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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.

ABSTRACT: 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.

SUBMITTER: Morpurgo L

PROVIDER: S-EPMC1162643 | biostudies-other | 1981 Feb

REPOSITORIES: biostudies-other

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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

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.

| S-EPMC1152154 | biostudies-other

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

Project description:The thermal denaturation of laccase from the Japanese lacquer tree (Rhus vernicifera) was studied by differential scanning calorimetry. The endotherms of holo-laccase, type 2-Cu-depleted laccase and apo-laccase were deconvoluted into two independent two-state transitions, providing evidence for a domain structure of the protein. The correlation of the two transitions with the bleaching of copper optical bands and the decrease of the transitions' enthalpy on Cu removal show that the process involves the denaturation of Cu sites. No detectable unfolding of secondary structure was observed, since the thermal transitions, characterized by low overall specific enthalpy, did not modify either the laccase c.d. spectra in the beta-fold region or the maximum wavelength of the fluorescence emission. On chemical denaturation, however, the emission was red-shifted by about 20 nm. The laccase behaviour is substantially different from that of stellacyanin, a protein containing a single blue Cu ion, in which the thermal transition had higher specific enthalpy and induced a large change of the c.d. spectrum in the beta-fold region. The laccase denaturation behaviour is similar to that of ascorbate oxidase from zucchini (courgette; Cucurbita pepo) [Savini, D'Alessio, Giartosio, Morpurgo and Avigliano (1990) Eur. J. Biochem. 190, 491-495], suggesting a structural analogy. In both proteins heating may cause a change of tertiary structure through modifications of Cu co-ordination with loosening of the bonds between the structural domains at the interface of which the trinuclear Cu cluster is located.

| S-EPMC1136577 | 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.

| S-EPMC1161802 | biostudies-other

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.

Project description: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.

| S-EPMC1161801 | biostudies-other

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.

Project description:The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions. This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion. In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).

| S-EPMC1154388 | biostudies-other

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

Project description:Anaerobic reactions of Rhus vernicifera laccase and its type-2 copper-depleted derivatives with hexacyanoferrate(II) were investigated by absorption and e.s.r. spectroscopy. When native laccase was treated with excess hexacyanoferrate(II), the type-1 and type-2 coppers were immediately reduced and the e.s.r. signal due to type-3 copper was transiently observed. After incubation, a novel e.s.r. signal (g parallel = 2.31, g perpendicular = 2.08) developed together with the type-1 copper signal. Only the novel e.s.r. signal was left after the sample had been treated with ascorbate. In the corresponding absorption spectrum, a new band was observed at around 490 nm. A similar new e.s.r. signal did not appear for the type-2-copper-depleted (T2D) laccase, in which the type-3 copper had been reduced during the procedure to deplete the type-2 copper. On the other hand, the novel e.s.r. signal emerged when the type-3 copper in T2D laccase had been previously reoxidized with H2O2. The novel e.s.r. signal was not significantly saturated even by 200 mV microwave power at 4 K. Quantitative estimations and a small molecule study for CuII-FeII(CN)6 and CuII-FeIII(CN)6 systems suggested that the novel e.s.r. signal corresponds to some sort of adduct involving the type-3 copper and hexacyanoferrate(II).

| S-EPMC1132592 | biostudies-other

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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Methods to promote seed germination in the lacquer tree, Toxicodendron vernicifluum (Stokes) F.A. Barkley.

Project description:The lacquer tree, Toxicodendron vernicifluum, is a common industrial crop in East Asia. However, T. vernicifluum seeds are extremely difficult to germinate, which poses a major obstacle to establishing seedlings for sap production. In this study, we examined the germination properties of T. vernicifluum seeds in order to establish an inexpensive and effective method to promote seed germination. The seeds are covered with a hard endocarp, which we degrade using conventional sulfuric acid-based methods. Although sulfuric acid was effective in promoting seed germination, the germination rate was less than 5%. In addition to treatment with sulfuric acid, co-treatment with cold temperatures or the phytohormone gibberellic acid increased the germination rate to 22-35%. Seed viability analysis combined with specific gravity-based seed selection revealed that more than half of the seeds housed embryos that were incapable of germination. In additions, specific gravity-based seed selection aided in the selection of seeds capable of germination and improved the germination rate to approximately 47%. Taken together, our results suggest that the low germination rate of T. vernicifluum seeds is due to deep seed dormancy-which is controlled by physical and physiological mechanisms-and low embryo viability. To improve the germination rate of T. vernicifluum seeds, we propose an effective method whereby seeds with good germination capacity are selected based on specific gravity, following which their physiological dormancy is inactivated through cold pretreatment.

| S-EPMC9371279 | biostudies-literature

Complete Chloroplast Genome Sequence of Chinese Lacquer Tree (<i>Toxicodendron vernicifluum</i>, Anacardiaceae) and Its Phylogenetic Significance.

Project description:Chinese lacquer tree (Toxicodendron vernicifluum) is an important commercial arbor species widely cultivated in East Asia for producing highly durable lacquer. Here, we sequenced and analyzed the complete chloroplast (cp) genome of T. vernicifluum and reconstructed the phylogeny of Sapindales based on 52 cp genomes of six families. The plastome of T. vernicifluum is 159,571 bp in length, including a pair of inverted repeats (IRs) of 26,511 bp, separated by a large single-copy (LSC) region of 87,475 bp and a small single-copy (SSC) region of 19,074 bp. A total of 126 genes were identified, of which 81 are protein-coding genes, 37 are transfer RNA genes, and eight are ribosomal RNA genes. Forty-nine mononucleotide microsatellites, one dinucleotide microsatellite, two complex microsatellites, and 49 long repeats were determined. Structural differences such as inversion variation in LSC and gene loss in IR were detected across cp genomes of the six genera in Anacardiaceae. Phylogenetic analyses revealed that the genus Toxicodendron is closely related to Pistacia and Rhus. The phylogenetic relationships of the six families in Sapindales were well resolved. Overall, this study providing complete cp genome resources will be beneficial for determining potential molecular markers and evolutionary patterns of T. vernicifluum and its closely related species.

| S-EPMC7011389 | biostudies-literature

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