Laccase, Polyporus

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 ... 

Eckenrode et al. demonstrated that copper oxidases could oxidize vindoline through the same sequence of intermediates found in the metabolism of the alkaloid by Streptomyces griseus. Laccase from Polyporus anceps, laccase from the lacquer tree, and the mammalian (human serum) equivalent copper oxidase... 

Scheme 19. Oxidation of cleavamine by Polyporus anceps laccase. 

Cleavamine Intramolecular cyclization Polyporus anceps laccase 171... 

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)... 

The other small blue proteins are only poorly characterized at present It is assumed their function is that of electron transfer. Rusticyanin from Thiobacillus ferrooxidans is thought to be the initial electron acceptor from iron(II) in the respiratory chain at pH 2. Rusticyanin contains 159 residues, with one cysteine, three methionine and five histidine residues. The protein is unusually stable at low pH, in accord with its presence in an acidophilic organism. The midpoint potential of rusticyanin is high (+680 mV), and is second in magnitude only to that of Polyporus laccase. 

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. 

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

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

In the case of Polyporus laccase, Malkin et al. (95) have differentially removed the non-blue Cu(II) from the protein. This inactivates the enzyme but leaves the intense blue color intact. The activity and original copper content can be restored by adding Cu(II) and ascorbate (95). Anions such as F" and CN" appear to inhibit by reacting with the non-blue copper (66). Fluoride, for example, appears to react exclusively with the non-blue Cu(II) since the super hyperfine lines from the fluoride nucleus appear exclusively on the non-blue Cu(II) hyperfine lines in the ESR spectrum, and the blue Cu(II) hyperfine lines remain unaltered (Figure 6) (96). Figure 6 is an ESR spectrum taken at a... 

Figure 5. Experimental and simulated ESR spectra at 9,2 GHz of Polyporus laccase (94)...

The function of the diamagnetic copper in the blue copper oxidases is not clear. Anaerobic titrations of Polyporus laccase with a number of... 

Figure 6. The reaction of Polyporus laccase with F as studied by ESR at 35 GHz. (A) Native laccase (B) laccase plus 1 equivalent of NaF (C) laccase plus 15 equivalents of NaF. The hyperfine splitting due to two forms of Cu(II) and the superhyperfine splitting due to F are indicated (96).

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]. 

Two other copper enzymes possess ascorbate oxidase activity, human ceruloplasm and Polyporus laccase (70,71). Ceruloplasm may function as an AA oxidase in vivo. Both ceruloplasm and laccase are 10 times less active toward AA oxidation than is ascorbate oxidase. However, the reaction is definitely enzymic, and water is produced. 

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). 

The microbial transformations of vindoline and its derivatives have been further examined. Human caeruloplasmin and laccases of Polyporus anceps and Rhus vernicifera converted vindoline (44) into the known enamine 270 and the dimer 266 (201), which have been encountered in previous microbiological studies on vindoline. The dimer 266 is also formed by the action of horseradish peroxidase on vindoline (202). 

FS Sariaslani, JM Beale Jr, P Rosazza. Oxidation of rotenone by Polyporus anceps laccase. J Nat Prod 47(4) 692- 697, 1984. 

B. Reinhammar, Kinetic Studies on Polyporus and Tree Laccases. In Mutti-Copper Oxidases] A. Messerschmidt, Ed. World Scientific Publishing Singapore, 1997 pp 167-200. 

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

Rhus vemicifera Laccase. The physical and chemical properties of Rhus laccase and fungal laccase may be compared in Table 2. It is clear that while there are differences in the gross physical properties such as molecular weight, carbohydrate content, and amino acid composition, nature has preserved a four-Cu complex capable of accepting four electrons (77—79) consisting of one Type 1 Cu2+, and one Type 2 Cu2+, and the Type 3 pair. There are other minor differences reflected in the comparable redox potentials, g values, and other detailed spectroscopic parameters but on the whole the state and function of Cu bound to Rhus laccase would seem to be identical with Polyporus laccase (cf. Fig. 3). 

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... 

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

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