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1 Supramolecular studies of Rigidoporus lignosus and other fungal laccases

Laccases (benzenediol oxygen oxidoreductase, EC are multicopper oxidases containing transition metal ions which are essential for electron transfer from substrate to T1 copper site. The redox potential Eo is one of the most important parameter that characterizes the behaviour of these enzymes. Despite having very similar metal site features, the T1 copper site span a large range of Eo, suggesting that the secondary coordination sphere influences are important. This problem is especially relevant because laccases can directly oxidize only compounds with ionization potential not higher than the Eo of their T1-Cu sites. Therefore, the E0 of T1 determines the efficiency of catalysis on substrate oxidation, making high-potential laccases promising for biotechnological and protein engineering applications.

The computational studies reported in one of our previous publications(Cambria et al., 2012) detected several structural factors that may influence the Eo of the examined laccases. Some of these factors are dependent on the nature of the coordination ligands of T1 site, but others can be ascribed to the hydrophobic residues, hydrogen bond network, stacking and electrostatic interactions not necessary interacting directly with the copper metal.

The length of the copper T1 ligation histidine bond carried out on laccases with different Eo, shows that the distances of the Cu1 coordinate histidine bond of high Eo laccases are longer then that of low Eo laccases. These longer distances render the copper site more electron deficient and destabilizes its higher oxidation states with a consequent increase of the Eo (see Figure 1).


Figure 1. View of coordination distances and amino acid residues around the T1-binding site in different laccases. Hydrogen bonding interactions (Å) are shown as dotted lines; amino acid residues are represented as a colour coded stick; T1 Cu is shown as a pale blue sphere..

Hydrophobic environment around the T1 copper site appears to be another major structural determinant in modulating the Eo, where the stronger hydrophobic environment correlates with higher Eo (see Figure 2).


Figure 2. (a, b, c). The graphs report a detailed visualization of hydropathy profiles by Kyte-Doolittle scale (slide window = 5) for remarkable regions surrounding the RlL, TvL, CcL, and MaL T1 copper. Hydropathy values and amino acid sequences are shown by a colour code and are expressed as logP.

Analysis of hydrogen bond network (HBN) demonstrates that the amino acids building the T1 site strongly interact with neighbouring atoms therefore contributing to the stabilization of protein folding. Changes in these HBN that modified the Cu1 preferred coordination geometry leads to an increase in Eo (see Table 1).


Table 1. Distances between T1 Cu and ligand atoms (Cu-ligand). Hydrogen bonding distance between atoms of the Cu binding residues and spatially neighbouring atoms is also reported. Data refer to both high and low Eo laccases. Active site residues are highlighted as bold.

Stacking interactions between amino acid aromatic and metal ion coordination histidine imidazole ring, in the second coordination shell, play important roles in modulating Eo of laccases (see Figure 3 and Table 2).


Figure 3. Superposition of RlL, TvL, CcL and MaL T1 proximal pairs involved in π-π interactions. Amino acid residues are represented as a colour coded stick; T1 copper is shown as a pale blue sphere.


Table 2. Stacking parameters of pairs detected in RlL, TvL,CcL and MaL T1 homologue shell.

The electrostatic interactions between the T1 copper site with backbone carbonyl ligand show that, in the high Eo laccases, the bond length are longer if compared with the homologues of low Eo laccases. This elongated bond give rise to a destabilization of the higher Cu1 oxidation states and consequently to an increase of Eo(see Figure 4).


Figure 4. Schematic representation of the interaction between the T1 Cu and a dipole moment associated to a neighbouring backbone carbonyl ligand, in the different laccases.

1.1 In silico mutation of Rigidoporus lignosus laccase (RlL) Leu 462

In this section an in silico approach was used for simulating site-directed mutagenesis for RlL Leu-462 residue, in order to investigate about the possibility to establish an additional H-bond with the thiolate sulphur of Cys-452 residue, which could lead to an increasing of the redox potential Eo as reported for the cupredoxin variants (Yanagisawa et al., 2006; Marshall et al, 2009)(see Fig. 5). Moreover, the presence of a third hydrogen bond to the thiolate sulphur of Cys-452 increases the E0 even by ~ 350 mV as stated for Plastocyanin and Rusticyanin, by using computational methods (Olsson et al., 2003).


Figure 5.a, Native azurin (PDB 4AZU). b, N47S/M121L azurin: N47S affects the rigidity of the copper binding site and, probably, the direct hydrogen bonds between the protein backbone and Cys 112. c, N47S/F114N azurin: introducing a hydrogen-bond donor at position 114 perturbs hydrogen-bonding near the copper binding site, possibly disrupting donor-acceptor interactions to His 117, or ionic interactions between the copper and the carbonyl oxygen of Gly 45. d, F114P/M121Q azurin: F114P deletes a direct hydrogen bond to Cys 112 resulting in a lower redox potential. The ultraviolet-visible spectroscopy of the F114P-containing variants shows a significant increase in the copper d → d absorbance range around 800 nm. This increased absorbance suggests slight rearrangement of the copper binding site, but is consistent with F114P Az and other T1 copper proteins, such as plastocyanin. In all panels copper is shown in green, carbon in cyan, nitrogen in blue, oxygen in red and sulphur in yellow. Hydrogen-bonding interactions are shown by dashed red lines..

1.1.1 Methods

Among the suitable natural amino acids tested in our simulation, only asparigine was considered as a good candidate for site-directed mutagenesis because of the favourable orientation of Nd, which can act as a donor of the Cys-452 S. According to this hypothesis, the molecular software PyMol (PyMOL) was used to perform the Leu462Asn mutant.

Then, we used the molecular package ABALONE (Abalone) to run a 1000 step energy optimization protocol for both the wild-type and the mutated 1V10 laccase enzymes, by using a Steepest Descendent algorithm under Amber96 Force Field by implicit solvation model, down to a tolerance of 0.1, setting a protein dielectric permittivity of 4, a long range cutoff of 8 Å and a short range cutoff of 2.6 Å.

1.1.2 Results

At the end of the minimization protocol, the measured distance between Asn-462 Nd and Cys-452 S of the mutated 1V10 laccase was 3.04 Å, in line with the H-bond range distance recognized by the HB plot program (HB Plot). Instead, the distance between Cys-452 S and T1 Cu remained unchanged. Thus, in the light of these results, an experimental investigations could be carried on in order to clarify whether the Leu462Asn substitution, followed by the possible addition of a third H-bond to Cys-452 S, might represents a valid strategy for contributing to the tuning of the RlL Eo.


· Cambria MT, Gullotto D, Garavaglia S, Cambria A. In silico study of structural determinants modulating the redox potential of Rigidoporus lignosus and other fungal laccases. JBSD, 30(1), 89 - 101 (2012).

· Marshall NM, Garner DK, Wilson TD, Gao YG, Robinson H, Nilges MJ, Lu Y. Rationally tuning the reduction potential of a single cupredoxin beyond the natural range.Nature 462, 113-116 (2009).

· Olsson MHM, Hong GJ, Warshel A. Frozen density functional free energy simulations of redox proteins: computational studies of the reduction potential of plastocyanin and rusticyanin. J Am Chem Soc 125, 5025-5039 (2003).

· Yanagisawa S, Banfield MJ, Dennison C. The role of hydrogen bonding at the active site of a cupredoxin : the Phe114Pro azurin variant. Biochemistry 45, 8812-8822 (2006).