Experimental insights into electrocatalytic [Cp*Rh(bpy)Cl]⁺ mediated NADH regeneration

NADH plays a crucial role in many enzymatically catalysed reactions. Due to the high costs of NADH a regeneration mechanism of this cofactor can enlarge the applications of enzymatic reactions dramatically. This paper gives a thorough system analysis of the mediated electrochemical regeneration of active NADH using cyclic voltammograms and potentiostatic measurements with varying pH, electrode potential, and electrolyte solution, highlighting the system's limiting conditions, elucidating optimal working parameters for the electrochemical reduction of NAD⁺, and bringing new insight on the oxidation of inactive reduction products. Using [Cp*Rh(bpy)Cl]⁺ as an electron mediator dramatically increases the percentage of enzymatically active electrochemically reduced NADH from 15% (direct) to 99% (mediated) with a faradaic efficiency of up to 86%. Furthermore, investigations of the catalytic mechanisms of [Cp*Rh(bpy)Cl]⁺ clarifies the necessary conditions for its functioning and questions the proposed reaction mechanism by two-step reduction where first the mediator is reduced and then brought in contact with NAD⁺.