In silico study of structural determinants modulating the redox potential of Rigidoporus lignosus and other fungal laccases

Laccases are multicopper oxidases in which substrate oxidation takes place at the type-1 (T1) copper site. The redox potential (E⁰) significantly varies amongst members of the family and is a key parameter for substrate specificity. Despite sharing highly conserved features at the T1 copper site, laccases span a large range of E⁰, suggesting that the influence of the metal secondary coordination sphere is important. In silico analysis of structural determinants modulating the E⁰of Rigidoporus lignosus and other fungal laccases indicated that different factors can be considered. First, the length of the T1 copper coordinating histidine bond is observed to be longer in high E⁰ laccases than in low E⁰ ones. The hydrophobic environment around the T1 copper site appeared as another important structural determinant in modulating the E⁰, with a stronger hydrophobic environment correlating with higher E⁰. The analysis of hydrogen bonding network (HBN) around the T1-binding pocket revealed that the amino acids building up the metal binding site strongly interact with neighbouring residues and contribute to the stabilization of the protein folds. Changes in these HBNs that modified the Cu1 preferred coordination geometry lead to an increase of E⁰. The presence of axial ligands modulates the E⁰ of T1 to different extent. Stacking interactions between aromatic residues located in the second coordination shell and the metal ion coordination histidine imidazole ring have also been identified as a factor that modulates the E⁰. The electrostatic interactions between the T1 copper site and backbone carbonyl oxygen indicate that Cu1-CO=NH distance is longer in the high E⁰ laccases. In short, the in silico study reported herein identifies several structural factors that may influence the E⁰ of the examined laccases. Some of these are dependent on the nature of the coordination ligands at the T1 site, but others can be ascribed to the hydrophobic effects, HBNs, axial ligations, stacking and electrostatic interactions, not necessary directly interacting with the copper metal.