Hydride Transfer for NADH Regeneration: From Nature, Beyond Nature

The transfer of energy through hydrides is of particular significance in the biocatalysis field. In nature, some enzymes proceed the cascaded hydride transfers via nicotinamide adenine dinucleotide (NADH) with high selectivity in mild conditions. Moreover, chemical strategies are developed to transfer hydrides in vitro for NADH regeneration, which often require noble metals as mediators to promote the regioselectivity of NADH reduction to bioactive 1,4‐NADH. Recently, the Mo3+ hydride generated in amorphous molybdenum sulfide under cathodic potential can work for selective hydrogenation of NAD+ to 1,4‐NADH, opening promising prospects for sustainable biocatalysis.


Enzymatic Hydride Transfer for NADH Regeneration in Nature
Hydride transfers are of particular significance in biocatalysis. The biological energy carrier NADH usually serves the purpose of such hydride transfer function to connect two or more enzymatic redox events, in which the NADH regeneration was usually accomplished through enzymatic regeneration routes. Take alcohol dehydrogenase (ADH) as an example; the Zn-dependent enzyme could reversibly oxidize alcohol to aldehyde by means of hydride transfer and the NADH could be regenerated. [9] For ADH, a Zn 2þ atom is coordinated in the active site by Cys-174, Cys-46, and His-67 and functions to position the alcohol group in the active site. Ser-48 and His-51 act as a charge-relay network to deprotonate the alcohol group for further oxidation to the aldehyde. As a Lewis acid, the zinc stabilizes the alkoxide intermediate, which forms a hydrogen bond with the Ser-48 hydroxyl group and is connected to His-51 through a proton relay channel. The hydride is transferred from the alkoxide ion to NAD þ , leading to NADH and a zinc-bound aldehyde (Figure 1a,b).

Chemocatalytic Hydride Transfer for Abiotic NADH Regeneration
Feasible abiotic NADH regeneration strategies should be established for sustainable biocatalysis. Photo-and electrochemical approaches have been developed to regenerate NADH, showing promises to electrify the chemical reactions with better sustainability. For example, Liu and Antonietti reported that a carbon nitride photocatalyst could work for direct NADH regeneration through the π-π interaction between the conjugated framework and the DOI: 10.1002/aesr.202200172 The transfer of energy through hydrides is of particular significance in the biocatalysis field. In nature, some enzymes proceed the cascaded hydride transfers via nicotinamide adenine dinucleotide (NADH) with high selectivity in mild conditions. Moreover, chemical strategies are developed to transfer hydrides in vitro for NADH regeneration, which often require noble metals as mediators to promote the regioselectivity of NADH reduction to bioactive 1,4-NADH. Recently, the Mo 3þ hydride generated in amorphous molybdenum sulfide under cathodic potential can work for selective hydrogenation of NAD þ to 1,4-NADH, opening promising prospects for sustainable biocatalysis. nicotinamide ring. [10] However, the poor selectivity under such metal-free condition toward bioactive 1,4-NADH necessitates the presence of noble metal-based homogeneous mediators. [11] Among this, the in situ-generated [Cp*Rh(bpy)(H)] þ is very successful for such selective conversion through the plausible ring-slippage mechanism. [12] Nonetheless, noble metal-based homogeneous mediators pose both economic and environmental challenges for sustainable enzymatic reactions. To mitigate these challenges, immobilized organometallic catalysts on different substrates have been explored, facilitating the recycle and reuse of noble metal catalysts. [13,14] Alternatively, various non-noble metals have been investigated as catalysts to electrochemically regenerate 1,4-NADH from NAD þ through metal hydride transfers, whereas may also suffer from the poor selectivity due to single-electron transfer or nonregioselective protoncoupled electron transfer events. [15] Therefore, the ideal catalysts for NADH regeneration from NAD þ would be capable of direct and regioselective hydride transfer.
In Nature Catalysis, Bau et al. reported that amorphous molybdenum sulfide (a-MoS x ) could work as the efficient electrochemical catalyst for selective NADH regeneration through the Mo 3þ hydride intermediate. [16] The electron paramagnetic resonance (EPR) spectroscopy was used for validating the existence of Mo 3þ hydride. The in situ-generated NADH could be coupled with alcohol dehydrogenase for the conversion of benzaldehyde to benzyl alcohol (Figure 1c).
Specifically, the existence of Mo 3þ hydride species in the a-MoS x was verified via EPR. The reduced a-MoS x sample under cathode potential exhibited an isotropic EPR spectrum, which displayed the characteristic g-value of Mo 3þ . The Mo 3þ EPR signal disappeared following oxidation and re-emerged after reduction. The difference in the peak-to-peak width in EPR spectrum between the original signal and the re-emerged one indicates the hyperfine coupling of Mo 3þ with a spin-active nuclei, that is, hydrogen atom. The results revealed the presence of a hydride directly bound to Mo 3þ . The trapped Mo 3þ hydride in the amorphous molybdenum sulfide works for NADH regeneration and direct biocatalysis with high yield under appropriate cathodic potentials. Consistent with electrochemical reduction of NAD analogue NMN, dimer species barely formed during the regeneration of NADH using the a-MoS x catalyst. Furthermore, NMR results confirmed that no 1,2-or 1,6-NADH isomers were emerged in NADH regeneration. While the reduction of NAD þ by Mo 3þ hydride was shown to be regiospecific for 1,4-NADH, the authors didn't specify the full mechanistic details for the important cofactor conversion. The detailed explanation for such site-specific hydrogenation should be clarified for future investigation. It should be noted that neither glassy carbon nor titanium catalyst reached quantitative NADH regeneration under the tested conditions, in contrast to the defective molybdenum sulfides.
The Mo 3þ hydride in molybdenum sulfide was also claimed as the origin for electrochemical H 2 evolution. The observation was in contrast with the primary thiol-based model, in which the evolution of H 2 was via the recombination of two active hydrogen species. However, the competition of H 2 evolution with NADH regeneration is a big challenge to use the molybdenum-based materials in biocatalysis because H 2 evolution decreases the Faraday efficiency for NADH formation and denatures the enzyme during catalytic reactions. Hence, efforts should be made to bring the nonnoble metal to practical applications. Nevertheless, the report unveils the hydride transfer mechanism of MoS x materials in electrocatalysis. Considering the numerous reports involving the Figure 1. Biological and bioinspired NADH regeneration. a,b) Schematic illustration of NAD þ /NADH equilibrium, H À transfer, substrate channel for ADH dehydrogenase. c) Mo 3þ hydride in the NADH regeneration and H 2 evolution. Under cathodic potentials, amorphous molybdenum sulfide could generate Mo 3þ hydride, which could be transferred to NAD þ to form NADH and protons to form H 2 , respectively. Reproduced with permission. [16] Copyright 2022, Springer Nature.
www.advancedsciencenews.com www.advenergysustres.com molybdenum, [17,18] the finding by Bau et al. paved the way for the economical application of electrocatalysis to biocatalysis, opening up new possibilities for practical biocatalysis. [16]

Conclusion
In conclusion, enzymatically catalyzed hydride transfers consistently inspire scientists to develop better catalysts for NADH regeneration beyond nature. The feasible abiotic regeneration through chemocatalytic strategies makes NADH a suitable candidate to bridge the biological and chemical systems via hydride transfers. The transfer hydrogeneration could be realized with high efficiency in mild conditions by coupling biocatalysis with different chemocatalytic categories, such as transition metal-, organo-, photo-, and electrocatalysis. Despite great achievements, most of the efficient approaches for regenerating bioactive NADH require noble metals as catalysts. The emergence of non-noble metal catalysts as reported by Bau et al. for highly selective NADH regeneration will lead to more sustainable multistep catalysis. Apart from NADH regeneration, hydride transfers through non-noble metal-based catalysts also contribute to other important catalytic hydrogenation reactions such as CO 2 reduction. [19] The integration of bio-and chemocatalysis can maximize the selectivity and efficiency for designed synthetic processes, providing an important pathway to energy conversion.