Kinetic and thermodynamic aspects of NAD-related enzyme-linked mediated bioelectrocatalysis

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.