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Optical properties of japanese-lacquer-tree (Rhus vernicifera) laccase depleted of type 2 copper(II). Involvement of type-2 copper(II) in the 330nm chromophore.

ABSTRACT: 1. Spectroscopic and functional properties of Japanese-lacquer-tree (Rhus vernicifera) laccase were re-investigated, with special emphasis on the relationships between the different types of copper centres (Types 1, 2, and 3). 2. On removal of the Type 2 Cu(II), a decrease of absorbance occurred in the wavelength region above 650 nm (delta epsilon 750 = 300 M-1 . cm-1) and around 330 nm (delta episom 330 up to 2200 M-1 . cm-1). 3. Reductive titrations with ascorbic acid or ferrocyanide showed that the electron-accepting capacity of the partial apoprotein is one electron-equivalent lower than that of the native protein, i.e. the protein two-electron acceptor is present in the oxidized state in spite of absorbance loss at 330 nm. 4. The 330 nm chromophore apparently depends on the presence of both the Type 2 and the Type 3 copper in the oxidized state. 5. This finding may have implications in the relative location of Type 2 and 3 copper centres and on the redox behaviour of laccase.

SUBMITTER: Morpurgo L

PROVIDER: S-EPMC1161801 | biostudies-other | 1980 May

REPOSITORIES: biostudies-other

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Low-temperature resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and H2O2-treated type-2-copper-depleted laccase.

Project description:Resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and the latter form treated with H2O2 were measured in liquid and frozen solution, on excitation into the 600 nm absorption band. Significant changes in intensity and/or frequency of the bands lying in the 370-430 cm-1 region were observed on freezing, indicating local structural rearrangements taking place at the blue copper site. These findings corroborate previous suggestions based on e.p.r. measurements and redox data [Morpurgo, Calabrese, Desideri & Rotilio (1981) Biochem. J. 193, 639-642]. They show the strong dependence of the physical properties of blue copper centres on local symmetry. Some conclusions on the origin of the Raman bands are also drawn.

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Heterogeneity of the Type 3 copper in Japanese-lacquer-tree (Rhus vernicifera) laccase.

Project description:The two steps of the titration of the Japanese-lacquer-tree (Rhus vernicifera) laccase with N3- [Morpurgo, Rotilio, Finazzi-Agrò & Mondovi (1974) Biochim. Biophys. Acta 336, 324-328; LuBien, Winkler, Thamann, Scott, Co, Hodgson & Solomon (1981) J. Am. Chem. Soc. 103, 7014-7016] were shown to be two distinct reactions, each involving one different portion of the native enzyme molecules. The difference consists in the oxidation state of the Type 3 Cu, which is reduced in the portion with higher affinity for N3- and oxidized in the portion with lower affinity for N3-. The difference is eliminated by treatment with oxidizing (H2O2) or reducing agents, and a single N3- adduct is then formed. The e.p.r. spectra of the H2O2-treated enzyme and of its F- derivatives support this interpretation of the results. The similarity of the spectroscopic properties of the high-affinity N3- adduct to those of the N3- adducts of half-met-haemocyanins and half-met-tyrosinase is discussed.

| S-EPMC1153910 | biostudies-other

Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites.

Project description:1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.

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Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism.

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Anaerobic reactions of Rhus vernicifera laccase and its type-2 copper-depleted derivatives with hexacyanoferrate(II).

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Dependence on freezing of the geometry and redox potential of type 1 and type 2 copper sites of Japanese-lacquer-tree (Rhus vernicifera) laccase.

Project description:The room-temperature e.p.r. spectrum of the Japanese-lacquer-tree (Rhus vernicifera) laccase shows A parallel (the hyperfine splitting constant) and g parallel values of both the Type 1 and Type 2 Cu appreciably different from those measured at liquid-N2 temperature. The geometry of the sites, as inferred from the room-temperature e.p.r. parameters, is more consistent with their redox properties. A rough correlation is found between A parallel and g parallel values and redox potential of the blue copper in several enzymes.

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Stability of Japanese-lacquer-tree (Rhus vernicifera) laccase to thermal and chemical denaturation: comparison with ascorbate oxidase.

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Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of native and type-2-copper-depleted Vietnamese-lacquer-tree and Japanese-lacquer-tree laccases.

Project description: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.

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Type 2-depleted fungal laccase.

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| S-EPMC1149334 | biostudies-other

Incorporation of copper ions into crystals of T2 copper-depleted laccase from Botrytis aclada.

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| S-EPMC4666473 | biostudies-literature

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