme by laser flash photolytically generated 5-deazariboflavin semiquinone occurs at the Type I Cu with a rate constant of 5 x 10(8) M(-1)s(-1). Subsequent to this initial reduction step, equilibration of the reducing equivalent between the Type I Cu and the trinuclear Type II, III copper cluster (TNC) occurs with rate constant of 430 s(-1). The 41% of the reduced Type I Cu is oxidized by this intramolecular electron transfer reaction. When these reactions are performed in the presence of Ag(+) equimolar to dimeric AO, the bimolecular reduction of the enzyme by the 5-deazariboflavin semiquinone is not affected. As in the case of the native enzyme, intramolecular electron transfer between the Type I Cu and the TNC occurs, which continues until 25% of the reducing equivalent has been transferred. At that point, the reducing equivalent is observed to more slowly return to the Type I Cu, resulting a second reduction phase whose rate constant (100 s(-1)) is protein and Ag(+) concentration independent. The data suggest that partial reduction of the TNC results in Ag(+) binding to the enzyme which causes the apparent midpoint potential of the TNC as a whole to decrease thereby reversing the direction of electron flow. These results are consistent with the inhibitory effect of Ag(+) on the steady-state activity of ascorbate oxidase [S. Maritano, E. Malusa, A. Marchesini, presented at The Meeting on Metalloproteins, SERC Daresbury Laboratory, Warrington, England, 1992; A. Marchesini, XIX Convegno Nazionale SICA, Italian Society of Agricultural Chemistry, Reggio Calabria, Italy, September 2001.].  相似文献   

19.
Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes.  相似文献   

20.
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.  相似文献   

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