Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2 degrees ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, beta-nicotinamide adenine dinucleotide phosphate ( NADP(+)), is more expensive and less stable than beta-nicotinamide adenine dinucleotide ( NAD(+)). PCR-based, site-directed mutagenesis was performed on 2 degrees ADH in an attempt to adjust the cofactor specificity toward NAD(+) by mutating Tyr(218) to Phe ( Y218F 2 degrees ADH). This mutation increased the K-m(app) for NADP(+) 200-fold while decreasing the K-m( app) for NAD(+) 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2 degrees ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD(+). The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD(+) was electrically recycled 6.7 x 10(4) times, suggesting that the Y218F 2 degrees ADH-modified bioelectronic interface is stable.