Copper proteins laccase

Reinhammar B (1984) Laccase. In Lontie R (ed) Copper proteins and copper enzymes, vol 3. CRC, Boca Raton, pp 1-36... 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

Bacterial hosts are inappropriate choices for expression of proteins such as the blue copper proteins stellacyanin, laccase, and ceruloplasmin which are extensively glycosylated. In these cases, it may be necessary to employ tissue cultures of appropriate origin to obtain the native protein. In this regard, the amino-terminal half of human serum transferrin, which lacks carbohydrate, has been expressed in high yield in baby hamster kidney cells by Funk et al. [13], while the glycosylated carboxyl-terminus has proved to be more problematic [103]. 

In order to investigate the active sites of these proteins, laccases I and III were subjected to ESR (electron spin resonance) spectroscopic analysis. The ESR spectra shown in Figure 5 indicate clear differences in peaks 2 and 6 which support the concept that the copper atoms in laccases I and III have different conformations in each molecule. Furthermore, immunological similarity between laccases I and III was also investigated. Antibody specific for laccase III was prepared from rabbit serum by conventional methods. When applied to Ouchterlony diffusion plates containing laccase I, no precipitation lines developed (Figure 6). This result showed that there were no conserved epitopes on the surfaces laccases I and III. 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

Copper proteins are involved in a variety of biological functions, including electron transport, copper storage and many oxidase activities. A variety of reviews on this topic are available (Sykes, 1985 Chapman, 1991). Several copper proteins are easily identified by their beautiful blue colour and have been labelled blue copper proteins. The blue copper proteins can be divided into two classes, the oxidases (laccase, ascorbate oxidase, ceruloplasmin) and the electron carriers (plastocyanin, stellacyanin, umecyanin, etc.). 

The second class consists of multidomain blue copper proteins composed of exclusively two or more BCB domains and includes nitrite reductase (Section IV, E), multicopper blue oxidases such as laccase, ascorbate oxidase, ceruloplasmin, and hephaestin (Section VII), and some sequences found in extreme halophilic archaea (see Section V, E). 

Fig. 3. Geometries of the type 1 copper sites of various blue copper proteins. The trigonal planar geometry is the type 1 site of laccase from Coprinus cinereus (PDB Code 1A65). The trigonal bipyramidal geometry shown is the copper site of azurin from Pseudomonas aeruginosa (PDB Code lAZU). The trigonal pyramidal/distorted tetrahedral sites are of the stellacyanin from Cucumis sativus (PDB Code IJER), NNSO site, and of the plastocyanin from Populus nigra (PDB Code IPLC) NNSS site.

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... 

A structurally based amino-acid sequence alignment strongly suggests a three-domain structure for laccase, closely related to ascorbate oxidase, and a six-domain structure for ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationship suggests that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and domain 3, and ceruloplasmin has mononuclear copper ions in domains 2, 4, and 6 and a trinuclear copper between domain 1 and domain 6. 

This paper summarizes briefly the physicochemistry and enzymology of plant copper oxidases with particular emphasis on polyphenol oxidase and laccase. A brief comparative discussion of other naturally occurring copper proteins and artificial copper proteins is appropriate when discussing the physicochemistry of the copper site itself. In the case of the copper proteins listed in Table I, we know a great deal more about the copper site than about the physicochemistry of the rest of the protein molecule. This is primarily a result of the availability of sophisticated spectroscopic techniques such as optical spectroscopy (both absorption and circular dichroism) and electron spin resonance which are applicable to the electronic transitions of the copper ion. On the other hand, protein chemistry has progressed more slowly. Many of the proteins are large and complex multisubunit enzymes, difficult to purify, and often unstable. There are several excellent reviews on this group of proteins (59, 60, 61, 62). 

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