Laccase 3

In nature, biological redox processes can be performed by multicopper blue proteins which catalyze the reduction of oxygen to water. One of the two metalloenzymes capable of such reaction is laccase. Laccase is located in lacquer tress, fungi, bacteria, plants, and some insects [1]. Laccases have four copper atoms that comprise the multicopper core each of which is coordinated to several amino acids. The following explores several properties of laccases and describes their uses in industry.

All laccase structures are multicopper oxidoreductases that are able to perform the reduction of oxygen (O₂) to water. Laccases have four copper atoms; one type-1 Cu atom, one type-2 Cu atom, and two type-3 Cu atoms (Figure 6). There are three classes of copper center atoms. Type-1 Cu centers are characterized by a strong signal in an electronic spectrum at approximately 600-610nm, and is paramagnetic. Type-2 Cu centers are also paramagnetic. Type 3 Cu centers are characterized by an electronic absorption signal at approximately 330nm and are diamagnetic when combined [2]. In laccase, the type-2 and type-3 Cu atoms are connected with trigonal coplanar geometry forming a trinuclear cluster (Figure 1). However, OH^- or O^- ions may coordinate with the nuclear cluster which attaches an oxygen ligand to all three Cu atoms in the center. Both type-3 Cu atoms are coordinated to three histidine residues through Cu-N linkages [4]. The type-2 Cu atom is coordinated to two histidine residues also through Cu-N linkages. The type-1 Cu atom is either above or below the trinuclear cluster. It is coordinated trigonally planar to two histidine residues and one cysteine residue; Cu-N and Cu-S linkages, respectively. The cysteine residue is attached to the histidine residues of the type-3 Cu atoms which links together the four copper centers. The crystal absorption spectrum showed a strong signal at 590nm for the Cu-S, which allows for the blue color of the crystal [4]. However, the mononuclear Cu atom may have a fourth ligand either a leucine or methionine residue in some laccase structures. In some laccases, the position of the coordinated oxygen or hydroxyl groups may or may not be symmetrically coordinated to the type-3 Cu atoms. Despite relatively subtle changes in position or addition of another ligand, the general structure of a four Cu-atom core remains for all laccases.

Considering the Cu atoms individually, each atom has its own unique point group relative to the entire laccase structure. Type-2 and type-3 Cu atoms are attached to four ligands, and in some laccases the type 1-Cu atom is also four-fold. Assuming each individual Cu atom has four-fold ligation and all of the ligands (L) were identical, then each Cu atom would have tetrahedral geometry and belong to the T_d point group, based on structures generated by Briganti et al (Figure 2) [5]. However, because the ligands must be taken into account, the point groups change with respect to the ligands. For each type-3 Cu atom, it is directly coordinated to three histidine residues, and one oxygen atom. As a result, the point group of either type-3 Cu atom is C_3v (Figure 3). Type-2 Cu atom is coordinated to two histidine residues, one oxygen atom, and a water molecule. Thus, the point group is C₁ since the geometry is tetrahedral, and the Cu atom is attached to two different nitrogen sites on both histidine residues which eliminates a possible mirror plane (Figure 4). Type-1 Cu is coordinated to one cysteine, one methionine/leucine, and two histidine residues, and belongs to the point group C₁ (Figure 5). However, since type-1 Cu atoms of some laccases are not coordinated to the fourth ligand of the methionine or leucine residue, the geometry could also be trigonal planar, based on structures by Matera et al, with C_2v point group [5]. All the Cu atoms contribute to the distorted tetragonal symmetry with the trinuclear cluster as the trigonal planar base. However, due to the different linkages to the nitrogen atom of the histidine residues, and the variety of amino acids, the entire molecule belongs to the C₁ point group.

Studies have been performed with IR and Raman spectroscopy of native laccases. Because of the numerous sites of Cu-N bonds from histidine as well the presence of the Cu-S bond from cysteine, at least two Raman stretches are expected for laccase despite the heterogeneity. These bands among others agree with studies carried out by Klis et al. Using Raman spectroscopy, Klis et al. found several additional bands for the native laccase structure, and reported the following bands: 384, 405, 263, 332, 468, and 423 cm^-1 [6].The band at 263 cm^-1 resulted from the Cu-N stretching, and bands at 405 and 468 cm^-1 resulted from the Cu-S stretching modes of cysteine. Stretching within the ligand molecule, specifically between of the C-S bond in cysteine signaled at 332 cm^-1. In addition, Cu-Cu interactions also resulted in stretching modes at band 384 cm^-1. All values, despite variations in signal intensity, were consistent with previously reported data by Nestor et al. in which Raman spectroscopy experiments were carried out without the crystal structure of laccase [7]. Another spectroscopy experiment performed was Fourier transform-Infrared Spectroscopy (FT-IR) on the fungal laccase from Rigidoporous lignosus. Due to the Cu-N coordination, this interaction allows for changes in dipole moment. Thus, bands would be expected for amide groups which are consistent with IR values observed by Ragusa et al. In water medium, it was reported that bands were observed in the region of 1600-1700 cm^-1, specifically: 1514.7, 1545.3, 1583.9, 1611.0, 1635.5, 1642.0, 1659.0, 1669.9, 1677.5, 1687.3, and 1692.0 cm^-1 [8]. The amino acid side chains contribute to the IR spectrum for bands below 1620 cm^-1, but the peak at 1545.3 cm^-1 corresponds with amide group to the Cu atom. All other bands were due to other protein structure interactions of the β-sheets [8]. Assuming that the overall structure of laccase belong to the C₁ point group in which no Raman or IR bands are listed in the C₁ character table, then the appearance of bands for the enzyme is inconsistent. However, because all metals and ligands are coordinated to each other through dative bonds, the structure of laccase, specifically, each Cu type to its respective groups has to its own point group, as previously predicted. Similarly, all the Cu copper atoms have a distorted tetrahedral geometry. In this way, dividing the structure of the enzyme allows for point groups which correspond to IR and Raman bands which may or may not be observable. Thus, the appearance of Raman and IR bands are explained through each component of laccase rather than the entire structure.

Each Cu atom of the enzyme partakes in the reduction of oxygen to water by transferring electrons among the Cu atoms, which allows for use of laccase in industry. However, in addition, to reducing oxygen, laccases also function as multicopper oxidoreductases for aromatic compounds, such as phenols. The following two reactions occur [3]:

O₂ + 4H⁺ + 4e^- <-> 2H₂O (Reaction A)

4RH + O₂ -> 4R· + 2H₂O (Reaction B)

The general hypothesized mechanism assumes that the type-1 Cu is the primary oxidation site of the organic substrate while the trinuclear cluster, type-2 and type-3 Cu atoms, is responsible for the reduction of oxygen to water due to the evidence of the bridged oxygen present in the trinuclear structure [9]. Because type-1 Cu is not part of the trinuclear cluster, it is more accessible to solvent and substrate which allows for its relatively low specificity compared to the trinuclear cluster. Initially, the organic substrate is oxidized at the type-1 Cu site (Reaction B). Electrons from the type-1 Cu active site are transferred to the trinuclear cluster through the histidine and cysteine linkages. As shown in Reaction A, these electrons reduce dioxygen to water [4,10]. Biologically, this catalysis is advantageous because there is no formation of toxic peroxide intermediates or products when water is released [2]. Because a radical was generated initially, the radical may continue to cleave covalent bonds in nature such as in lignin. In order to decolorize paper pulp, conventional methods use chlorine- or oxygen-based chemical oxidants that have negative environmental consequences related to the release of chlorinated by-products. Conversely, because laccase generates radical substrates that can catalytically cleave bonds, laccases have been viewed as an alternative method to pretreat and degrade lignin [11]. Loosening the structure, allows for more efficient depulping techniques. Furthermore, the low substrate-specificity of laccase has its uses in waste detoxification. Aromatic xenobiotics among other pollutants are prevalent in polluted water and soil sites. Using laccase for its catalytic functions could potentially degrade chlorophenols and aromatic rings, or polymerize contaminants to nontoxic substances that can later be removed. In the medical industry, laccases have come up as a possible source of generating iodine, a disinfectant, from oxidizing iodide to iodine [11]. Storing iodide as a starting material has advantages in terms of stability, storage, and transport; thus, conversion to iodine would be catalyzed by laccase. Similarly, laccase as a catalyst for polymerization has possible uses in synthesizing medicinal compounds such as vinblastine as an antitumor agent, penicillin X dimer, or cephalosporin antibiotics [11]. Using laccase has an additional advantage in its environmentally safe disposal. Laccase is a versatile enzyme that has a variety applications in industry attributed to its multicopper structural properties.

Not only are laccases isolated from different organisms, they also have numerous applications which utilize their ability to reduce dioxygen to water and generate radical organic compounds. Assigning point groups to each Cu atom, Cu-ligand site, and the overall structure further elucidated the physical properties of the enzyme observed from FT-IR and Raman spectroscopy methods. Concomitantly, analysis of the geometry of each component of laccase complemented the proposed mechanism with regards to substrate specificity and reduction of dioxygen. Despite the different metalloenzymes present in nature, the ability of laccase to generate radical substrates while reducing dioxygen to water has found potential uses in several fields.

Figures are in attached file.

References

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