Electrochemical evaluation of the reaction rate between methyl viologen mediator and diaphorase enzyme for the electrocatalytic reduction of NAD+ and digital simulation for its voltammetric responses

The electrocatalytic reduction of NAD⁺ using diaphorase enzyme was studied. Methyl viologen was used as an electron transfer mediator between an electrode and the enzyme. A catalytic wave for the reduction of NAD⁺ when all the species were in the solution was measured with cyclic voltammetry at a gold-amalgam electrode which showed low background currents at negative potentials. Steady-state currents could be obtained under the conditions of slow scan rate, low methyl viologen concentration, and high NAD⁺ concentration as the electrode reaction was converted to an electrochemical-catalytic (EC′) reaction. The bimolecular rate constant for the reaction of the reduced methyl viologen with the oxidized diaphorase was estimated as 7.5×10³ M^?1 s^?1 from the slope of the current versus MV²⁺] plot. Another slope of the current against the square root of the enzyme concentration also gave a close value of 6.7×10³ M^?1 s^?1. In the calculation of the rate constant, the stoichiometric factor when it is not one-to-one was considered. With the evaluated rate constant, digital simulation using the suggested reaction mechanism was compared with the experimentally obtained voltammograms. Satisfactory agreement indicates that the evaluation methods of the rate constant and the suggested mechanism are appropriate for the mediated enzyme-catalyzed electrochemical reactions.