biostudies-literatureGiang PDBasic Energy SciencesAustralian Research Council6035-6049https://www.ebi.ac.uk/biostudies/studies/S-EPMC11891784biostudies-literatureUnknown16(14)Formate dehydrogenase (FdsDABG) from <i>Cupriavidus necator</i> 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 (<i>K</i> _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 <i>K</i> _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.Chemical scienceReversible enzyme-catalysed NAD&lt;sup&gt;+&lt;/sup&gt;/NADH electrochemistry.PMC11891784DE-SC0010666DP220103268Niks DHille RBernhardt PVGiang PDHakopian SfalseReversible enzyme-catalysed NAD&lt;sup&gt;+&lt;/sup&gt;/NADH electrochemistry.Formate dehydrogenase (FdsDABG) from <i>Cupriavidus necator</i> 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 (<i>K</i> _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 <i>K</i> _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.2025-01-01T00:00:00Z2025 Apr2025-06-27T03:05:39.256Z2025-04-07T07:49:47.005ZS-EPMC118917844007047210.1039/d5sc00570a