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

ABSTRACT: 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.

SUBMITTER: Agostinelli E

PROVIDER: S-EPMC1136577 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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

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| S-EPMC1153910 | biostudies-other

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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.

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A Fungal Ascorbate Oxidase with Unexpected Laccase Activity.

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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).

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