Electroreduction of O 2 to water at 0.6 V (SHE) at pH 7 on the ‘wired’ Pleurotus ostreatus laccase cathode Scott Calabrese Barton a, *, Michael Pickard b , Rafael Vazquez-Duhalt c , Adam Heller d a Department of Chemical Engineering, Columbia University, New York, NY 10027, USA b Department of Biological Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2E9 c Instituto de Biotecnologia UNAM, Cuernavaca, Morelos 62250, Mexico d Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, USA Received 15 November 2001; received in revised form 20 December 2001; accepted 13 March 2002 Dedicated to Dr Frieder W. Scheller on his 60th birthday. Abstract O 2 was electroreduced to water at 0.6 V (SHE) near neutral pH on the ‘wired’ Pleurotus ostreatus laccase cathode. We previously reported high-current density (5 mA cm 2 ), four-electron electroreduction of O 2 to water on a ‘wired’ Coriolus hirsutus laccase electrode at  /0.7 V (SHE) in pH 5 in citrate buffer. Since the enzyme was inhibited by chloride and because its activity declined steeply when the pH was raised to neutral, the rate of O 2 electroreduction in a physiological buffer solution was only /1% of that at pH 5 in absence of chloride. Here we show that substitution of the C. hirsutus laccase by laccase from P. ostreatus allows the upward extension of the pH range of O 2 electroreduction. The current density of the electrode made with laccase from P. ostreatus in pH 7 citrate buffer was /100 mA cm 2 and at pH 7 and in phosphate buffered NaCl (PBS, 20 mM phosphate, 0.1 M NaCl) it still retained 6% of its maximal (1 mA cm 2 ) current density at pH 5 in citrate buffer. The electrocatalyst consisted of the crosslinked P. ostreatus laccase and the electron conducting redox polymer PVI-Os(dmebpy)(tpy) 2/3 [PVI /poly(N -vinyl imidazole) with about 1/5th of the rings complexed with (Os-dmebpy-tpy) 2/3 ; dmebpy /4,4?-dimethyl-2,2?-bipyridine; tpy /2,2?,6?,2ƒ-terpyr- idine]. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Enzymes; Oxygen electrode; Biofuel cells; Sensors 1. Introduction Fungal laccases have been considered as electrocata- lysts for the four-electron electroreduction of O 2 to water at positive electrode potentials (Calabrese Barton et al., 2001b; Palmore and Kim, 1999). Earlier we have immobilized and electrically connected (‘wired’) laccase from Coriolus hirsutus in a redox hydrogel of poly-N - vinyl imidazole with 1/5th of its rings coordinated to [Os(tpy)(dmebpy)] 2/3 (where tpy is 2,2?:6?,2ƒ-terpyr- idine and dmebpy is 4,4?-dimethyl-2,2?-bipyridine). In well-stirred pH 5 citrate buffer and at 37 8C the electroreduction current density exceeded 1 mA cm 2 for smooth vitreous carbon electrodes and 5 mA cm 2 for porous carbon paper electrodes (Calabrese Barton et al., 2001b). Use of the cathodes in miniature implanted sensors or biofuel cells requires that they retain their activity under physiological conditions, i.e. pH 7 and chloride concen- trations near 100 mM. Most fungal laccases are inactive under these conditions. Trudeau et al. (1997) reported an optimal pH value of 4.7 for the activity of the laccase II isoenzyme from Trametes versicolor , when mediated by the redox couple Os(bpy) 2 Cl 2 2/3 in acetate buffer. O 2 is, however, not electroreduced to water by the T. versicolor laccase and Os(bpy) 2 Cl 2 2/3 in pH 7 PBS (Trudeau et al., 1997). The activity of laccase from C. hirsutus in PBS (20 mM phosphate, 100 mM NaCl) is reported here to be about 1% of its activity in pH 5 citrate buffer free of chloride. Xu has reported substrate dependence in recombinant laccases, showing optimal pH values of 5  /8 for the homogeneous oxidation of * Corresponding author. Fax:  /1-212-854-3054 E-mail address: scb2001@columbia.edu (S.C. Barton). Biosensors and Bioelectronics 17 (2002) 1071 /1074 www.elsevier.com/locate/bios 0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0956-5663(02)00100-8