Electrochemical Studies of Immobilized Laccases on the Modified-Gold Electrodes

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 Lccla) covalently attached to a gold electrode. An irreversible peak at 0.47V vs. SCE is observed in the cyclic voltammorams of LCCI. In contrast, the cyclic voltammograms of LCCIa contain a quasi-reversible peak at 0.18V vs. SCE and an irreversible peak at 0.50V vs. SCE. We find that the replacement of the eleven amino acids a the C-terminus with a single cysteine residue <TEX>(i.e., \;LCCI→LCCIa)</TEX> influences the rate of heterogeneous electron transfer between an electrode and the copper containing active sites <TEX>(Kₕₑₜ\;for\;LCCI=1.0×10⁻²\;s⁻¹\;and\;Kₕₑₜ\;for\;LCCIₐ= 1.0\;times10⁻¹\;s⁻¹\'at\;0.18V\;versus\;SCE\;and\;4.0×10⁻²\;s⁻¹\;at\;0.50V\; versus\;SCE)</TEX>. 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.