Electrochemical Studies of Immobilized Laccases on the Modified -Gold Electrodes

Abstract

The direct electrochemical studies of four laccases (plant and fungal laccases) have been investigated on a gold electrode functionalized with a new tether of 2.2'-dithiosalicylic aldehyde. Results from these studies indicate that the redox potential of the active site of plant laccase from Rhus vernificera is shifted to a more negative value (255 mV versus SCE) than that of fungal laccase from Pyricularia oryzae (480 mV versus SCE). Mechanistic studies indicate that the reduction of type-1 Cu precedes the reduction of type-2 and type-3 Cu ions when the electrode is poised initially at different potentials. Also a new tether, 2.2'-dithiosalicylic aldehyde, has been used to study the redox properties of two laccases (LCCI and LCCIa) covalently attached to a gold electrode. An irreversible peak at 0.47 V vs. SCE is observed in the cyclic voltammograms of LCCI. In contrast, the cyclic voltammograms of LCCIa contain a quasi-reversible peak at 0.18 V vs. SCE and an irreversible peak at 0.50 V vs. SCE. We find that the replacement of the eleven amino acids a the C-terminus with a single cysteine residue (i.e., LCCIçLCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper containing active sites (Khet for LCCI = 1.0 Ý10-2 s-1 and Khet for LCCIa = 1.0Ý10-1 s-1 at 0.18 V versus SCE and 4.0Ý10-2 s-1 at 0.50 V versus SCE). These results show for the first time that the change of the primary structure of a protein via site-directed mutagenesis influences both the redox potentials of the copper ions in the active site and the rate of heterogeneous electron transfer.