In order to investigate the mechanism of oxygen electroreduction by fungal laccase combined as well as stand alone spectral and electrochemical approaches have been exploited. Several homogeneous preparations of laccases from different basidiomycetes have been used, which were recently biochemically characterized in detail [1]. Currently it is believed that the redox potentials of the T2 and T3 copper sites in laccase are very close to each other. These potentials are approximately equal to 400 mV in low redox potential laccases and to about 800 mV in high potential laccases [2]. For the first time we report [3] that the redox potential of the T2 copper site of a high redox potential laccase from the basidiomycete Trametes hirsuta is close to 400 mV vs. NHE. Electrochemical studies of laccases from different sources show that these enzymes establish different heterogeneous ET pathways on carbon and gold electrodes [3-5]. It is determined that in the case of carbon electrodes a "normal" ET pathway can be observed, i.e., electron donor (electrode or substrate) - T1 site - T2/T3 cluster [4,5]. Due to this reason laccases adsorbed on carbon electrodes usually show well-pronounced electrocatalytic reduction of oxygen [4,5]. However, in the case of gold electrodes the heterogeneous ET pathway from the electrode to the copper centres in the enzyme globule is different [3,5]. We found that Trametes hirsuta laccase on gold electrodes electronically communicate with the electrode surface through the T2/T3 cluster. We observed that the electrons from the electrode to the T1 copper flow through the T2/T3 coppers. We confirm that T1 site has a redox potential of 780 mV [1,3,5], however, the redox potential of one copper ion from the T2/T3 cluster is close to 400 mV [3,5]. From the data about direct ET reactions of laccase on carbon and gold electrodes, as well as from the spectral results a possible modification of the catalytic cycle [6] of the enzyme is suggested and will be discussed.

[1] S. Shleev et al., Biochimie, 86 (2004) 693.
[2] B. Reinhammar, Biochim. Biophys. Acta, 275 (1972) 245.
[3] S. Shleev et al., Biochem. J., 385 (2005) 745.
[4] S. Shleev et al., Bioelectrochem., 67 (2005) 115.
[5] S. Shleev et al., Biosens. Bioelectron., 20 (2005) 2517.
[6] E. Solomon et al., Chem. Rev., 96 (1996) 2563.

Acknowledgement: This work was financially supported by the European Commission (ICA2-CT-2000-10050) and the Swedish Research Council. The Swedish Institute (SI) is acknowledged for the support of a postdoctoral fellowship for Sergey Shleev.

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