Reversible enzyme-catalysed NAD⁺/NADH electrochemistry.

ABSTRACT: Formate dehydrogenase (FdsDABG) from Cupriavidus necator is a Mo-containing enzyme capable of catalysing both formate oxidation to CO₂ and the reverse CO₂ 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/CO₂-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 (K _M,NADH = 1.7 × 10² 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 K _M,NAD⁺ = 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.

SUBMITTER: Giang PD 

PROVIDER: S-EPMC11891784 | biostudies-literature | 2025 Apr

REPOSITORIES: biostudies-literature