Powering fuel cells through biocatalysis - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 393

type 1 (T1), type 2 (T2), and type 3 (T3) [44]. The T2 and T3 coppers form a tri-nuclear
cluster for reduction of oxygen, whereas successive one-electron oxidation of the sub-
strates occur at the so-called “blue” T1 site, approximately 1.3 nm distal to the T2/T3
cluster. The key characteristic of the “blue” multi-copper oxygenases is the standard
reduction potential of the T1 site. The catalytic effi ciency of the laccase reaction with
its substrate has been shown to depend on the thermodynamic driving force for elec-
tron transfer: the potential difference between substrate and T1 copper site [34, 44-48].
Laccases possessing T1 sites of relatively high reduction potentials can drive oxidation
of otherwise recalcitrant organic or biopolymeric substrates, fi nding application in biore-
mediation and dye and pulp bleaching. In addition, the oxidation of substrates coupled to
intra-molecular electron transfer to the T2/T3 cluster can result in the reduction of oxy-
gen at relatively high potentials. The reduction potential of the T1 site can be determined
by redox titrimetry [44-49].
The copper-containing redox enzymes have also been shown to transfer electrons
directly with electrode materials, allowing determination of the reduction potentials of
the active site using voltammetry, and possible correlation with structure and activity.
Direct electron transfer to a laccase was fi rst reported by Yaropolov's group at car-
bon electrodes [50]. Subsequent studies [8, 9, 51, 52] have investigated direct electron
transfer to the copper active sites of the multi-copper oxidases as a means to classify
the oxidases. The laccases can thus be classifi ed, as suggested recently by Shleev et al.
[9, 10], into three separate groups, based upon the reduction potential of the T1 copper
site. The plant laccases have a low T1 potential of 0.43 V vs NHE, while fungal

laccases possess T1 sites of, either middle potential of 0.47 to 0.71 V vs NHE, or

high potential of 0.78 V vs NHE.

While direct electron transfer to laccases may help elucidate the mechanism of
action of these enzymes it is unlikely that this process will supply suffi cient power for
a viable implantable biocatalytic fuel cell, because of diffi culties associated with the
correct orientation of the laccase and the two-dimensional nature of the biocatalytic
layer on the surface. However, a recent attempt to immobilize laccase in a carbon dis-
persion, to provide electrodes with correctly oriented laccase for direct electron trans-
fer, and a higher density of electrode material shows promise [53].
Mediated reduction of oxygen by laccase, particularly from fungal sources with
high T1 potentials, as demonstrated by Palmore and Kim [26], does show great prom-
ise for the development of biocatalytic fuel cell cathodes. Immobilization of both
mediator and laccase provides a biocatalytic cathode for oxygen reduction that may
be used in a miniaturized membraneless biocatalytic fuel cell. Trudeau et al. [30] were
the fi rst to report on oxygen reduction by fi lms of immobilized mediator and laccase,
formed by cross-linking laccase to an osmium-based redox polymer fi lm on carbon
electrodes. The redox polymer structure, shown in Fig. 12.5, was prepared by substitu-
tion of one of the chloride ligands of an Os(2,2

-bipyridine) 2 Cl 2 complex, with every
tenth imidazole monomeric unit of the polyvinylimidazole polymer backbone.
Co-immobilization of this redox polymer with a fungal laccase from Trametes
versicolor, possessing a T1 copper site reduction potential of 0.57 V vs Ag/AgCl

( 0.77 vs NHE), was achieved using a diepoxide cross-linker, in an approach