The Electrical Contacting of NAD P -dependent Enzymes

For the efficient electrooxidation of NAD(P)H, mediated electrocatalysis is necessary [22, 170, 171], and a wide range of diffusional mediators has been studied [188-193], Organic compounds that undergo two-electron reduction-oxidation processes and also function as proton acceptors-donors upon their redox transformations (such as ortho- and para-derivatives of quinones, phenylenedi-amines and aminophenols) have been found to be ideal for the mediation of NAD(P)H oxidation, although single-electron-transfer mediators (e.g. ferrocene derivatives) are also capable of oxidizing NAD(P)H [190, 191], Some compounds demonstrate very high rates for the mediated oxidation of NAD(P)H in aqueous solutions [188,189,194,195], 

NAD (P)+-dependent enzymes, electrically contacted with electrode surfaces, can provide efhcient bioelectrocatalysis for the NAD(P)H oxidation. For example, diaphorase (DI) was applied to oxidize NADFl, using a variety of quinone compounds, several kinds of flavins, or viologens as mediators between the enzyme and electrode [217, 218]. The bimolecular reaction rate constants between the enzyme and mediators whose redox potentials are more positive than -0.28 V at pH 8.5 can be as high as 10 s , suggesting that the reac- 

Direct, nonmediated electrochemical reduction of NADIP) at modified electrode surfaces has been used to produce the en2ymatically active NAD(P)H and even to couple the NAD(P)H regeneration process with some biocatalytic reactions [228]. The modifier molecules used for these purposes are not redox active and they do not mediate the electron-transfer process between an electrode and NAD(P)+ however, they can effectively decrease the required overpotential and prevent formation of the nonenzymatically active dimer product [228]. For example, the efficiency of the direct electrochemical regeneration of NADH from NAD was enhanced by the use of a cholesterol-modified gold amalgam electrode that hinders the dimerization of the NAD-radicals on its modified-surface [228]. This direct electrochemical NAD+ reduction process was used favorably to drive an enzymatic reduction of pyruvate to D-lactate in the presence of lactate dehydrogenase. The turnover number for NAD was estimated as 1400 s k Other modifiers that enhance formation of the enzymatically active NAD(P)H include L-histidine [229] and benzimidazole [230], immobilized as monolayers on silver electrodes. CycKc voltammetric experiments demonstrated that these modified electrodes can catalyze the reduction of NAD+ to enzymatically active NADH at particularly low overpotentials. 

The regeneration of NAD(P)H with the participation of mediator-contacted enzymes ensures that NAD] ) reduction proceeds selectively and that only enzymatically active NAD(P)H is produced. Many enzymes have been used in this context to provide the bio-electrocatalytic reduction of NAD(P) , for example, ferredoxin-NADP reductase (FNR) [245-249], lipoamide dehydrogenase [250-254], formate dehydrogenase 

Immobilized low potential electron-transfer mediators (e.g. viologens) are more promising than diffusional mediators for the practical regeneration of NAD(P)H coupled with further biocat-alytic reactions. The immobiKzation of viologens usually results in significant positive potential shift of their redox potential [269-272], which however, badly affects their efficiency. The potential shift 

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