NADH and NMNH Regeneration Using Palladium Hydride Electrocatalysts

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Regeneration of noncanonical redox cofactors (NRCs) has received increased attention due to their ability to improve the overall selectivity and activity of biochemical production processes. Given the universality of canonical redox cofactors such as nicotinamide adenine dinucleotide (NAD⁺/NADH) and nicotinamide adenine diphosphate (NADP⁺/NADPH), a variety of undesired byproducts can form during enzymatic reactions. NRCs provide a unique capability when coupled with genetically modified enzymes to insulate desired metabolic pathways, however, in situ regeneration is challenging and synthesizing these cofactors is often costly. This study explores the direct electrocatalytic regeneration of the NRC nicotinamide mononucleotide (NMN⁺/NMNH) using palladium hydride (PdH 0.5 ). In addition to adsorbing protons on the surface, PdH 0.5 stores interstitial hydrogen (H 0 ) within the palladium lattice which can promote direct hydride transfer to NMN⁺. The desired mechanism involves proton adsorption (H⁺ + e - → H ads ) followed by a concerted electron-proton transfer (CEPT) to NMN⁺ (NMN⁺ + H ads + e - → NMNH). An alternative pathway leads to the formation of a radical species via a single electron transfer (NMN⁺ + e - → NMN*) followed by the protonation of the radical (NMN* + H ads → NMNH) or a dimerization reaction (NMN*+NMN * → NMN 2 ). During this study, the applied voltage was varied to investigate the optimum reaction conditions that favor the CEPT mechanism and minimize dimer formation. Cyclic voltammetry (CV), linear sweep voltammetry (LSV), and chronoamperometry (CA) were used to characterize redox activity, identify onset potentials, and evaluate catalytic performance. Preliminary experiments revealed the inhibition of the characteristic hydrogen underpotential deposition (H upd ) peak upon the addition of 1 mM NMN⁺, suggesting competitive adsorption. Additionally, CA scans at negative potentials suggest a direct relationship between potential magnitude and NMNH yield based on the absorbance at 340 nm using UV-Vis. This suggests higher overpotentials are required for sufficient NMN⁺ reduction. Enzymatic assays were performed to compare the activity of diaphorase from Geobacillus stearothermophilus (GsDI) in the presence of the electrochemically regenerated NMNH and pure NMNH. Further experimentation is needed to determine the overall selectivity and faradaic efficiency of NMN⁺ reduction to the enzymatically active 1,4-NMNH isomer. A significant challenge with this method is product quantification given the instability of reduced cofactors in neutral pH and room temperature conditions. Product stability can be improved using alkaline solutions and the influence of high pH on the catalytic performance of PdH 0.5 will be examined. These findings highlight the potential of metal hydrides to perform heterogeneous electrocatalysis for cofactor regeneration. Understanding the relationships between proton adsorption, electron transfer, and selective hydride delivery on PdH 0.5 surfaces will provide fundamental insight regarding selective electrochemical reductions involving biological and noncanonical redox cofactors. In addition to developing a more robust quantification protocol, future work includes varying catalyst material (PdH 0.5 /C vs Pd/C) and cofactor species (NMN + vs NAD + ) to confirm the improved reduction capabilities of the interstitial hydrogen source and determine whether NRCs have a higher affinity for direct electrocatalytic regeneration. The development of this regeneration method could significantly reduce cofactor production costs, enable renewable-energy-driven biocatalysis, and expand the integration of electrochemical and biochemical systems for sustainable chemical manufacturing. Figure 1

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