Polyporus versicolor Laccase

Polyporus versicolor Laccase. There are two electrophoretic forms, A and B, of fungal laccase (50—52) which otherwise seem to be identical as enzymes. 

The general properties of this protein are summarized in Table 2. Four copper atoms are bound per molecular weight 62—64,000, and the work of Butzow (53) suggests that if the enzyme is polymeric it does not consist of subunits which are easily dissociated. Indeed it appears that this protein, like transferrin, (MW 80,000), may consist of only one polypeptide chain [cf. (54)). 

There has been relatively little work done on the chemical properties of the individual amino acids. Briving and Deinum (55) have examined the reactivity of the cysteine SH groups. The native oxidized enzyme has no reactive SH while the enzyme denatured under anaerobic conditions in the presence of appropriate trapping agents for Cu2+, exhibits a single SH group. The remaining cysteine appears to exist in two disulfide bonds. 

Also assembled in Table 2 are the spectroscopic and magnetic properties of the associated Cu chromophores. The characteristic features of the optical absorption spectrum are an intense absorption band at 610 nm (e=4900 cm ) and a 

Electron paramagnetic resonance and magnetic susceptibility measurements have been essential in detecting the presence of the uniquely different Cu ions bound to the enzyme. Early measurements (57) demonstrated that only 50 % of the total Cu was detectible by double integration of the EPR signal, and subsequent 

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Polyporus versicolor Laccase. Under all studied conditions the Type 1 Cu2+ is rapidly reduced by substrates. With ferrocyanide the reaction follows a second-order law during the initial phase when substrate concentration is below... 

On adding dioxygen to the fully reduced laccase of the lacquer tree Rhus vemicifera, the type-1 Cu and the type-3 Cu-pair were oxidized in the ms range and an optical intermediate was observed at 360 nm At liquid helium temperatures an EPR signal was observed, which was tentatively interpreted as due to O ", as a result of its very short relaxation time and of the increase of its linewidth when the reduced laccase of the fungus Polyporus versicolor was treated with 0 A similar paramagnetic oxygen intermediate was also observed with the laccase of another lacquer tree Rhus succedanea and with ceruloplasmin. The decay of the intermediate at 25 °C (tj = 1 s at pH 5.5 with R. succedanea laccase) was accompanied by the reoxidation of the type-2 Cu >. One would expect, however, such an intermediate to be extremely reactive (See Sect. 3.3), while it was stable in tree laccase depleted of type-2 Cu(II)... 

We illustrate these aspects of metalloprotein dynamics at surfaces by two specific proteins. One is the two-centre bacterial di-heme protein cyt c4 from Pseudomonas stutzeri, and the other is the fungal four-centre redox enzyme laccase from Polyporus versicolor. 

Laccase Polyporus versicolor 02 Absorption maximum at 360 nm, new EPR signal at low temperature (lO K) 0" radical 6l5-nm band, 5 x 10 Af sec"330-nm band, 5 > lO M sec 360-nm absorption maximum. 5 x 10 Msec" type-2 copper EPR signal, 20-8 halftime Absorption maximum at 360 nm. 20-s halftime, first-order reaction [Pg.161]

Laccase Polyporus versicolor) obscured by blue copper ... 

Figure 1. Visible CD (A) and absorption spectra (B) of azurin from Pseudomonas aeruginosa (left) and laccase from Polyporus versicolor...

Laccase, 1.10.3.2, Rhus vernicifera, Coriolus hirsitus, Polyporus versicolor... 

The redox potential of the Tl Cu-site has been determined using potentiometric titrations with redox mediators for a large number of different laccases and varies between 410 mV vs. NHE for Rhus vernicifera [67] and 790 mV for laccases from Polyporus versicolor and Coriolus hirsutus [244,251]. The T2 and T3 sites have higher potentials [251]. 

Faced with the problem of elucidating the individual roles of the diflFerent copper centers in the blue oxidases, the researcher has naturally focused in recent years on the laccases (9). Being easier to isolate, better characterized, and containing fewer copper atoms than cemloplasmin or ascorbate oxidase, the laccases from the Japanese lacquer tree Rhus vernicifera and the fungus Polyporus versicolor have been the subject of several transient kinetic studies in the millisecond range, that is, studies using stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy (9,49,50). 

Fungal laccase A from Polyporus versicolor, a copper-containing oxidase... 

Extended quinones. A soln. of laccase from Polyporus versicolor added to a soln. of 2-methyl-1-naphthol in 0.01 M acetate buffer of pH 5.4, and kept 4 days at 30° 3,3 -dimethyl-1,1 -binaphthyl-4,4 -quinone. Y 63%. Also with K-ferricyanide, and formation of dinaphthones s. B. R. Brown and A. H. Todd, Soc. 1963, 5564 with K-ferricyanide and lead dioxide s. F. R. Hewgill and B. S. Middleton, Soc. 1965, 2914. 

As with soil, diphenol oxidases extracted from forest litter also can be fractionated to yield components essentially free of co-extracted humic compounds. A comparison of humic-free laccases (p-diphenol oxidases) purified from soil and litter extracts with those from Polyporus versicolor indicated that the former preparations were strongly electronegative and were only weakly adsorbed to humic colloids. By contrast other enzymically-active fractions from soil extracts were associated with humic compounds in complexes which were not separable by chromatography or electrophoresis. ... 

MAYAUDON J. and SARKAR J.M. 1974. Etude des diphenol oxydases extraites d une litiere de foret. Soil Biology and Biochemistry, 6, 269-274. MAYAUDON J. and SARKAR J.M. 1974. Chromatographie et purification des diphenol oxydases du sol. Soil Biology and Biochemistry, 6, 275-285. MAYAUDON J. and SARKAR J.M. 1975. Laccases de Polyporus versicolor dans le sol et la litiere. Soil Biology and Biochemistry, 7, 31-34. 

Laccase from Polyporus versicolor (E.C. 1.10.3.2., 180 000 U/ml) was obtained from the Institute of Biochemistry, Academy of Sciences, Armenian SSR. Peroxidase (POD) from milk (E.C. 1.11.1.7., 340 pmol/l) was a ift from the Institute of Physiological Chemistry, University of Umea (Sweden). Other reagents used were of analytical grade. The background solution was a Sorenson phosphate buffer of pH 5.0 - 7.0 or 0.1 M acetate buffer of pH 5.5. 

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