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Updated: May 23, 2025
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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
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Reversible enzyme-catalysed NAD+/NADH electrochemistry
Peter D Giang1, Dimitri Niks2, Sheron Hakopian2
1School of Chemistry and Molecular Biosciences, University of Queensland Brisbane 4072 Australia p.bernhardt@uq.edu.au.
Chemical Science
|March 12, 2025
Summary
Formate dehydrogenase subcomplex FdsBG from Cupriavidus necator reversibly interconverts NAD+/NADH. Electrochemical analysis determined kinetic properties and rate constants for this NADH dehydrogenase enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Bioenergetics
Background:
- Formate dehydrogenase (FdsDABG) from Cupriavidus necator is a molybdenum-containing enzyme.
- It catalyzes formate oxidation to CO2 and CO2 reduction to formate using NAD+ or NADH.
- FdsDABG belongs to the NADH dehydrogenase superfamily.
Purpose of the Study:
- To electrochemically characterize the FdsBG subcomplex, which lacks the Mo-cofactor but contains FMN and iron-sulfur clusters.
- To determine the redox potentials of the cofactors within FdsBG.
- To elucidate the kinetic and mechanistic properties of NAD+/NADH interconversion.
Main Methods:
- UV-vis spectroelectrochemistry to determine cofactor redox potentials (pH 6-8).
- Cyclic voltammetry to investigate oxidation and reduction half-reactions.
- Mediated enzyme electrochemistry with redox mediators (methylene blue, methyl viologen) to determine kinetic parameters.
Main Results:
- Redox potentials of FMN, [2Fe-2S], and [4Fe-4S] clusters were determined.
- Michaelis constant for NADH oxidation (KM,NADH) was 1.7 × 10^2 μM.
- Michaelis constant for NAD+ reduction (KM,NAD) was 1.2 mM.
- Electrochemical simulations reproduced experimental data, yielding self-consistent rate constants.
Conclusions:
- This study provides the first electrochemical kinetic analysis of a reversible NADH dehydrogenase enzyme.
- The findings offer new insights into the catalytic mechanism of the FdsDABG formate dehydrogenase holoenzyme.
- The kinetic data are crucial for understanding the enzyme's role in cellular redox balance.
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