Reversible enzyme-catalysed NAD/NADH electrochemistry.

Formate dehydrogenase (FdsDABG) from is a Mo-containing enzyme capable of catalysing both formate oxidation to CO and the reverse CO reduction to formate by utilising NAD or NADH, respectively. This enzyme is part of the NADH dehydrogenase superfamily. Its subcomplex, FdsBG, lacking the formate oxidizing/CO-reducing Mo-cofactor, but harbouring an FMN as well as [2Fe-2S] and [4Fe-4S] clusters, reversibly interconverts the NAD/NADH redox pair. UV-vis spectroelectrochemistry across the range 6 < pH < 8 determined the redox potentials of these three cofactors. Cyclic voltammetry was used to explore mechanistic and kinetic properties of each oxidation- and reduction-half reaction. Through mediated enzyme electrochemistry experiments, the Michaelis constant for NADH oxidation ( = 1.7 × 10 μM) was determined using methylene blue as a redox mediator. For the reverse NAD reduction reaction using methyl viologen as electron donor a similar analysis yielded the value of = 1.2 mM. All experimental voltammetry data were reproduced by electrochemical simulations furnishing a set of self-consistent rate constants for the catalytic FdsBG system for both NAD reduction and NADH oxidation. This comprises the first electrochemical kinetic analysis of its kind for a reversible NADH dehydrogenase enzyme and provides new insight to the function of the FdsDABG formate dehydrogenase holoenzyme.