Feature Article Kinetic and Thermodynamic Aspects of NAD-Related Enzyme- Linked Mediated Bioelectrocatalysis Riccarda Antiochia, Albino Gallina, Irma Lavagnini, Franco Magno* Dipartimento di Chimica Inorganica, Metallorganica ed Analitica, Universita ¡ di Padova, Via Marzolo 1, I-35171 Padova, Italy e-mail: Magno@chin.unipd.it Received: October 16, 2001 Final version January 15, 2002 Abstract The diaphorase-catalyzed electrochemical oxidation of NADH with the aid of quinones (MAP) or ferrocene derivatives (FMCA) as electron transfer mediators was studied by cyclic voltammetry under variable conditions of concentration polarization of NADH near the electrode surface. The suppression of this polarization was obtained by the redox L-lactate dehydrogenase-catalyzed reaction between L-lactate and NAD  . The addition of pyruvate inhibits the formation of NADH owing to the thermodynamically unfavorable (uphill) electron transfer from L-lactate to NAD  . The role played by the uphill/downhill character of the last step of the catalytic chain was illustrated by regenerating NADH using the redox couple gluconolactone/glucose with glucose dehydrogenase as catalyst. The influence on the catalytic wave of the presence of a downhill or uphill step between diaphorase and NAD  /NADH couple was also rationalized via the digital simulation technique using experimental data reported in the literature. Keywords: Bioelectrocatalysis, Mediator, Diaphorase, NADH-dependent enzyme 1. Introduction Intheyearsfollowing1990muchattentionhasbeenfocused on the use of dehydrogenase enzymes to develop ampero- metric biosensors and bioreactors [1, 2]. Dehydrogenases requiring nicotinamide adenine dinucleotide (NAD  , oxi- dized form; NADH, reduced form) coenzyme as co- substrate are more than 250 [3] and in principle are of special interest because of the enormous variety of substrates that can be determined. The major problem in practical applications of dehydrogenases is the difficulty in the regeneration of the nicotinamide coenzyme. Although theformalpotentialoftheNADH/NAD  coupleis  0.32 V versus the normal hydrogen electrode (NHE) at pH 7.0, a large overpotential is required for the direct oxidation of NADH at solid electrodes [4 ± 7]. A large number of redox mediators such as quinones [8, 9] or inorganic molecules [10 ± 12] was used in order to reduce the overpotential but their use was limited by a slow response and/or low stability. Another approach was the use of suitably modified electro- des [13, 14]. A good method to accelerate the NADH oxidation is the use of the diaphorase (DI) reaction coupled with an electron transfer mediator such as ferrocene and its derivatives [15] or quinone compounds [16]. Moreover, owing to the reversibility characteristics of the reaction involved, diaphorase coupled with organic mediators can also catalyze the reduction of NAD  [17]. This reversibility property is profitably applied in NAD-related two-enzyme- linked bioelectrocatalysis (Figure 1) and the study of the reaction rates of the various steps of the global reaction, in particularbetweenDIandmediators,isthematterofrecent investigations [18±20]. In the present work we investigated the kinetic and thermodynamic aspects of a bioelectrocatalytic system based on the DI reaction mediated by p-methylaminophe- nol (MAP) or ferrocenemonocarboxylic acid (FMCA) linked to two different NAD-dependent dehydrogenase systems, namely L-lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH), in order to enlighten the role of the thermodynamically unfavorable last step (uphill) Fig. 1. Scheme for NAD-related two-enzyme -linked bioelectrocatalysis (ox  oxidized form; red  reduced form). 1256 Electroanalysis 2002, 14, No. 18 ¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 1040-0397/02/1809-1256 $ 17.50+.50/0