Reduction of NAD and NMN on mineral surfaces with H2 reveals a functional role for the adenosine moiety in prebiotic evolution

Reduction of NAD and NMN on mineral surfaces with H2 reveals a functional role for the adenosine moiety in prebiotic evolution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reduction of NAD and NMN on mineral surfaces with H2 reveals a functional role for the adenosine moiety in prebiotic evolution Martina Preiner, Delfina Henriques Pereira, Xiulan Xie, Zainab Subrati, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6214903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract Many cofactors share a molecular structure – adenosine – that otherwise occurs in nucleic acids. The presence of adenosine in cofactors has presented an evolutionary puzzle. Is it a biochemical ‘handle’ that allows proteins to bind the cofactor more tightly, or is it a relic from a time when cofactors arose from the building blocks of genes? Using the example of nicotinamide adenine dinucleotide (NAD), we find a surprising and previously unknown property of its adenosine monophosphate (AMP) handle. Reduction experiments with hydrogen gas (H 2 ) on mineral surfaces show that the handle-free nicotinamide mononucleotide (NMN) overreduces quickly, while NAD gets reduced specifically. The AMP handle allows NAD to function in a hydrothermal, mineral-based setting, indicating that it is a form of protection against a harsh chemical environment in which biochemistry and life arose. Our findings uncover a specific functional role for the AMP moiety of NAD under environmental conditions capable of nonenzymatic NAD reduction, thereby identifying a structural element of a redox cofactor that is older than the enzymes that use it. Biological sciences/Biochemistry/Biocatalysis Biological sciences/Chemical biology/Metals adenosine cofactors hydrogen emergence of life mineral catalysis protometabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In many theories for the origin of metabolism, organic cofactors such as NAD + , flavin adenine dinucleotide (FAD), S-adenosyl methionine (SAM) and coenzyme A (CoA) are seen as crucial intermediates in the transition from inorganic to enzymatic catalysis 1 – 7 . These cofactors catalyse ancient biochemical reactions with a wide variety of substrates, are often active in the absence of enzymes 8 – 12 , and share a ‘handle’ consisting of the nucleoside adenosine, a central component of nucleic acids such as RNA. The catalytic and nucleoside moieties of cofactors like NAD + thus unite “metabolism first” and “information first” views for the origin of life 1 , 7 , 13 in a single molecule (Fig. 1). In all enzymatic reactions known to date, the adenosine moiety is catalytically inert, in non-enzymatic reactions its phosphate groups have been shown to enabling association to metal ions 9 , 11 . Some cofactors that do not possess the moiety, such as pterins or folates, nonetheless derive from nucleotides in their biosynthesis 5 , 14 – 16 , indicating close connections between cofactor catalysis and RNA bases in early biochemical evolution. The evolutionary rationale behind the conserved presence of an adenosine moiety to NAD + is traditionally viewed either functionally, in terms of a biochemical handle that allows enzymes to bind the cofactor more efficiently 17 , 18 , or historically also as a holdover from an earlier phase of evolution in which RNA preceded enzymes and cofactors were active in ribozymes 1 . But there is, however, a third possibility. NAD + functions exclusively as a redox cofactor and neither traditional view takes into account the environmental source of electrons required to generate NADH or reduced substrates for NAD + dependent reactions. NAD is an ancient redox cofactor that is essential in metabolism and traces back to the last universal common ancestor, LUCA 2 , 19 – 21 . Several prebiotic routes of NAD synthesis have been proposed, including via mineral-assisted synthesis under hydrothermal conditions 6 , 22 or via nitrile-dependent syntheses 13 , 23 , 24 . Recent work has shown how NAD + can be non-enzymatically reduced under a variety of conditions 8 , 9 , 12 , 25 , 26 . Additionally, the reducing abilities of NADH have been demonstrated both with and without metal ions as catalysts 11 , 27 . What is a good possible primary electron source and thus geochemical setting? In this study, we are investigating the abilities of water-rock-interaction systems, where protons of water are being reduced to hydrogen (H 2 ) gas by electrons of Fe(II) containing minerals (serpentinizing systems). Previous studies have shown that NAD + readily reacts with H 2 and metal powders (Ni, Co, Fe) to specifically form the biologically relevant form of reduced NAD (1,4-NADH) 12 . An independent study also reported NAD + reduction without H 2 , using iron sulfides as reductant at lower yields 25 . The conditions of serpentinizing systems not only reduce NAD + but also carbon dioxide (CO 2 ) with the electrons provided by H 2 20,21,28,29 . They are rich in Fe and, depending on the system, Ni as well 30 , just like enzymes of the acetyl CoA pathway, which H 2 dependent anaerobic autotrophs use to reduce CO 2 in their carbon and energy metabolism 31 , 32 . Hotter systems tend to feature Ni-Fe-alloys with higher nickel composition, while cooler ones are richer in iron 30 . Nickel-containing intermetallic compounds such as awaruite (Ni 3 Fe) or taenite (NiFe 3 to Ni 2 Fe) are products of the reaction of H 2 with Ni(II) compounds in serpentinizing systems 30 , 33 , 34 and are also found in meteorites 35 . Native metals occur naturally in these highly reducing systems 36 , 37 . Here, we investigate the ability of naturally occurring Ni- and Fe-containing allows to replace enzymes for H 2 dependent NAD + reduction. We then compare NAD to its AMP-lacking homologue NMN to test how the AMP moiety impacts the nature of products obtained from non-enzymatic (prebiotic) NAD + reduction with H 2 . Results Screening naturally occurring iron and nickel containing minerals. Ni-Fe containing minerals found in hydrothermal settings were tested for the reduction of NAD + under conditions comparable to those found in mild serpentinizing hydrothermal systems (40°C, 0.133 M phosphate buffer pH 8.5, 5 bar H 2 , Supplementary Scheme S1 ). These nanoparticular mineral powders were synthesized via the nano-casting method by using tea leaves as a template and were previously characterized 38 , 39 . The metal content in these reactions is equivalent to that of the cofactor (1 metal atom per cofactor). The resulting H 2 concentration at our conditions is 3.6 mM (using Henry’s law, s. Supplementary Equations S1–3 and references 19 , 40 ), which is comparable to the H 2 concentrations found in the effluent of serpentinizing systems 41 , 42 . The buffer was bubbled with N 2 for 1 h and handled inside a glove box to approximate the anoxic conditions on early Earth. Several controls were implemented, including controls without metal and H 2 , respectively. The liquid phase was analysed by 1 H-NMR. After 4 h under H 2 , 1,4- and 1,6-NADH formed in all samples at different yields, the reaction with nanoparticular NiFe 3 (nNiFe 3 ) yielding the most NADH (Fig. 2 ). Control experiments starting from 1,4-NADH showed that 1,6-NADH is very likely a product of rearrangement from 1,4-NADH – with only a marginal influence of the used metals ( Supplementary Scheme S2, Tables S4 and S5, Figure S3 ). Samples under Ar also produced NADH with a Fe-rich minerals (nNiFe 3 , nFe). In addition to transferring hydrides from H 2 to NAD + , iron can oxidize, donating its own electrons either by producing nascent H 2 gas, ultimately reducing NAD + or by direct electron transfer to NAD + . This process can also be used as a proxy for the constant H 2 -production in serpentinizing systems 12 . Ni by itself is H 2 -dependent in the promotion of NAD + reduction. Scanning transmission electron microscopy (STEM) imaging before and after the reactions (the latter including a washing and dilution step to assure true surface alteration) confirms that Fe, both under Ar and H 2 , gets associated with phosphate ions in ratio that suggests the formation of iron phosphate. Ni does not associate with phosphate, suggesting it stays in its native form ( Supplementary Methods and Figures S4–7 ). In Ni-rich minerals, the Fe is expected to slowly reduce NAD under Ar conditions. However, it is likely that the resulting products do not reach the detection threshold within 4 h. Overall, bimetallic minerals are significantly more efficient than the individual transition metals when hydrogen is available. Introducing one Ni atom to a Fe atom increases the yield by 300% (nNiFe vs. nFe). Their properties, already observed in a previous study 12 , seem to complement each other for the reduction of NAD + with H 2 : Fe being mostly an electron donor, while Ni promotes hydride transfer from H 2 . These complementary roles have also been described in other publications, suggesting charge transfers from Fe (more electropositive) to Ni could increase the electron density in Ni 38 , 43 . The “universal” adenine nucleotide in organic cofactors. Many central cofactors share an AMP “handle” (Fig. 1) attached to the catalytically active moiety. In the case of NAD, NMN is the hydride-transferring nicotinamide, AMP is inert. NAD is stable in water, with its pH range depending on the reduction state of the nicotinamide: NADH is more stable at pH > 7, while NAD + is more stable under acidic conditions 12 . To investigate the role of the AMP-tail in a prebiotic context, several experiments were designed to compare NAD and NMN. We initially focussed on nNiFe 3 , the most efficient of the Ni-Fe minerals in the above described NAD experiment (Fig. 2 ). All other reaction conditions (buffer, pH, temperature, metal to cofactor ratio) were maintained ( Supplementary Scheme S3 ). Products were quantified via 1 H-NMR spectroscopy. Without metals, NMN does not react and remained stable ( Supplementary Figures S8 and S9 ). Under Ar, NMN still got reduced due to the abundant iron in the mineral compound, but more slowly than under H 2 ( Supplementary Tables S6 and S7 ). In additional to the 2 h experiment with NAD + under H 2 showed the increase of 1,4- and 1,6-NADH to be steady and inversely proportional to the decrease of NAD + in solution (Fig. 3C). After 4 h with nNiFe 3 , on average 57% of NAD + was reduced with 26% remaining oxidized. The remaining 17% can in part be attributed to nicotinamide formation but also unassigned degradation reactions and loss via surface absorption 44 . NMN, however, shows a completely different reaction profile (Fig. 3D). After only 1 h, 69% of the starting NMN had been converted, and a variety of products was observed in 1D 1 H-NMR (Fig. 3A). 2D-NMR spectroscopy facilitated the identification of the overreduction of NMN’s nicotinamide ring with two and three hydrogenation sites, so 1,4,6-NMNH 3 ( 2c ), and 1,2,4,6-NMNH 5 ( 2d ) respectively (Fig. 3A, B and D). While the fully reduced species 2d formed quickly and its concentration remained constant over time, the concentration of twice reduced 2c increased with once reduced 1,4-NMNH decreasing. This indicates that not all reductions are a step-wise process (s. Figure 3B), especially in the case of 2d . Under Ar, 2d did not form at all, demonstrating that H 2 is necessary for the full hydrogenation of the nicotinamide ring ( Supplementary Table S7 and Figure S9 ). 2c , however, also formed under Ar, albeit in far lower yields (3%) than under H 2 (25%) after 4h. The yield of 1,4-NMNH was relatively similar in both atmospheres (7% under Ar; 10% under H 2 ). Transferring these observations to environmental conditions suggests that less reducing conditions could be favourable for specific NMN reduction. After 1 h under H 2 , 1,4-NMNH was the main product (Fig. 3A and D). Other side products formed at a comparable rate, rapidly depleting the reagent NMN. Consequently, the production of 1,4-NMNH seems to have stopped after 2 h and subsequently began to decrease in concentration. The concentration of 2c continuously increased over time. Even though the concentration of 1,4-NMNH decreased from 35–9% in 2 h, the total amount of reduced NMN remained relatively stable, exceeding 60%. This suggests that 1,4-NMNH is the first and main product of NMN reduction, which can subsequently undergo further reduction to other species, mainly 2c . We were able to exclude two products commonly found in NAD + reduction, where C2 or C6 of the nicotinamide ring is reduced 26 , 45 , 46 . Reduction products presumably starting with these two one-time reduced products could be excluded ( Supplementary Schemes S8, S25–28 ). In the case of NADH, its 1,2-reduced form is known to be unstable, so it is likely this is the case with 1,2-NMNH as well, leading to its absence in our reaction 47 . In the case of 1,4-NMNH loss over time, several routes exist: i) mainly the further reduction to 2c , ii) 1,4-NMNH becoming hydrolysed at C5 or C6, and iii) 1,4-NMNH engaging in various dimerization reactions with 1,6-NMNH (Diels-Alder type reactions; Supplementary Scheme S5 ). Via Liquid Chromatography Mass Spectroscopy (LC-MS) we were able to exclude such products and confirmed the presence of a hydration product, so an OH - being added to 1,4-NMNH (Fig. 4 B, Supplementary Figures S20–22 ). We were able to assign the hydration product NMNH 2 OH ( 2b ) to a peak in the 1 H-NMR ( Supplementary Figures S18 and S19 ). Both 2b and 2c have several conformational isomers, which result in several peaks within the 1 H-NMR spectrum as indicated in Fig. 4 A. An often reported side-product of NAD reduction (e.g. via cyclic voltammetry) is a 4,4’-linked NAD dimer 48 , which also qualifies as a possible side reaction of NMN reduction. Here, after careful interpretation of our 2D NMRs of the 1 h and 4 h reaction with NMN and the 4h reaction with NAD, we can exclude the presence of such dimers ( Supplementary Figure S14 ; no peak at 40 to 50 ppm in 13 C of a bridging methine corresponding to the linkage). This was also confirmed via LC-MS (no double charged molecules were detected). As these dimers are a direct result of radical-forming 1e - transfers onto NAD 49 , we can draw the conclusion that direct hydride or 2e - transfer is the present mechanism in our reactions. After quantification of all identified species, we can account for at least 70.7% of transformed NMN for all reactions, often more. Unidentified species encountered in lower yields can also stem from the differently reduced versions of the degradation product nicotinamide 50 . It is furthermore possible, as mentioned above, that some NMN was lost due to interaction with the mineral surface. Overall, there is a notable and surprising difference between the reduction profile of the dinucleotide and the mononucleotide, the origin of which will be addressed in the discussion. Different metals, different mechanisms Starting from the observation that during NMN reduction, 1,4-NMNH is a main product decreasing with the length of the reaction, we hypothesized that less efficient catalysts might help to avoid overreduction and thus reduce NMN more specifically than the previously used rather efficient nanoparticular NiFe-alloys. As both nNi and nFe visibly worked less efficiently for NAD + reduction, but Fe promoted reduction in higher yields, we decided to work with the same nanoparticular NiFe and Fe powder (nNiFe, nFe; 1:1 ratio to the cofactor) powder used for NAD + reduction (Fig. 4 A). Reducing the ratio of Fe (nNiFe) also reduces the amount of converted NMN, while keeping the ratio of 1,4-NMNH to side products quite similar. The nFe powder by itself converted far less NMN but also did not promote the formation of three times reduced 2d , while twice reduced 2c is only produced in almost untraceable amounts. The hydration product 2b visible as a peak at 7.35 ppm still accumulated over the 4 h reaction time. We repeated experiments with NMN and H 2 over commercially available Fe and Ni micropowder (µFe, particle size: <150 µm; µNi, particle size: 3–7 µm). This separates the metal dependency from the general reduction efficiency of nanoparticular powders due to a large surface area. The metal-cofactor ratio was 200:1 to guarantee the detection of even low concentration side products. The results (Fig. 4 B) show a remarkable trend to overreduction with µNi, while µFe mostly displays two main products: 1,4-NMNH and 2b , the latter being the hydrolysis product of the former. Comparing the spectra of NMN reduction with µNi only and µFe only with those of nNiFe, the distinct product patterns of each metal becomes apparent. Nickel has long been recognized as a hydrogenation catalyst 51 , – but why does it, when not combined with Fe, only reluctantly reduce NAD (Figs. 2 and 12 ) and yet overreduce NMN, not leaving any traceable amount of single-reduced species? The answer, we suggest, lies again in the structural differences between NAD and NMN. NMN can be more easily absorbed to a hydrogenated Ni surface, possibly over the entirety of its nicotinamide ring ( Supplementary Scheme S9 ). This could also explain the fast formation of its fully reduced product 2d shown in Fig. 4 C. NAD in a staggered formation could only absorb partly on the surface, avoiding overreduction 52 . If so, why does Fe not overreduce NMN as readily as Ni? Here, we can reflect on the mechanisms postulated by us in Pereira et al. 12 , that Fe both serves as a (less effective) hydrogenation catalyst and a strong electron donor, either via direct electron transfer to the nicotinamide cofactor or the formation of nascent hydride groups on its surface. Assuming that Fe predominantly reduces NMN through direct electron transfer, the reduction process prioritizes the species with the most favourable redox potential first—namely, 1,4-NMNH (and 1,4-NADH, in the case of NAD). This hypothesis was substantiated by cyclic voltammetry (CV) measurements, which revealed that 1,4-NMNH exhibits the highest oxidation potential among all the reduction products obtained from NMN ( Supplementary Table S12, Figures S30–33 ). Another possible explanation could be that the Ni catalyst does not alter as much as the Fe surface, meaning there would be a constant supply of hydrides available. For Fe, the previously described association with phosphate from the buffer could block active centres, which further prevent overreduction. While the combination of nickel’s hydrogenation strengths and iron’s electron donation increases the yield of 1,4-NADH immensely compared to Fe or Ni separately (Fig. 2 ), the addition Ni does not increase the directed reduction of NMN to 1,4-NMNH. Competing reactions. The addition of an AMP handle to the functional nicotinamide group could harbour an advantage for specific reduction in a mineral-based environment. To test this hypothesis, we conducted experiments with both NMN and NAD + in the same reaction mixture using µNi and µFe as metal promoters at pH 8.5 to explore the reduction of both cofactors in direct competition (Table 1 ). As controls, we reduced NAD + and NMN separately. For the mixed experiments, both cofactors (12 mM ea.) were combined with 600 mM of metal powder, leading to a 25:1 metal to cofactor ratio. In all cases, the 1,4-NADH concentration exceeded that of 1,4-NMNH (Table 1 ). The results indicate that NAD, while delivering comparable reduction yields for itself in all experiments, seems to have a dampening effect on NMN (over)reduction when both cofactors are in the mixture. Ultimately, reducing NAD and NMN with the help of H 2 and metal catalysts is just one part of these cofactors’ role in the prebiotic path towards the first functioning cells – being able to act as a reductant is equally important. The reduction capability of NMNH and NADH. Investigating the redox potential of both 1,4-NADH and 1,4-NMNH standards via cyclic voltammetry helped comparing their oxidation potential with that of the reaction mixtures of NiFe-assisted reduction of NAD + and NMN with H 2 ( Supplementary Table S12, Figures S30–33 ). In the case of NiFe-assisted NAD + reduction, the resulting mixture shows only the oxidation potential of 1,4-NADH, while in the case of NMN reduction, the oxidation potential of both 1,4-NMNH and that of a second reduced species (most likely the species with the 2nd highest concentration, 2c ) is measured. As the second signal has a lower oxidation potential (meaning is harder to oxidize), the 1,4-NMN species is the most relevant reductant, not only in a biological but also in a prebiotic context 53 – 55 . One could argue that it is possible that the 1,4 position of an overreduced species would show a similar oxidation potential as a single reduced 1,4-species. However, as the oxidation of the latter leads to the aromatization of the nicotinamide ring, this reaction would be energetically favourable. This theoretically also applies to the single-reduced 1,6-NADH, but we could not isolate this side product to test it as we did for 1,4-NADH and 1,4-NMNH in the following. Table 1. Overview of yields of mixtures of NAD + and NMN in comparison to separate reduction. Row 1 shows the quantification of NAD + (n=3) and NMN (n=3) in individual reactions with µNi and µFe, row 2 shows reactions of mixtures of NAD + and NMN (for all n=3). In all experiments, the metal powder concentration lies at 600 mM, so for row 1, the metal to cofactor ratio is 50:1, for row 2 it is 25:1. All yields are calculated per to 12 mM of starting cofactor. Unpaired t-tests were used to evaluate whether the differences in concentration between 1,4-NADH and 1,4-NMNH in the competition experiments are significant: *two-tailed P value = 0.0166, significant difference; ***two-tailed P value = 0.0008, very significant difference. All additional data for these experiments can be found in Supplementary Tables S13–16, Figures S34–37 and Schemes S10–12 . It was recently shown that Fe 3+ ions (among other metal ions and also minerals) can promote the reaction of 1,4-NADH with pyruvate to lactate abiotically ( Supplementary Scheme S13 ) 11 . Here, we used this reaction as a proxy to compare the reducing capabilities of 1,4-NMNH and 1,4-NADH, showing that both molecules can reduce pyruvate to equal amounts under aqueous conditions with Fe 3+ in 17 h at 40°C (pH < 5), based on recently published experiments by Mayer and Moran 11 . These results underline that both 1,4-NMNH and 1,4-NADH are equally good hydride donors and thus that the adenosine nucleotide tail does not – or at least not strongly – influence the efficiency of the catalysed hydride transfer (Fig. 5 ). The conditions used for reduction and for oxidation in this paper diverge – while for reduction slightly alkaline conditions are used, oxidation is conducted under acidic conditions. 1,4-NADH is known to hydrolyse under acidic conditions 56 . We performed qualitative experiments at pH 5.5 over µFe and µNi with both NAD + and NMN ( Supplementary Scheme S14 and S15, Figures S40 and S41 ) to confirm this applies to both nicotinamides in a similar manner. These experiments show the formation of hydrolysis products of the 1,4-species of both cofactors in the case of Ni, while, over Fe, also 1,4-NMNH and 1,4-NADH can be detected probably due to the increase in pH (up to pH 8) during the latter experiments. Although NAD + and NMN will be reduced under acidic conditions, they are hydrolysed quickly. The oxidation of 1,4-NADH and 1,4-NMNH, however, seems to work preferably under acidic conditions 11 . Discussion In this study, we have shown that both nicotinamide mono- and dinucleotide can be reduced under conditions found in serpentinizing systems, i.e. with H 2 gas promoted by Fe and Ni containing minerals. Relative to NMN, the presence of adenosine (in the form of AMP) in NAD + influences the reduction product spectrum associated with the nicotinamide ring. We demonstrated that NMN is much more reactive than NAD + in a time course experiment with NiFe 3 nanopowder. Within 1 h, a lot more NMN is consumed than NAD + in 4 h, under the same experimental conditions. The first and main product of both reactions seems to be 1,4-NADH/NMNH. However, while 1,4-NADH remains stable in solution, 1,4-NMNH quickly undergoes further reduction to form increasingly reduced products. From previous studies 12 , we know that 1,4-NADH is not overreduced and remains stable even when the experimental conditions are more reducing or a higher metal to cofactor ratio is employed. Where does this specificity for 1,4-NADH come from? It is known and well-described that NAD(H) in aqueous solution alternates between a folded (Fig. 6 ) and open conformation 57 – 61 . This could shield the nicotinamide ring from excessive overreduction – and possibly also from side reactions such as hydrolysis. The role of Fe and Ni individually in NMN reduction revealed that Ni tends to generate overreduction products in NMN reduction while Fe promotes the formation of 1,4-NMNH, the second main side-product being the hydrolysis product 2b . However, experiments with less Ni to cofactor ratio (e.g. Table 1 , row 1) reveal that Ni also promotes the formation of 2b , suggesting that the hydrolysis product forms whenever 1,4-NMNH is not overreduced very quickly. The immediate presence of 2d , the fully hydrogenated from of NMN, in Ni-assisted reactions indicates a direct association to the mineral surface that the folded conformation of NAD could likely prevent ( Supplementary Scheme S9 ). Based on these results, one can discuss how environmental conditions such as metal availability could have influenced the prebiotic selection process of redox cofactors. The stabilization of NAD’s functional nicotinamide part by the AMP moiety means that the redox properties of NAD could have been maintained within a broader variety of environmental conditions than without the moiety. We validated this hypothesis further by performing experiments with both NMN and NAD + in the same reaction mixture using µNi and µFe as metal promoters. Here, the concentration of 1,4-NAD always surpasses that of NMN, the latter forming more side products. In addition, NAD decreases NMN (over)reduction, lowering the interaction of NMN with the metal surfaces and metal-cofactor interactions leading to full hydrogenation of the nicotinamide ring (Table 1 ). Cyclic voltammetry experiments showed that the oxidation potential of single-reduced species, while all further side (overreduction) products fall behind. Concerning the single-reduced side product 1,6-NADH, we assume it to have a comparable redox potential as 1,4-NADH, although we cannot account for possible steric hindrances during actual reduction reactions. In a biological context, only oxidation at the 1,4-position of the nicotinamide ring is observed. Prebiotically, a 1,6-species could also be relevant for reduction, but has not yet been reported in an experimental setup. The higher reducing strength of single-reduced nicotinamide species creates a mechanistic bottleneck for the back reaction – both overreduction and hydrolysis products could likely not compete as reducing agents in a prebiotic scenario. Additionally, we have shown here that both 1,4-NMNH and 1,4-NADH act equally well as a hydride source in a non-enzymatic context, using a protocol established by colleagues 11 . These results substantiate how the adenosine moiety can be essential for the targeted reduction of the 1,4-position, as well as for the stability of this specific reduction product. In other words: NAD is functional in a wider variety of environments than NMN, ensuring specific nicotinamide reduction and thus maintaining a steady redox potential in the form of single-reduced NADH. Assuming that redox cofactors present a way to detach hydrides from a mineral surface under certain conditions 29 to expose them to different environmental conditions, a molecular structure stabilizing the optimal reducing form (NAD) would be preferable over one that does not (NMN, Fig. 6 ). So far, NAD reduction and oxidation were not shown to occur under the same conditions. Slightly alkaline conditions promote reduction as the reduced forms of NMN and NAD are prone to hydrolyse under acidic conditions; acidic conditions promote oxidation as 1,4-NADH is known to hydrolyse more efficiently then 56 . Slightly fluctuating conditions are likely necessary to direct oxidation and reduction. As NAD effectively detaches hydrides from mineral surfaces, it enables the transport to other geochemical conditions and thus a separation of conditions for reduction and oxidation. Necessary fluctuations in pH would thereby be a natural mechanism to facilitate the role of organic hydride carriers (Fig. 6 ). Serpentinizing systems exist in both acidic and alkaline conditions, though geologically separated from each other 40 , 41 . Alternating physicochemical conditions on a micro-compartment level within serpentinizing systems have been observed and further hypothesized as driving force for prebiotic reactions 62 – 65 . The real effect of such alternations still needs to be investigated in both laboratory and natural settings – including a variety of different geochemical settings. In summary, our findings suggest a new evolutionary rationale behind the tenacious conservation of the AMP handle in NAD. Its presence reflects a prebiotic functional constraint that mediated specific reduction of the hydride carrier under environmental conditions where H 2 was the electron donor, made accessible via mineral surfaces. If the first nicotinamide-dependent enzymes arose in such an environment, they would have required the adenosine moiety not as a handle, but as an inherent structural property of the NAD cofactor that permitted its function with H 2 as the reductant on metal catalysts. In that sense, adenosine in NAD is not so much a handle as it is an insulator that protected the cofactor from overreduction. These observations of a stabilizing function of adenosine-derived tails could be applied other cofactors such as FAD, CoA or SAM. It seems feasible that also there, the extended structure could have been of merit in a prebiotic setting prior to a biological function 18 , 66 . Materials and Methods Metal preparation. The synthesis of Ni 0 , Fe 0 and Ni-Fe Nanoparticles was carried out as described by Beyazay et al. 38 . Commercially bought Fe 0 (reduced, < 149 µm, Carl Roth, referred to as µFe 0 ) and Ni 0 micropowders (3–7 micron, Thermo Scientific, referred to as µNi 0 ) were treated under 5 bar of H 2 , at 50°C for 16 h before being used. A detailed characterization of these metals can be found in the same publication. Experimental setup with Ni-Fe alloys. Under anaerobic conditions, using a glovebox (JACOMEX), 3 mL of anoxic 0.133 M phosphate buffer solution (PBS; pH 8.5; potassium phosphate monobasic and sodium phosphate dibasic, Sigma-Aldrich, in HPLC-grade water); bubbled with N 2 for 1 h containing 36 or 18 µmol of the organic nicotinamide (Nam) compound NAD + (> 95.0%, TCI) or NMN (100% Uthever, MoleQlar; >98.0%, TCI; Supplementary Figure S42 ) were placed in 5 mL glass vials (beaded rim) with a polytetrafluoroethylene (PTFE)-coated stirring bar. Equimolar amounts (relative to the cofactor) of metal atoms of Fe, NiFe 3 , NiFe, Ni 3 Fe, or Ni nanopowders were added to the bottom of each vial, with the exception of a metal-free control ( Supplementary Table 18 ). Alternatively, experiments with Fe and Ni micropowders had 1.8 or 7.2 mmols of metal and 36 µmol of cofactor (metal-cofactor ratio 50 or 200:1) in 0.5 M PBS (pH 8.5 or pH 5.5). The vials were sealed with a crimp cap with a PTFE-coated membrane. To allow gas exchange between the interior and the exterior of the glass vial, a syringe needle was inserted through the crimp cap membrane before the vials were placed in the high-pressure reactor. Standard high-pressure reaction. After pressurizing the reactor (Berghof Reactor 300) with either 5 bar of Ar gas (99.999%, Air Liquid) or 5 bar of H 2 gas (99.9% Nippon Gases), the reactions were started and regulated by a controlled reactor heating system (Berghof Products + Instruments). Reactions were performed from 1 h to 4 h at 40°C and 400 rpm, in a Berghof Reactor Heating System (BR-HS). Afterward, reactors were depressurized under anaerobic conditions and the samples (metal powders and solution) were transferred to 2 mL Eppendorf tubes and centrifuged for 20 min, at 4°C, and 13,000 rpm (Fresco 17 Microcentrifuge). The supernatants were subjected to different analyses, which are described below. Reduction of pyruvate with Fe 3+ and 1,4-NMNH or 1,4-NADH. These experiments followed the protocol described in Supplementary Tables S15 and S16 of the paper Mayer et al. 2024 11 . An aqueous mixture of 0.1 mL with 0.1 M pyruvate (Pyruvic acid, Carl Roth), 0.2 M 1,4-NADH (95%, Thermo Scientific), and 0.06 M FeCl 3 (98% anhydrous, Grüssing GmbH) reacted overnight at 40°C and 400 rpm (pH < 5). For the removal of metal ions, it was added 0.2 mL of a thiolate/phosphate solution (100 mg NaSH, 100 mg NaOH in 10 mL saturated aqueous Na 3 PO 4 ), and left to settle in the fridge (4°C) for 3 h. Instead of a DMSO standard as used in the referenced protocol, 0.1 mL of a 7mM DSS stock solution was added at the end of the experiment. To reach a certain volume, 0.2 mL of D 2 O were also added before the sample was measured. 1 H-NMR spectra were obtained by an AV III HD 250 MHz Spectrometer with a Double Resonance Broad Band (BBOF) probe head. The same experiment was repeated with 1,4-NMNH (97%, AmBeed) instead of 1,4-NADH. Quantitative proton nuclear magnetic resonance (qNMR) analysis. To monitor reactions, as well as detect and quantify the formation of reduced NADH and side products we established a protocol for quantitative proton nuclear magnetic resonance ( 1 H-NMR) 67 , 68 . The internal standard was a 7 mM solution of sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS, CH 3 peak at 0 ppm; >98.0%, TCI) in deuterium oxide (D 2 O 99.8 atom%D, AcroSeal, Thermo Scientific), mixed 1:6 with the supernatant of our samples. qNMR spectra were obtained on a Bruker AVANCE-NEO 600 MHz spectrometer equipped with a 5 mm iprobe TBO with z-gradient. Thirty-two scans were made for each sample with a relaxation delay of 40 s (600 MHz) and a spectral width from − 3 to 13. Analysis and integration were performed using MestReNova (v.15.0.1). Metal-free controls (ran under the same conditions as the quantified, metal-containing samples) were used as references to the initial amount of NAD/NMN in the sample to account for evaporation and possible degradation under the given pH, temperature and time. The average initial amount of cofactor in the controls was used as t = 0 h and to normalize the reaction yields. Standards. 1 H-NMR standards were prepared with 24 mM of the compound and 1 mM of DSS dissolved in D2O ( Supplementary Figure S43 ). The spectra were obtained by a AV III HD 250 MHz Spectrometer with a BBOF probe head. Product Characterization through 2D-NMR. 2D 1 H-NMR enabled the assignment of peaks for 1,4-NMNH and NMN in accordance with literature and in comparison to the pair NAD/NADH ( Supplementary Tables S19 and S20, Figures S44–58 ) 69 . Reduction products were also characterized through different 2D-NMR correlation spectra ( Supplementary Scheme S4, Figures S11–19 ). 3 mL of sample from a 1h and 4 h reduction of NMN with equimolar amounts of NiFe 3 (5 bar H 2 , 40°C, 400 rpm) were dried using a vacuum concentrator (SpeedVac DNA 130, Savant). The remaining solution and pellet were suspended in 500 µl of D 2 O to increase the concentration of the products and resolution of the NMR spectra. The same procedure was performed for a NAD + sample after a 4 h reaction with equimolar amounts of NiFe 3 (5 bar H 2 , 40°C, 400 rpm). Two-dimensional correlation spectra of 1 H, 1 H DQF-COSY (Double-Quantum Filtered COrrelated SpectroscopY), 1 H, 1 H TOCSY (Total COrrelated SpectroscopY), 1 H, 13 C HMBC (Heteronuclear Multiple Bond Correlation spectroscopy) were recorded with standard pulse programs 70 . Edited HSQC (Heteronuclear Single Quantum Coherence spectroscopy) spectra were recorded using sensitivity improvement with echo/anti-echo gradient selection and multiplicity editing during selection step 6 , 7 . NOESY (Nuclear Overhauser Effect SpectroscopY) spectrum was recorded with mixing time of 1.5 s. Chemical shifts are referenced with sodium salt of trimethylsilylpropanesulfonic acid (DSS). Spectra were obtained with the same instrument as qNMR and compared to a list of possible products. Declarations Supporting Information All Supporting Information (methods, figures showing analytic data, tables, schemes) is comprehensibly presented in a single pdf file. Competing Interest Statement: Disclose any competing interests here. Author Contributions: Conceptualization: M.P. & D.P.H.P. Methodology: M.P. & D.P.H.P. Investigation: D.P.H.P., M.P., Z.S, T.B. Validation: D.P.H.P., Z.S. & X.X. Formal analysis: X.X., J.B., N.P., D.P.H.P. & M.P. Resources: H.T., K.V. Writing Original Draft: M.P. & D.P.H.P. Writing Review & Editing: all authors. Visualization: M.P., D.P.H.P. & X.X. Supervision: M.P. Acknowledgements D.P.H.P and M.P. thank Bill Martin for critical reading and discussions. D.P.H.P and M.P thank Alicia Casitas and team for providing access and help to cyclic voltammetry measurements. X.X. thanks Armin Geyer for discussions and the DFG funding for NMR spectrometer NEO600 (Forschungsgroßgeräte project number 508097909). M.P. thanks the Max Planck Society (MPG) and the International Max Planck Research School ‘Principles of microbial life’ for funding. H.T. thanks MPG, the Volkswagen Foundation (96_742) and Deutsche Forschungsgemeinschaft (TU 315/8 − 1 / TU 315/8 − 3). This project was supported by the European Regional Development Fund (ERDF) and the Recovery Assistance for Cohesion and the Territories of Europe (REACT-EU). Data Availability Statement The data that support the findings of this study are available in the SI Appendix of this article. Original analysis files (LC-MS, NMR) will be provided by the corresponding authors upon reasonable request. References Goldman, A. D. & Kacar, B. Cofactors are remnants of life’s origin and early evolution. Journal of Molecular Evolution 89, 127–133 (2021). Xavier, J. C., Hordijk, W., Kauffman, S., Steel, M. & Martin, W. F. Autocatalytic chemical networks at the origin of metabolism. 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Nature Ecology and Evolution 2, 1019–1024 (2018). Zapata-Pérez, R. et al. Reduced nicotinamide mononucleotide is a new and potent NAD + precursor in mammalian cells and mice. The FASEB Journal 35, e21456 (2021). Berger, S. & Braun, S. 200 and More NMR Experiments: A Practical Course . (WILEY-VCH, Weinheim, 2011). Additional Declarations There is NO Competing Interest. Supplementary Files PereiraSICommChem.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6214903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430879894,"identity":"687cee94-a2ec-4d41-a554-13df2a4991be","order_by":0,"name":"Martina Preiner","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5137-5556","institution":"Max-Planck-Institute for Terrestrial Microbiology","correspondingAuthor":true,"prefix":"","firstName":"Martina","middleName":"","lastName":"Preiner","suffix":""},{"id":430879895,"identity":"c76c2da1-6811-44ca-84e7-3a7f5957385e","order_by":1,"name":"Delfina Henriques Pereira","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Delfina","middleName":"Henriques","lastName":"Pereira","suffix":""},{"id":430879896,"identity":"e4e5345a-6dc9-40ca-9d17-1bf6a1cacd29","order_by":2,"name":"Xiulan Xie","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiulan","middleName":"","lastName":"Xie","suffix":""},{"id":430879897,"identity":"32a58585-c3a4-41a2-8073-ab604578a8ae","order_by":3,"name":"Zainab Subrati","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zainab","middleName":"","lastName":"Subrati","suffix":""},{"id":430879898,"identity":"93d3dd54-1746-42a0-9697-098c29531da2","order_by":4,"name":"Tuğçe Beyazay","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tuğçe","middleName":"","lastName":"Beyazay","suffix":""},{"id":430879899,"identity":"7881a328-a811-45ca-ad95-c9fd723b494f","order_by":5,"name":"Nicole Paczia","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Paczia","suffix":""},{"id":430879900,"identity":"ede2f2a5-6383-4279-b704-bf882a1d6bdf","order_by":6,"name":"Jürgen Belz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jürgen","middleName":"","lastName":"Belz","suffix":""},{"id":430879901,"identity":"c578f529-ad9e-4595-ab62-5d9121fba9f8","order_by":7,"name":"Kerstin Volz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kerstin","middleName":"","lastName":"Volz","suffix":""},{"id":430879902,"identity":"614f8722-68fe-474f-a2e1-80923b13c304","order_by":8,"name":"Harun Tüysüz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Harun","middleName":"","lastName":"Tüysüz","suffix":""}],"badges":[],"createdAt":"2025-03-12 21:15:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6214903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6214903/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-025-01752-3","type":"published","date":"2025-10-30T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79465125,"identity":"6f32566f-3578-42fa-abcf-0e8fe1b45552","added_by":"auto","created_at":"2025-03-28 18:42:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123136,"visible":true,"origin":"","legend":"\u003cp\u003eA selection of central organic cofactors with an adenosine-based handle (blue) connected to a functional part that determines the role of these cofactors in metabolism (black): Electron/hydride transfer, methyl transfer or acetyl-transfer. In grey, the function-associated half of nicotinamide dinucleotide (NAD) is highlighted: nicotinamide mononucleotide (NMN).\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/118ef0c7c74e9b5aa998873a.jpeg"},{"id":79465414,"identity":"f42f47f2-dcad-492f-80fe-4de700d26857","added_by":"auto","created_at":"2025-03-28 18:50:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106636,"visible":true,"origin":"","legend":"\u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e reduction at 40 °C over 4 h with equimolar amounts (normalized to 1 metal atom per NAD molecule) of several Ni-Fe-alloys and 5\u0026nbsp;bar of H\u003csub\u003e2\u003c/sub\u003e (or Ar). A) Segment of the \u003csup\u003e1\u003c/sup\u003eH-NMR spectra where the chemical shift of the hydrogen on the 2\u003csup\u003end\u003c/sup\u003e nicotinamide carbon is visible upon reduction. 1,4-NADH features a characteristic peak at δ = 6.9 ppm and 1,6-NADH at δ = 7.1 ppm (\u003cem\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e\u003c/em\u003e). B) Yield of 1,4-NADH obtained for several metals after 4 h under H\u003csub\u003e2\u003c/sub\u003e and Ar. Reduction under Ar is detected only with minerals whose metal content is ≥ 75% Fe. With 5 bar of H\u003csub\u003e2\u003c/sub\u003e, all metals can facilitate 1,4-NADH synthesis. Mixed alloys are more efficient than the pure metals. All spectra and yields can be found in \u003cem\u003e\u003cstrong\u003eSupplementary Tables S2–3, Figures S1 and S2\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/3ed0ff9231bfb084bc84f54d.jpeg"},{"id":79465126,"identity":"29cf0ea8-c578-465d-b140-ffed06d5c122","added_by":"auto","created_at":"2025-03-28 18:42:02","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198693,"visible":true,"origin":"","legend":"\u003cp\u003eThe reduction of NMN and NAD\u003csup\u003e+\u003c/sup\u003e promoted with equimolar quantities of NiFe\u003csub\u003e3\u003c/sub\u003e nanopowder (normalized to the number of metal atoms) and 5 bar of H\u003csub\u003e2\u003c/sub\u003e (40 °C) was monitored for 4 h (\u003cem\u003e\u003cstrong\u003eSupplementary Schemes S2 and S3\u003c/strong\u003e\u003c/em\u003e). Both reactions were carried out in oxygen-free, aqueous solutions at a pH of 8.5 (0.133 M phosphate buffer). A) \u003csup\u003e1\u003c/sup\u003eH-NMR (600MHz) spectra monitoring NMN reduction over time. Dashed lines mark the peaks used for identifying and quantifying reduced NMN species previously identified via 2D NMR (\u003cem\u003e\u003cstrong\u003eSupplementary Table S8)\u003c/strong\u003e\u003c/em\u003e. Due to the complexity of the mixture not all peaks could be assigned (\u003cem\u003e\u003cstrong\u003eSupplementary Scheme S4 and S5, Figures S11–22\u003c/strong\u003e\u003c/em\u003e) B) Proposed mechanism for NMN reduction with H\u003csub\u003e2\u003c/sub\u003e and Ni-Fe minerals. The reduction site and additional protons are highlighted in red. Full arrows represent proposed reaction mechanisms supported by the data obtained, while the dashed arrow is an additional reaction that could not be entirely excluded. Grey circles indicate the proton providing the NMR signal for quantification. In bold font numeral assignments for NMN reduction products are made. C) Time course of NAD\u003csup\u003e+\u003c/sup\u003e reduction (\u003cem\u003e\u003cstrong\u003eSupplementary Schemes S6\u003c/strong\u003e\u003c/em\u003e). Reduced NAD species are plotted as relative percentage to a metal-free control sample (SI Methods) – all time points represent the mean and SD of at least triplicates of the same reaction. The ring chart represents the distribution of products after 4\u0026nbsp;h, percentage in the center indicates the entirety of assigned products. D) Time course of NMN reduction (\u003cem\u003e\u003cstrong\u003eSupplementary Schemes S7\u003c/strong\u003e\u003c/em\u003e). Reduced NMN species are plotted as relative percentage to a metal-free control sample – all time points represent the mean and SD of at least n=3 of the same reaction. The ring chart represents the distribution of products after 4\u0026nbsp;h, percentage in the center indicates the entirety of assigned products. Yields shown in C) and D) are also listed in detail in \u003cem\u003e\u003cstrong\u003eSupplementary Tables S9 and S10, Supplementary Figures S23 and\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/ba4905067577b357436b1348.jpeg"},{"id":79465129,"identity":"94388d9f-226c-461d-9e84-e54150ec97e8","added_by":"auto","created_at":"2025-03-28 18:42:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93060,"visible":true,"origin":"","legend":"\u003cp\u003eSelectivity and efficiency of NMN reduction with H\u003csub\u003e2\u003c/sub\u003e gas is influenced by the type of metal prevalent. (A) Product spectra of NMN reduction with decreasing Ni ratio. The proportion of overreduction products decreases abruptly when no Ni is used (nFe), but so does the overall NMN conversion (s. also \u003cem\u003e\u003cstrong\u003eSupplementary Table S6 and Figure S8\u003c/strong\u003e\u003c/em\u003e). (B) The reduction of NMN assisted by nNiFe alloys (cofactor ratio 1:1) yields products that directly correlate with the products obtained when utilizing the metals separately in micropowder form (metal-cofactor ratio 200:1). µNi\u003csup\u003e0\u003c/sup\u003e promotes the accumulation of \u003cstrong\u003e2c\u003c/strong\u003e and \u003cstrong\u003e2d\u003c/strong\u003e (purple) and other side products. 1,4-NMNH (grey) seems to not react further when using µFe\u003csup\u003e0\u003c/sup\u003e, accumulating over time, but hydrolyse in parts further to \u003cstrong\u003e2b\u003c/strong\u003e. Note that this comparison is a qualitative one as it would not be appropriate to compare the yields between nano- and micropowder. Yields for micropowder reactions shown in this figure are listed in \u003cem\u003e\u003cstrong\u003eSupplementary Table S11\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/88ebdf486a832be72d8d1ba5.jpg"},{"id":79465130,"identity":"fa8ea032-a40f-454a-b81f-060f75349f19","added_by":"auto","created_at":"2025-03-28 18:42:02","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":57059,"visible":true,"origin":"","legend":"\u003cp\u003eReduction of pyruvate to lactate with 1,4-NMNH (n=4) and 1,4-NADH (n=4).\u003cstrong\u003e \u003c/strong\u003eThe method of the reaction was performed according to \u003csup\u003e11 \u003c/sup\u003ewith the help of Fe(III) chloride as a catalyst in 17\u0026nbsp;h. Both nicotinamides perform equally well in this reaction. In both cases, lactate forms in comparable amounts (for original NMR spectra and quantification s. \u003cem\u003e\u003cstrong\u003eSupplementary Table S17 and Figures S38 and S39\u003c/strong\u003e\u003c/em\u003e). Unpaired t-test\u003csup\u003e \u003c/sup\u003e(two-tailed P value = 0.1303) shows the difference between NADH and NMNH-dependent pyruvate reduction to be not significant (ns).\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/e08384a1dbd7baf5c3bd6e58.jpeg"},{"id":79465134,"identity":"92b3ffeb-a032-4808-9e94-18d9b109445d","added_by":"auto","created_at":"2025-03-28 18:42:02","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":158688,"visible":true,"origin":"","legend":"\u003cp\u003eSummarizing figure showing all educts and products detected during simultaneous reduction of NAD\u003csup\u003e+\u003c/sup\u003e and NMN. NMN produces more side products than NAD, possibly creating a mechanistic bottleneck for the back reaction, for which only one-time reduced nicotinamides have a comparable reducing strength – both overreduction products such as NMNH\u003csub\u003e3\u003c/sub\u003e and hydrolysis products such as NMNH\u003csub\u003e2\u003c/sub\u003eOH could likely not compete as reducing agents in a prebiotic scenario. Consequently, only single-reduced species such 1,4-NADH and 1,4-NMNH could be coupled for reduction reactions, thus detaching the reduction from a mineral surface. Based on assessments of the redox potential, we assume 1,6-NADH being able to reduce equally well as 1,4-NADH – not accounting for possible steric hindrances.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/e320cabb6b7f1ffca4422e99.jpeg"},{"id":94830121,"identity":"2bd1ffd2-d781-4c9b-817b-09065f1b4aa2","added_by":"auto","created_at":"2025-10-31 07:14:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2253597,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/e5c22273-7b56-4e7a-9d64-743be5b84111.pdf"},{"id":79465907,"identity":"5d81bc32-03bf-4856-9442-dd0a41ab4c88","added_by":"auto","created_at":"2025-03-28 19:06:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6059055,"visible":true,"origin":"","legend":"","description":"","filename":"PereiraSICommChem.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6214903/v1/a97ba86fdc4bbcbba84ff2ba.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Reduction of NAD and NMN on mineral surfaces with H2 reveals a functional role for the adenosine moiety in prebiotic evolution","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003c/p\u003e \u003cp\u003eIn many theories for the origin of metabolism, organic cofactors such as NAD\u003csup\u003e+\u003c/sup\u003e, flavin adenine dinucleotide (FAD), S-adenosyl methionine (SAM) and coenzyme A (CoA) are seen as crucial intermediates in the transition from inorganic to enzymatic catalysis\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These cofactors catalyse ancient biochemical reactions with a wide variety of substrates, are often active in the absence of enzymes\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and share a \u0026lsquo;handle\u0026rsquo; consisting of the nucleoside adenosine, a central component of nucleic acids such as RNA. The catalytic and nucleoside moieties of cofactors like NAD\u003csup\u003e+\u003c/sup\u003e thus unite \u0026ldquo;metabolism first\u0026rdquo; and \u0026ldquo;information first\u0026rdquo; views for the origin of life\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e in a single molecule (Fig.\u0026nbsp;1). In all enzymatic reactions known to date, the adenosine moiety is catalytically inert, in non-enzymatic reactions its phosphate groups have been shown to enabling association to metal ions\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSome cofactors that do not possess the moiety, such as pterins or folates, nonetheless derive from nucleotides in their biosynthesis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, indicating close connections between cofactor catalysis and RNA bases in early biochemical evolution. The evolutionary rationale behind the conserved presence of an adenosine moiety to NAD\u003csup\u003e+\u003c/sup\u003e is traditionally viewed either functionally, in terms of a biochemical handle that allows enzymes to bind the cofactor more efficiently\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, or historically also as a holdover from an earlier phase of evolution in which RNA preceded enzymes and cofactors were active in ribozymes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. But there is, however, a third possibility. NAD\u003csup\u003e+\u003c/sup\u003e functions exclusively as a redox cofactor and neither traditional view takes into account the environmental source of electrons required to generate NADH or reduced substrates for NAD\u003csup\u003e+\u003c/sup\u003e dependent reactions.\u003c/p\u003e \u003cp\u003eNAD is an ancient redox cofactor that is essential in metabolism and traces back to the last universal common ancestor, LUCA\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Several prebiotic routes of NAD synthesis have been proposed, including via mineral-assisted synthesis under hydrothermal conditions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e or via nitrile-dependent syntheses\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Recent work has shown how NAD\u003csup\u003e+\u003c/sup\u003e can be non-enzymatically reduced under a variety of conditions\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, the reducing abilities of NADH have been demonstrated both with and without metal ions as catalysts\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhat is a good possible primary electron source and thus geochemical setting? In this study, we are investigating the abilities of water-rock-interaction systems, where protons of water are being reduced to hydrogen (H\u003csub\u003e2\u003c/sub\u003e) gas by electrons of Fe(II) containing minerals (serpentinizing systems). Previous studies have shown that NAD\u003csup\u003e+\u003c/sup\u003e readily reacts with H\u003csub\u003e2\u003c/sub\u003e and metal powders (Ni, Co, Fe) to specifically form the biologically relevant form of reduced NAD (1,4-NADH)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. An independent study also reported NAD\u003csup\u003e+\u003c/sup\u003e reduction without H\u003csub\u003e2\u003c/sub\u003e, using iron sulfides as reductant at lower yields\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe conditions of serpentinizing systems not only reduce NAD\u003csup\u003e+\u003c/sup\u003e but also carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) with the electrons provided by H\u003csub\u003e2\u003c/sub\u003e \u003csup\u003e20,21,28,29\u003c/sup\u003e. They are rich in Fe and, depending on the system, Ni as well\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, just like enzymes of the acetyl CoA pathway, which H\u003csub\u003e2\u003c/sub\u003e dependent anaerobic autotrophs use to reduce CO\u003csub\u003e2\u003c/sub\u003e in their carbon and energy metabolism\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Hotter systems tend to feature Ni-Fe-alloys with higher nickel composition, while cooler ones are richer in iron \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Nickel-containing intermetallic compounds such as awaruite (Ni\u003csub\u003e3\u003c/sub\u003eFe) or taenite (NiFe\u003csub\u003e3\u003c/sub\u003e to Ni\u003csub\u003e2\u003c/sub\u003eFe) are products of the reaction of H\u003csub\u003e2\u003c/sub\u003e with Ni(II) compounds in serpentinizing systems\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and are also found in meteorites\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Native metals occur naturally in these highly reducing systems\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Here, we investigate the ability of naturally occurring Ni- and Fe-containing allows to replace enzymes for H\u003csub\u003e2\u003c/sub\u003e dependent NAD\u003csup\u003e+\u003c/sup\u003e reduction. We then compare NAD to its AMP-lacking homologue NMN to test how the AMP moiety impacts the nature of products obtained from non-enzymatic (prebiotic) NAD\u003csup\u003e+\u003c/sup\u003e reduction with H\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eScreening naturally occurring iron and nickel containing minerals.\u003c/b\u003e Ni-Fe containing minerals found in hydrothermal settings were tested for the reduction of NAD\u003csup\u003e+\u003c/sup\u003e under conditions comparable to those found in mild serpentinizing hydrothermal systems (40\u0026deg;C, 0.133 M phosphate buffer pH 8.5, 5 bar H\u003csub\u003e2\u003c/sub\u003e, \u003cb\u003eSupplementary Scheme S1\u003c/b\u003e). These nanoparticular mineral powders were synthesized via the nano-casting method by using tea leaves as a template and were previously characterized\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The metal content in these reactions is equivalent to that of the cofactor (1 metal atom per cofactor). The resulting H\u003csub\u003e2\u003c/sub\u003e concentration at our conditions is 3.6 mM (using Henry\u0026rsquo;s law, s. \u003cb\u003eSupplementary Equations S1\u0026ndash;3\u003c/b\u003e and references\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e), which is comparable to the H\u003csub\u003e2\u003c/sub\u003e concentrations found in the effluent of serpentinizing systems\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The buffer was bubbled with N\u003csub\u003e2\u003c/sub\u003e for 1 h and handled inside a glove box to approximate the anoxic conditions on early Earth. Several controls were implemented, including controls without metal and H\u003csub\u003e2\u003c/sub\u003e, respectively. The liquid phase was analysed by \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 4 h under H\u003csub\u003e2\u003c/sub\u003e, 1,4- and 1,6-NADH formed in all samples at different yields, the reaction with nanoparticular NiFe\u003csub\u003e3\u003c/sub\u003e (nNiFe\u003csub\u003e3\u003c/sub\u003e) yielding the most NADH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Control experiments starting from 1,4-NADH showed that 1,6-NADH is very likely a product of rearrangement from 1,4-NADH \u0026ndash; with only a marginal influence of the used metals (\u003cb\u003eSupplementary Scheme S2, Tables S4 and S5, Figure S3\u003c/b\u003e). Samples under Ar also produced NADH with a Fe-rich minerals (nNiFe\u003csub\u003e3\u003c/sub\u003e, nFe). In addition to transferring hydrides from H\u003csub\u003e2\u003c/sub\u003e to NAD\u003csup\u003e+\u003c/sup\u003e, iron can oxidize, donating its own electrons either by producing nascent H\u003csub\u003e2\u003c/sub\u003e gas, ultimately reducing NAD\u003csup\u003e+\u003c/sup\u003e or by direct electron transfer to NAD\u003csup\u003e+\u003c/sup\u003e. This process can also be used as a proxy for the constant H\u003csub\u003e2\u003c/sub\u003e-production in serpentinizing systems\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Ni by itself is H\u003csub\u003e2\u003c/sub\u003e-dependent in the promotion of NAD\u003csup\u003e+\u003c/sup\u003e reduction.\u003c/p\u003e \u003cp\u003eScanning transmission electron microscopy (STEM) imaging before and after the reactions (the latter including a washing and dilution step to assure true surface alteration) confirms that Fe, both under Ar and H\u003csub\u003e2\u003c/sub\u003e, gets associated with phosphate ions in ratio that suggests the formation of iron phosphate. Ni does not associate with phosphate, suggesting it stays in its native form (\u003cb\u003eSupplementary Methods and Figures S4\u0026ndash;7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn Ni-rich minerals, the Fe is expected to slowly reduce NAD under Ar conditions. However, it is likely that the resulting products do not reach the detection threshold within 4 h. Overall, bimetallic minerals are significantly more efficient than the individual transition metals when hydrogen is available. Introducing one Ni atom to a Fe atom increases the yield by 300% (nNiFe vs. nFe). Their properties, already observed in a previous study\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, seem to complement each other for the reduction of NAD\u003csup\u003e+\u003c/sup\u003e with H\u003csub\u003e2\u003c/sub\u003e: Fe being mostly an electron donor, while Ni promotes hydride transfer from H\u003csub\u003e2\u003c/sub\u003e. These complementary roles have also been described in other publications, suggesting charge transfers from Fe (more electropositive) to Ni could increase the electron density in Ni \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe \u0026ldquo;universal\u0026rdquo; adenine nucleotide in organic cofactors.\u003c/b\u003e Many central cofactors share an AMP \u0026ldquo;handle\u0026rdquo; (Fig.\u0026nbsp;1) attached to the catalytically active moiety. In the case of NAD, NMN is the hydride-transferring nicotinamide, AMP is inert. NAD is stable in water, with its pH range depending on the reduction state of the nicotinamide: NADH is more stable at pH\u0026thinsp;\u0026gt;\u0026thinsp;7, while NAD\u003csup\u003e+\u003c/sup\u003e is more stable under acidic conditions\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. To investigate the role of the AMP-tail in a prebiotic context, several experiments were designed to compare NAD and NMN. We initially focussed on nNiFe\u003csub\u003e3\u003c/sub\u003e, the most efficient of the Ni-Fe minerals in the above described NAD experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All other reaction conditions (buffer, pH, temperature, metal to cofactor ratio) were maintained (\u003cb\u003eSupplementary Scheme S3\u003c/b\u003e). Products were quantified via \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR spectroscopy.\u003c/p\u003e \u003cp\u003eWithout metals, NMN does not react and remained stable (\u003cb\u003eSupplementary Figures S8 and S9\u003c/b\u003e). Under Ar, NMN still got reduced due to the abundant iron in the mineral compound, but more slowly than under H\u003csub\u003e2\u003c/sub\u003e (\u003cb\u003eSupplementary Tables S6 and S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn additional to the 2 h experiment with NAD\u003csup\u003e+\u003c/sup\u003e under H\u003csub\u003e2\u003c/sub\u003e showed the increase of 1,4- and 1,6-NADH to be steady and inversely proportional to the decrease of NAD\u003csup\u003e+\u003c/sup\u003e in solution (Fig.\u0026nbsp;3C). After 4 h with nNiFe\u003csub\u003e3\u003c/sub\u003e, on average 57% of NAD\u003csup\u003e+\u003c/sup\u003e was reduced with 26% remaining oxidized. The remaining 17% can in part be attributed to nicotinamide formation but also unassigned degradation reactions and loss via surface absorption \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNMN, however, shows a completely different reaction profile (Fig.\u0026nbsp;3D). After only 1 h, 69% of the starting NMN had been converted, and a variety of products was observed in 1D \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR (Fig.\u0026nbsp;3A). 2D-NMR spectroscopy facilitated the identification of the overreduction of NMN\u0026rsquo;s nicotinamide ring with two and three hydrogenation sites, so 1,4,6-NMNH\u003csub\u003e3\u003c/sub\u003e (\u003cb\u003e2c\u003c/b\u003e), and 1,2,4,6-NMNH\u003csub\u003e5\u003c/sub\u003e (\u003cb\u003e2d\u003c/b\u003e) respectively (Fig.\u0026nbsp;3A, B and D). While the fully reduced species \u003cb\u003e2d\u003c/b\u003e formed quickly and its concentration remained constant over time, the concentration of twice reduced \u003cb\u003e2c\u003c/b\u003e increased with once reduced 1,4-NMNH decreasing. This indicates that not all reductions are a step-wise process (s. Figure\u0026nbsp;3B), especially in the case of \u003cb\u003e2d\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eUnder Ar, \u003cb\u003e2d\u003c/b\u003e did not form at all, demonstrating that H\u003csub\u003e2\u003c/sub\u003e is necessary for the full hydrogenation of the nicotinamide ring (\u003cb\u003eSupplementary Table S7 and Figure S9\u003c/b\u003e). \u003cb\u003e2c\u003c/b\u003e, however, also formed under Ar, albeit in far lower yields (3%) than under H\u003csub\u003e2\u003c/sub\u003e (25%) after 4h. The yield of 1,4-NMNH was relatively similar in both atmospheres (7% under Ar; 10% under H\u003csub\u003e2\u003c/sub\u003e). Transferring these observations to environmental conditions suggests that less reducing conditions could be favourable for specific NMN reduction.\u003c/p\u003e \u003cp\u003eAfter 1 h under H\u003csub\u003e2\u003c/sub\u003e, 1,4-NMNH was the main product (Fig.\u0026nbsp;3A and D). Other side products formed at a comparable rate, rapidly depleting the reagent NMN. Consequently, the production of 1,4-NMNH seems to have stopped after 2 h and subsequently began to decrease in concentration. The concentration of \u003cb\u003e2c\u003c/b\u003e continuously increased over time. Even though the concentration of 1,4-NMNH decreased from 35\u0026ndash;9% in 2 h, the total amount of reduced NMN remained relatively stable, exceeding 60%. This suggests that 1,4-NMNH is the first and main product of NMN reduction, which can subsequently undergo further reduction to other species, mainly \u003cb\u003e2c\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe were able to exclude two products commonly found in NAD\u003csup\u003e+\u003c/sup\u003e reduction, where C2 or C6 of the nicotinamide ring is reduced\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Reduction products presumably starting with these two one-time reduced products could be excluded (\u003cb\u003eSupplementary Schemes S8, S25\u0026ndash;28\u003c/b\u003e). In the case of NADH, its 1,2-reduced form is known to be unstable, so it is likely this is the case with 1,2-NMNH as well, leading to its absence in our reaction\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the case of 1,4-NMNH loss over time, several routes exist: i) mainly the further reduction to \u003cb\u003e2c\u003c/b\u003e, ii) 1,4-NMNH becoming hydrolysed at C5 or C6, and iii) 1,4-NMNH engaging in various dimerization reactions with 1,6-NMNH (Diels-Alder type reactions; \u003cb\u003eSupplementary Scheme S5\u003c/b\u003e). Via Liquid Chromatography Mass Spectroscopy (LC-MS) we were able to exclude such products and confirmed the presence of a hydration product, so an OH\u003csup\u003e-\u003c/sup\u003e being added to 1,4-NMNH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cb\u003eSupplementary Figures S20\u0026ndash;22\u003c/b\u003e). We were able to assign the hydration product NMNH\u003csub\u003e2\u003c/sub\u003eOH (\u003cb\u003e2b\u003c/b\u003e) to a peak in the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR (\u003cb\u003eSupplementary Figures S18 and S19\u003c/b\u003e). Both \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e2c\u003c/b\u003e have several conformational isomers, which result in several peaks within the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR spectrum as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eAn often reported side-product of NAD reduction (e.g. via cyclic voltammetry) is a 4,4\u0026rsquo;-linked NAD dimer\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, which also qualifies as a possible side reaction of NMN reduction. Here, after careful interpretation of our 2D NMRs of the 1 h and 4 h reaction with NMN and the 4h reaction with NAD, we can exclude the presence of such dimers (\u003cb\u003eSupplementary Figure S14\u003c/b\u003e; no peak at 40 to 50 ppm in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC of a bridging methine corresponding to the linkage). This was also confirmed via LC-MS (no double charged molecules were detected). As these dimers are a direct result of radical-forming 1e\u003csup\u003e-\u003c/sup\u003e transfers onto NAD\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, we can draw the conclusion that direct hydride or 2e\u003csup\u003e-\u003c/sup\u003e transfer is the present mechanism in our reactions.\u003c/p\u003e \u003cp\u003eAfter quantification of all identified species, we can account for at least 70.7% of transformed NMN for all reactions, often more. Unidentified species encountered in lower yields can also stem from the differently reduced versions of the degradation product nicotinamide\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. It is furthermore possible, as mentioned above, that some NMN was lost due to interaction with the mineral surface. Overall, there is a notable and surprising difference between the reduction profile of the dinucleotide and the mononucleotide, the origin of which will be addressed in the discussion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferent metals, different mechanisms\u003c/b\u003e Starting from the observation that during NMN reduction, 1,4-NMNH is a main product decreasing with the length of the reaction, we hypothesized that less efficient catalysts might help to avoid overreduction and thus reduce NMN more specifically than the previously used rather efficient nanoparticular NiFe-alloys. As both nNi and nFe visibly worked less efficiently for NAD\u003csup\u003e+\u003c/sup\u003e reduction, but Fe promoted reduction in higher yields, we decided to work with the same nanoparticular NiFe and Fe powder (nNiFe, nFe; 1:1 ratio to the cofactor) powder used for NAD\u003csup\u003e+\u003c/sup\u003e reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Reducing the ratio of Fe (nNiFe) also reduces the amount of converted NMN, while keeping the ratio of 1,4-NMNH to side products quite similar. The nFe powder by itself converted far less NMN but also did not promote the formation of three times reduced \u003cb\u003e2d\u003c/b\u003e, while twice reduced \u003cb\u003e2c\u003c/b\u003e is only produced in almost untraceable amounts. The hydration product \u003cb\u003e2b\u003c/b\u003e visible as a peak at 7.35 ppm still accumulated over the 4 h reaction time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe repeated experiments with NMN and H\u003csub\u003e2\u003c/sub\u003e over commercially available Fe and Ni micropowder (\u0026micro;Fe, particle size: \u0026lt;150 \u0026micro;m; \u0026micro;Ni, particle size: 3\u0026ndash;7 \u0026micro;m). This separates the metal dependency from the general reduction efficiency of nanoparticular powders due to a large surface area. The metal-cofactor ratio was 200:1 to guarantee the detection of even low concentration side products. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) show a remarkable trend to overreduction with \u0026micro;Ni, while \u0026micro;Fe mostly displays two main products: 1,4-NMNH and \u003cb\u003e2b\u003c/b\u003e, the latter being the hydrolysis product of the former. Comparing the spectra of NMN reduction with \u0026micro;Ni only and \u0026micro;Fe only with those of nNiFe, the distinct product patterns of each metal becomes apparent.\u003c/p\u003e \u003cp\u003eNickel has long been recognized as a hydrogenation catalyst\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, \u0026ndash; but why does it, when not combined with Fe, only reluctantly reduce NAD (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e) and yet overreduce NMN, not leaving any traceable amount of single-reduced species? The answer, we suggest, lies again in the structural differences between NAD and NMN. NMN can be more easily absorbed to a hydrogenated Ni surface, possibly over the entirety of its nicotinamide ring (\u003cb\u003eSupplementary Scheme S9\u003c/b\u003e). This could also explain the fast formation of its fully reduced product \u003cb\u003e2d\u003c/b\u003e shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. NAD in a staggered formation could only absorb partly on the surface, avoiding overreduction\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIf so, why does Fe not overreduce NMN as readily as Ni? Here, we can reflect on the mechanisms postulated by us in Pereira et al.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, that Fe both serves as a (less effective) hydrogenation catalyst and a strong electron donor, either via direct electron transfer to the nicotinamide cofactor or the formation of nascent hydride groups on its surface.\u003c/p\u003e \u003cp\u003eAssuming that Fe predominantly reduces NMN through direct electron transfer, the reduction process prioritizes the species with the most favourable redox potential first\u0026mdash;namely, 1,4-NMNH (and 1,4-NADH, in the case of NAD). This hypothesis was substantiated by cyclic voltammetry (CV) measurements, which revealed that 1,4-NMNH exhibits the highest oxidation potential among all the reduction products obtained from NMN (\u003cb\u003eSupplementary Table S12, Figures S30\u0026ndash;33\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAnother possible explanation could be that the Ni catalyst does not alter as much as the Fe surface, meaning there would be a constant supply of hydrides available. For Fe, the previously described association with phosphate from the buffer could block active centres, which further prevent overreduction.\u003c/p\u003e \u003cp\u003eWhile the combination of nickel\u0026rsquo;s hydrogenation strengths and iron\u0026rsquo;s electron donation increases the yield of 1,4-NADH immensely compared to Fe or Ni separately (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the addition Ni does not increase the directed reduction of NMN to 1,4-NMNH.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompeting reactions.\u003c/b\u003e The addition of an AMP handle to the functional nicotinamide group could harbour an advantage for specific reduction in a mineral-based environment. To test this hypothesis, we conducted experiments with both NMN and NAD\u003csup\u003e+\u003c/sup\u003e in the same reaction mixture using \u0026micro;Ni and \u0026micro;Fe as metal promoters at pH 8.5 to explore the reduction of both cofactors in direct competition (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As controls, we reduced NAD\u003csup\u003e+\u003c/sup\u003e and NMN separately. For the mixed experiments, both cofactors (12 mM ea.) were combined with 600 mM of metal powder, leading to a 25:1 metal to cofactor ratio. In all cases, the 1,4-NADH concentration exceeded that of 1,4-NMNH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results indicate that NAD, while delivering comparable reduction yields for itself in all experiments, seems to have a dampening effect on NMN (over)reduction when both cofactors are in the mixture.\u003c/p\u003e \u003cp\u003eUltimately, reducing NAD and NMN with the help of H\u003csub\u003e2\u003c/sub\u003e and metal catalysts is just one part of these cofactors\u0026rsquo; role in the prebiotic path towards the first functioning cells \u0026ndash; being able to act as a reductant is equally important.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe reduction capability of NMNH and NADH.\u003c/b\u003e Investigating the redox potential of both 1,4-NADH and 1,4-NMNH standards via cyclic voltammetry helped comparing their oxidation potential with that of the reaction mixtures of NiFe-assisted reduction of NAD\u003csup\u003e+\u003c/sup\u003e and NMN with H\u003csub\u003e2\u003c/sub\u003e (\u003cb\u003eSupplementary Table S12, Figures S30\u0026ndash;33\u003c/b\u003e). In the case of NiFe-assisted NAD\u003csup\u003e+\u003c/sup\u003e reduction, the resulting mixture shows only the oxidation potential of 1,4-NADH, while in the case of NMN reduction, the oxidation potential of both 1,4-NMNH and that of a second reduced species (most likely the species with the 2nd highest concentration, \u003cb\u003e2c\u003c/b\u003e) is measured. As the second signal has a lower oxidation potential (meaning is harder to oxidize), the 1,4-NMN species is the most relevant reductant, not only in a biological but also in a prebiotic context\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. One could argue that it is possible that the 1,4 position of an overreduced species would show a similar oxidation potential as a single reduced 1,4-species. However, as the oxidation of the latter leads to the aromatization of the nicotinamide ring, this reaction would be energetically favourable. This theoretically also applies to the single-reduced 1,6-NADH, but we could not isolate this side product to test it as we did for 1,4-NADH and 1,4-NMNH in the following.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eOverview of yields of mixtures of NAD\u003csup\u003e+\u003c/sup\u003e and NMN in comparison to separate reduction. Row 1 shows the quantification of NAD\u003csup\u003e+\u003c/sup\u003e (n=3) and NMN (n=3) in individual reactions with \u0026micro;Ni and \u0026micro;Fe, row 2 shows reactions of mixtures of NAD\u003csup\u003e+\u003c/sup\u003e and NMN (for all n=3). In all experiments, the metal powder concentration lies at 600 mM, so for row 1, the metal to cofactor ratio is 50:1, for row 2 it is 25:1. All yields are calculated per to 12 mM of starting cofactor. Unpaired t-tests were used to evaluate whether the differences in concentration between 1,4-NADH and 1,4-NMNH in the competition experiments are significant: *two-tailed P value = 0.0166, significant difference; ***two-tailed P value = 0.0008, very significant difference. All additional data for these experiments can be found in \u003cstrong\u003e\u003cem\u003eSupplementary Tables S13\u0026ndash;16, Figures S34\u0026ndash;37 and Schemes S10\u0026ndash;12\u003c/em\u003e.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e \u003cp\u003eIt was recently shown that Fe\u003csup\u003e3+\u003c/sup\u003e ions (among other metal ions and also minerals) can promote the reaction of 1,4-NADH with pyruvate to lactate abiotically (\u003cb\u003eSupplementary Scheme S13\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Here, we used this reaction as a proxy to compare the reducing capabilities of 1,4-NMNH and 1,4-NADH, showing that both molecules can reduce pyruvate to equal amounts under aqueous conditions with Fe\u003csup\u003e3+\u003c/sup\u003e in 17 h at 40\u0026deg;C (pH\u0026thinsp;\u0026lt;\u0026thinsp;5), based on recently published experiments by Mayer and Moran\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These results underline that both 1,4-NMNH and 1,4-NADH are equally good hydride donors and thus that the adenosine nucleotide tail does not \u0026ndash; or at least not strongly \u0026ndash; influence the efficiency of the catalysed hydride transfer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe conditions used for reduction and for oxidation in this paper diverge \u0026ndash; while for reduction slightly alkaline conditions are used, oxidation is conducted under acidic conditions. 1,4-NADH is known to hydrolyse under acidic conditions\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We performed qualitative experiments at pH 5.5 over \u0026micro;Fe and \u0026micro;Ni with both NAD\u003csup\u003e+\u003c/sup\u003e and NMN (\u003cb\u003eSupplementary Scheme S14 and S15, Figures S40 and S41\u003c/b\u003e) to confirm this applies to both nicotinamides in a similar manner. These experiments show the formation of hydrolysis products of the 1,4-species of both cofactors in the case of Ni, while, over Fe, also 1,4-NMNH and 1,4-NADH can be detected probably due to the increase in pH (up to pH 8) during the latter experiments. Although NAD\u003csup\u003e+\u003c/sup\u003e and NMN will be reduced under acidic conditions, they are hydrolysed quickly. The oxidation of 1,4-NADH and 1,4-NMNH, however, seems to work preferably under acidic conditions\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we have shown that both nicotinamide mono- and dinucleotide can be reduced under conditions found in serpentinizing systems, i.e. with H\u003csub\u003e2\u003c/sub\u003e gas promoted by Fe and Ni containing minerals. Relative to NMN, the presence of adenosine (in the form of AMP) in NAD\u003csup\u003e+\u003c/sup\u003e influences the reduction product spectrum associated with the nicotinamide ring. We demonstrated that NMN is much more reactive than NAD\u003csup\u003e+\u003c/sup\u003e in a time course experiment with NiFe\u003csub\u003e3\u003c/sub\u003e nanopowder. Within 1 h, a lot more NMN is consumed than NAD\u003csup\u003e+\u003c/sup\u003e in 4 h, under the same experimental conditions. The first and main product of both reactions seems to be 1,4-NADH/NMNH. However, while 1,4-NADH remains stable in solution, 1,4-NMNH quickly undergoes further reduction to form increasingly reduced products. From previous studies\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, we know that 1,4-NADH is not overreduced and remains stable even when the experimental conditions are more reducing or a higher metal to cofactor ratio is employed.\u003c/p\u003e \u003cp\u003eWhere does this specificity for 1,4-NADH come from? It is known and well-described that NAD(H) in aqueous solution alternates between a folded (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e) and open conformation\u003csup\u003e\u003cspan additionalcitationids=\"CR58 CR59 CR60\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. This could shield the nicotinamide ring from excessive overreduction \u0026ndash; and possibly also from side reactions such as hydrolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe role of Fe and Ni individually in NMN reduction revealed that Ni tends to generate overreduction products in NMN reduction while Fe promotes the formation of 1,4-NMNH, the second main side-product being the hydrolysis product \u003cb\u003e2b\u003c/b\u003e. However, experiments with less Ni to cofactor ratio (e.g. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, row 1) reveal that Ni also promotes the formation of \u003cb\u003e2b\u003c/b\u003e, suggesting that the hydrolysis product forms whenever 1,4-NMNH is not overreduced very quickly. The immediate presence of \u003cb\u003e2d\u003c/b\u003e, the fully hydrogenated from of NMN, in Ni-assisted reactions indicates a direct association to the mineral surface that the folded conformation of NAD could likely prevent (\u003cb\u003eSupplementary Scheme S9\u003c/b\u003e). Based on these results, one can discuss how environmental conditions such as metal availability could have influenced the prebiotic selection process of redox cofactors.\u003c/p\u003e \u003cp\u003eThe stabilization of NAD\u0026rsquo;s functional nicotinamide part by the AMP moiety means that the redox properties of NAD could have been maintained within a broader variety of environmental conditions than without the moiety. We validated this hypothesis further by performing experiments with both NMN and NAD\u003csup\u003e+\u003c/sup\u003e in the same reaction mixture using \u0026micro;Ni and \u0026micro;Fe as metal promoters. Here, the concentration of 1,4-NAD always surpasses that of NMN, the latter forming more side products. In addition, NAD decreases NMN (over)reduction, lowering the interaction of NMN with the metal surfaces and metal-cofactor interactions leading to full hydrogenation of the nicotinamide ring (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCyclic voltammetry experiments showed that the oxidation potential of single-reduced species, while all further side (overreduction) products fall behind. Concerning the single-reduced side product 1,6-NADH, we assume it to have a comparable redox potential as 1,4-NADH, although we cannot account for possible steric hindrances during actual reduction reactions. In a biological context, only oxidation at the 1,4-position of the nicotinamide ring is observed. Prebiotically, a 1,6-species could also be relevant for reduction, but has not yet been reported in an experimental setup. The higher reducing strength of single-reduced nicotinamide species creates a mechanistic bottleneck for the back reaction \u0026ndash; both overreduction and hydrolysis products could likely not compete as reducing agents in a prebiotic scenario. Additionally, we have shown here that both 1,4-NMNH and 1,4-NADH act equally well as a hydride source in a non-enzymatic context, using a protocol established by colleagues\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese results substantiate how the adenosine moiety can be essential for the targeted reduction of the 1,4-position, as well as for the stability of this specific reduction product. In other words: NAD is functional in a wider variety of environments than NMN, ensuring specific nicotinamide reduction and thus maintaining a steady redox potential in the form of single-reduced NADH. Assuming that redox cofactors present a way to detach hydrides from a mineral surface under certain conditions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e to expose them to different environmental conditions, a molecular structure stabilizing the optimal reducing form (NAD) would be preferable over one that does not (NMN, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, NAD reduction and oxidation were not shown to occur under the same conditions. Slightly alkaline conditions promote reduction as the reduced forms of NMN and NAD are prone to hydrolyse under acidic conditions; acidic conditions promote oxidation as 1,4-NADH is known to hydrolyse more efficiently then\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Slightly fluctuating conditions are likely necessary to direct oxidation and reduction.\u003c/p\u003e \u003cp\u003eAs NAD effectively detaches hydrides from mineral surfaces, it enables the transport to other geochemical conditions and thus a separation of conditions for reduction and oxidation. Necessary fluctuations in pH would thereby be a natural mechanism to facilitate the role of organic hydride carriers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Serpentinizing systems exist in both acidic and alkaline conditions, though geologically separated from each other\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Alternating physicochemical conditions on a micro-compartment level within serpentinizing systems have been observed and further hypothesized as driving force for prebiotic reactions\u003csup\u003e\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The real effect of such alternations still needs to be investigated in both laboratory and natural settings \u0026ndash; including a variety of different geochemical settings.\u003c/p\u003e \u003cp\u003eIn summary, our findings suggest a new evolutionary rationale behind the tenacious conservation of the AMP handle in NAD. Its presence reflects a prebiotic functional constraint that mediated specific reduction of the hydride carrier under environmental conditions where H\u003csub\u003e2\u003c/sub\u003e was the electron donor, made accessible via mineral surfaces. If the first nicotinamide-dependent enzymes arose in such an environment, they would have required the adenosine moiety not as a handle, but as an inherent structural property of the NAD cofactor that permitted its function with H\u003csub\u003e2\u003c/sub\u003e as the reductant on metal catalysts. In that sense, adenosine in NAD is not so much a handle as it is an insulator that protected the cofactor from overreduction. These observations of a stabilizing function of adenosine-derived tails could be applied other cofactors such as FAD, CoA or SAM. It seems feasible that also there, the extended structure could have been of merit in a prebiotic setting prior to a biological function\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eMetal preparation.\u003c/b\u003e The synthesis of Ni\u003csup\u003e0\u003c/sup\u003e, Fe\u003csup\u003e0\u003c/sup\u003e and Ni-Fe Nanoparticles was carried out as described by Beyazay et al.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Commercially bought Fe\u003csup\u003e0\u003c/sup\u003e (reduced, \u0026lt;\u0026thinsp;149 \u0026micro;m, Carl Roth, referred to as \u0026micro;Fe\u003csup\u003e0\u003c/sup\u003e) and Ni\u003csup\u003e0\u003c/sup\u003e micropowders (3\u0026ndash;7 micron, Thermo Scientific, referred to as \u0026micro;Ni\u003csup\u003e0\u003c/sup\u003e) were treated under 5 bar of H\u003csub\u003e2\u003c/sub\u003e, at 50\u0026deg;C for 16 h before being used. A detailed characterization of these metals can be found in the same publication.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental setup with Ni-Fe alloys.\u003c/b\u003e Under anaerobic conditions, using a glovebox (JACOMEX), 3 mL of anoxic 0.133 M phosphate buffer solution (PBS; pH 8.5; potassium phosphate monobasic and sodium phosphate dibasic, Sigma-Aldrich, in HPLC-grade water); bubbled with N\u003csub\u003e2\u003c/sub\u003e for 1 h containing 36 or 18 \u0026micro;mol of the organic nicotinamide (Nam) compound NAD\u003csup\u003e+\u003c/sup\u003e (\u0026gt;\u0026thinsp;95.0%, TCI) or NMN (100% Uthever, MoleQlar; \u0026gt;98.0%, TCI; \u003cb\u003eSupplementary Figure S42\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e were placed in 5 mL glass vials (beaded rim) with a polytetrafluoroethylene (PTFE)-coated stirring bar. Equimolar amounts (relative to the cofactor) of metal atoms of Fe, NiFe\u003csub\u003e3\u003c/sub\u003e, NiFe, Ni\u003csub\u003e3\u003c/sub\u003eFe, or Ni nanopowders were added to the bottom of each vial, with the exception of a metal-free control (\u003cb\u003eSupplementary Table\u0026nbsp;18\u003c/b\u003e). Alternatively, experiments with Fe and Ni micropowders had 1.8 or 7.2 mmols of metal and 36 \u0026micro;mol of cofactor (metal-cofactor ratio 50 or 200:1) in 0.5 M PBS (pH 8.5 or pH 5.5). The vials were sealed with a crimp cap with a PTFE-coated membrane. To allow gas exchange between the interior and the exterior of the glass vial, a syringe needle was inserted through the crimp cap membrane before the vials were placed in the high-pressure reactor.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStandard high-pressure reaction.\u003c/b\u003e After pressurizing the reactor (Berghof Reactor 300) with either 5 bar of Ar gas (99.999%, Air Liquid) or 5 bar of H\u003csub\u003e2\u003c/sub\u003e gas (99.9% Nippon Gases), the reactions were started and regulated by a controlled reactor heating system (Berghof Products\u0026thinsp;+\u0026thinsp;Instruments). Reactions were performed from 1 h to 4 h at 40\u0026deg;C and 400 rpm, in a Berghof Reactor Heating System (BR-HS). Afterward, reactors were depressurized under anaerobic conditions and the samples (metal powders and solution) were transferred to 2 mL Eppendorf tubes and centrifuged for 20 min, at 4\u0026deg;C, and 13,000 rpm (Fresco 17 Microcentrifuge). The supernatants were subjected to different analyses, which are described below.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReduction of pyruvate with Fe\u003c/b\u003e \u003csup\u003e \u003cb\u003e3+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eand 1,4-NMNH or 1,4-NADH.\u003c/b\u003e These experiments followed the protocol described in Supplementary Tables S15 and S16 of the paper Mayer et al. 2024\u003csup\u003e11\u003c/sup\u003e. An aqueous mixture of 0.1 mL with 0.1 M pyruvate (Pyruvic acid, Carl Roth), 0.2 M 1,4-NADH (95%, Thermo Scientific), and 0.06 M FeCl\u003csub\u003e3\u003c/sub\u003e (98% anhydrous, Gr\u0026uuml;ssing GmbH) reacted overnight at 40\u0026deg;C and 400 rpm (pH\u0026thinsp;\u0026lt;\u0026thinsp;5). For the removal of metal ions, it was added 0.2 mL of a thiolate/phosphate solution (100 mg NaSH, 100 mg NaOH in 10 mL saturated aqueous Na\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), and left to settle in the fridge (4\u0026deg;C) for 3 h. Instead of a DMSO standard as used in the referenced protocol, 0.1 mL of a 7mM DSS stock solution was added at the end of the experiment. To reach a certain volume, 0.2 mL of D\u003csub\u003e2\u003c/sub\u003eO were also added before the sample was measured. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR spectra were obtained by an AV III HD 250 MHz Spectrometer with a Double Resonance Broad Band (BBOF) probe head. The same experiment was repeated with 1,4-NMNH (97%, AmBeed) instead of 1,4-NADH.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative proton nuclear magnetic resonance (qNMR) analysis.\u003c/b\u003e To monitor reactions, as well as detect and quantify the formation of reduced NADH and side products we established a protocol for quantitative proton nuclear magnetic resonance (\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. The internal standard was a 7 mM solution of sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS, CH\u003csub\u003e3\u003c/sub\u003e peak at 0 ppm; \u0026gt;98.0%, TCI) in deuterium oxide (D\u003csub\u003e2\u003c/sub\u003eO 99.8 atom%D, AcroSeal, Thermo Scientific), mixed 1:6 with the supernatant of our samples. qNMR spectra were obtained on a Bruker AVANCE-NEO 600 MHz spectrometer equipped with a 5 mm iprobe TBO with z-gradient. Thirty-two scans were made for each sample with a relaxation delay of 40 s (600 MHz) and a spectral width from \u0026minus;\u0026thinsp;3 to 13. Analysis and integration were performed using MestReNova (v.15.0.1). Metal-free controls (ran under the same conditions as the quantified, metal-containing samples) were used as references to the initial amount of NAD/NMN in the sample to account for evaporation and possible degradation under the given pH, temperature and time. The average initial amount of cofactor in the controls was used as t\u0026thinsp;=\u0026thinsp;0 h and to normalize the reaction yields.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStandards.\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR standards were prepared with 24 mM of the compound and 1 mM of DSS dissolved in D2O (\u003cb\u003eSupplementary Figure S43\u003c/b\u003e). The spectra were obtained by a AV III HD 250 MHz Spectrometer with a BBOF probe head.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProduct Characterization through 2D-NMR.\u003c/b\u003e 2D \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR enabled the assignment of peaks for 1,4-NMNH and NMN in accordance with literature and in comparison to the pair NAD/NADH (\u003cb\u003eSupplementary Tables S19 and S20, Figures S44\u0026ndash;58\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Reduction products were also characterized through different 2D-NMR correlation spectra (\u003cb\u003eSupplementary Scheme S4, Figures S11\u0026ndash;19\u003c/b\u003e). 3 mL of sample from a 1h and 4 h reduction of NMN with equimolar amounts of NiFe\u003csub\u003e3\u003c/sub\u003e (5 bar H\u003csub\u003e2\u003c/sub\u003e, 40\u0026deg;C, 400 rpm) were dried using a vacuum concentrator (SpeedVac DNA 130, Savant). The remaining solution and pellet were suspended in 500 \u0026micro;l of D\u003csub\u003e2\u003c/sub\u003eO to increase the concentration of the products and resolution of the NMR spectra. The same procedure was performed for a NAD\u003csup\u003e+\u003c/sup\u003e sample after a 4 h reaction with equimolar amounts of NiFe\u003csub\u003e3\u003c/sub\u003e (5 bar H\u003csub\u003e2\u003c/sub\u003e, 40\u0026deg;C, 400 rpm). Two-dimensional correlation spectra of \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH DQF-COSY (Double-Quantum Filtered COrrelated SpectroscopY), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH TOCSY (Total COrrelated SpectroscopY),\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC HMBC (Heteronuclear Multiple Bond Correlation spectroscopy) were recorded with standard pulse programs\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Edited HSQC (Heteronuclear Single Quantum Coherence spectroscopy) spectra were recorded using sensitivity improvement with echo/anti-echo gradient selection and multiplicity editing during selection step\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. NOESY (Nuclear Overhauser Effect SpectroscopY) spectrum was recorded with mixing time of 1.5 s. Chemical shifts are referenced with sodium salt of trimethylsilylpropanesulfonic acid (DSS). Spectra were obtained with the same instrument as qNMR and compared to a list of possible products.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eSupporting Information\u003c/h2\u003e \u003cp\u003eAll Supporting Information (methods, figures showing analytic data, tables, schemes) is comprehensibly presented in a single pdf file.\u003c/p\u003e \u003ch2\u003eCompeting Interest Statement:\u003c/h2\u003e \u003cp\u003eDisclose any competing interests here.\u003c/p\u003e \u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eConceptualization: M.P. \u0026amp; D.P.H.P. Methodology: M.P. \u0026amp; D.P.H.P. Investigation: D.P.H.P., M.P., Z.S, T.B. Validation: D.P.H.P., Z.S. \u0026amp; X.X. Formal analysis: X.X., J.B., N.P., D.P.H.P. \u0026amp; M.P. Resources: H.T., K.V. Writing Original Draft: M.P. \u0026amp; D.P.H.P. Writing Review \u0026amp; Editing: all authors. Visualization: M.P., D.P.H.P. \u0026amp; X.X. Supervision: M.P.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eD.P.H.P and M.P. thank Bill Martin for critical reading and discussions. D.P.H.P and M.P thank Alicia Casitas and team for providing access and help to cyclic voltammetry measurements. X.X. thanks Armin Geyer for discussions and the DFG funding for NMR spectrometer NEO600 (Forschungsgro\u0026szlig;ger\u0026auml;te project number 508097909). M.P. thanks the Max Planck Society (MPG) and the International Max Planck Research School \u0026lsquo;Principles of microbial life\u0026rsquo; for funding. H.T. thanks MPG, the Volkswagen Foundation (96_742) and Deutsche Forschungsgemeinschaft (TU 315/8\u0026thinsp;\u0026minus;\u0026thinsp;1 / TU 315/8\u0026thinsp;\u0026minus;\u0026thinsp;3). This project was supported by the European Regional Development Fund (ERDF) and the Recovery Assistance for Cohesion and the Territories of Europe (REACT-EU).\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available in the SI Appendix of this article. Original analysis files (LC-MS, NMR) will be provided by the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoldman, A. D. \u0026amp; Kacar, B. Cofactors are remnants of life\u0026rsquo;s origin and early evolution. Journal of Molecular Evolution 89, 127\u0026ndash;133 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXavier, J. C., Hordijk, W., Kauffman, S., Steel, M. \u0026amp; Martin, W. F. 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The FASEB Journal 35, e21456 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerger, S. \u0026amp; Braun, S. \u003cem\u003e200 and More NMR Experiments: A Practical Course\u003c/em\u003e. (WILEY-VCH, Weinheim, 2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"adenosine cofactors, hydrogen, emergence of life, mineral catalysis, protometabolism","lastPublishedDoi":"10.21203/rs.3.rs-6214903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6214903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany cofactors share a molecular structure – adenosine – that otherwise occurs in nucleic acids. The presence of adenosine in cofactors has presented an evolutionary puzzle. Is it a biochemical ‘handle’ that allows proteins to bind the cofactor more tightly, or is it a relic from a time when cofactors arose from the building blocks of genes? Using the example of nicotinamide adenine dinucleotide (NAD), we find a surprising and previously unknown property of its adenosine monophosphate (AMP) handle. Reduction experiments with hydrogen gas (H\u003csub\u003e2\u003c/sub\u003e) on mineral surfaces show that the handle-free nicotinamide mononucleotide (NMN) overreduces quickly, while NAD gets reduced specifically. The AMP handle allows NAD to function in a hydrothermal, mineral-based setting, indicating that it is a form of protection against a harsh chemical environment in which biochemistry and life arose. Our findings uncover a specific functional role for the AMP moiety of NAD under environmental conditions capable of nonenzymatic NAD reduction, thereby identifying a structural element of a redox cofactor that is older than the enzymes that use it.\u003c/p\u003e","manuscriptTitle":"Reduction of NAD and NMN on mineral surfaces with H2 reveals a functional role for the adenosine moiety in prebiotic evolution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-28 18:41:57","doi":"10.21203/rs.3.rs-6214903/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dbacc9da-f6f0-4092-87f7-bdaad63e5e44","owner":[],"postedDate":"March 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45891323,"name":"Biological sciences/Biochemistry/Biocatalysis"},{"id":45891324,"name":"Biological sciences/Chemical biology/Metals"}],"tags":[],"updatedAt":"2025-10-31T07:14:12+00:00","versionOfRecord":{"articleIdentity":"rs-6214903","link":"https://doi.org/10.1038/s42004-025-01752-3","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2025-10-30 04:00:00","publishedOnDateReadable":"October 30th, 2025"},"versionCreatedAt":"2025-03-28 18:41:57","video":"","vorDoi":"10.1038/s42004-025-01752-3","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01752-3","workflowStages":[]},"version":"v1","identity":"rs-6214903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6214903","identity":"rs-6214903","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}