Abstract

Hydrogen gas, H-2, is generated in serpentinizing hydrothermal systems, where it has supplied electrons and energy for microbial communities since there was liquid water on Earth. In modern metabolism, H-2 is converted by hydrogenases into organically bound hydrides (H-), for example, the cofactor NADH. It transfers hydrides among molecules, serving as an activated and biologically harnessed form of H-2. In serpentinizing systems, minerals can also bind hydrides and could, in principle, have acted as inorganic hydride donors-possibly as a geochemical protoenzyme, a 'geozyme'- at the origin of metabolism. To test this idea, we investigated the ability of H-2 to reduce NAD(+) in the presence of iron (Fe), cobalt (Co) and nickel (Ni), metals that occur in serpentinizing systems. In the presence of H-2, all three metals specifically reduce NAD(+) to the biologically relevant form, 1,4-NADH, with up to 100% conversion rates within a few hours under alkaline aqueous conditions at 40 degrees C. Using Henry's law, the partial pressure of H-2 in our reactions corresponds to 3.6 mm, a concentration observed in many modern serpentinizing systems. While the reduction of NAD(+) by Ni is strictly H-2-dependent, experiments in heavy water ((H2O)-H-2) indicate that native Fe can reduce NAD(+) both with and without H-2. The results establish a mechanistic connection between abiotic and biotic hydride donors, indicating that geochemically catalysed, H-2-dependent NAD(+) reduction could have preceded the hydrogenase-dependent reaction in evolution.


Authors

Pereira, Delfina P. Henriques;  Leethaus, Jana;  Beyazay, Tugce;  Vieira, Andrey do Nascimento;  Kleinermanns, Karl;  Tuysuz, Harun;  Martin, William F.;  Preiner, Martina

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  • pre-publication peer review (FINAL ROUND)
    Decision Letter
    2021/12/15

    The FEBS Journal manuscript FJ-21-1142.R1
    Editor: Nigel Scrutton

    Dear Dr Preiner,

    Thank you for submitting your Original Article entitled "Specific abiotic hydride transfer by metals to the biological redox cofactor NAD+" to The FEBS Journal. I am pleased to inform you that the Editor-in-Chief has recommended acceptance of your manuscript.

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    Decision letter by
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    Author Response
    2021/12/09

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    Dear Prof. Martin,

    thank you for the opportunity to revise our paper entitled "Specific abiotic hydride transfer by metals to the biological redox cofactor NAD+", which you will find enclosed in this transmission. We have accommodated all of the comments of the referees and want to thank you, the editor Nigel Scrutton and the referees for the very speedy processing of our paper. We really appreciate it.

    On behalf of all coauthors,

    Yours sincerely, Martina Preiner



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  • pre-publication peer review (ROUND 1)
    Decision Letter
    2021/12/03

    03-Dec-2021

    Ref: FJ-21-1142
    Title: Specific abiotic hydride transfer by metals to the biological redox cofactor NAD+
    Authors: Henriques Pereira, Delfina Patricia; Leethaus, Jana; Beyazay, Tugce; do Nascimento Vieira , Andrey; Kleinermanns, Karl; Tüysüz, Harun; Martin, William; Preiner, Martina
    Editor: Nigel Scrutton

    Dear Dr Preiner

    Thank you for submitting your manuscript for consideration for publication in The FEBS Journal.

    I am pleased to inform you that your manuscript has received favourable comments from the referees, who consider the work acceptable for publication in the Journal, provided that you satisfactorily address the concerns raised (see referees' reports below).

    Please note that acceptance of a revised version of your manuscript, which cannot be guaranteed at this stage, is contingent upon satisfactory responses to the issues raised.

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    Thank you for your support of The FEBS Journal and we look forward to receiving a revised version of your manuscript in due course.

    Yours sincerely

    Seamus Martin
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    EDITOR'S COMMENTS

    Editor
    Comments to the Author:
    The manuscript has received supportive reviews. Minor revisions are requested to address the reviewers' comments.

    REFEREES' COMMENTS

    Referee: 1

    Comments for Transmission to Authors
    Henriques Pereira et al. describe an experimental study of the hydride-reduction of NAD+ by the metals Fe, Co and Ni in aqueous solutions containing H2; conditions aimed to mimic geochemical serpentizing systems. They show that all 3 metals are able to reduce NAD+ to NADH at 40 oC and suggest that metal-containing minerals may have been used by earlier life to hydride-reduce organic molecules prior to the emergence of hydrogenase enzymes.
    This is not my area of research, but I found the paper easy to read and engaging and I expect it may be of interest to FEBS J's readership.

    Minor points:

    How do Eq1 or Eq2 led to a change in pH? No protons are consumed during the reaction.

    Fig.3 One would expect the completely deuterated NADH to contain one each 1H and 2H at the NADH C4 position. Integration of the two CH4-H peaks (with normalisation to another proton peak) would help with interpretation of these data and allow quantification of amount of hydride originating from water cf. H2.

    pp.15 ‘The first cells or cell-like compartments such as LUCA6, were possibly still dependent upon metal hydrides from their geochemical surroundings...' This hypothesis relies on the organic hydride acceptor to be membrane permeable. Perhaps the authors should expand on this point, as these molecules probably require a transporter.

    Referee: 2

    Comments for Transmission to Authors
    The authors tested the abiotic hydride transfer to NAD with elemental Fe, Co, and Ni (powdered metal), either from H2 or from H2O. They aimed at imitating hydrothermal vent conditions and the geological process of serpentinization.
    The calculated Nernst potential of the tested metals under the given conditions (Me/Me(OH)2 are -550 mV for Fe0, -407 mV for Co0, and -390 mV for Ni0. The hydrogen potential is near -510 mV, and the NADH potential at - 390 mV. This means that - based on thermodynamics - the metals possibly would be able to reduce NAD directly by water (thereby being oxidized to metal hydroxydes), in addition to catalyzing H2 oxidation and hydride transfer to NAD. The relative contribution of these two hydride sources was tested with D2O. The results are as follows:
    FTIR experiments showed that Fe oxidized most easily, as expected. The source of the hydride, i.e. either protons from water plus electrons from metal, or hydrides from hydrogen gas, was studied by proton-NMR. It confirmed that Fe reduces D2O and transfers D- to NAD; the results with Co were less clear and a matter of dispute. In contrast, Ni exclusively behaved as catalyst for hydride transfer from H2. In all cases 1,4-NADH was the main product.
    Fe behaves ambivalent. In the absence of hydrogen, it is oxidized and transfers hydride from water to NAD. In the presence of hydrogen gas, its oxidation is outcompeted, the hydride from hydrogen gas is transferred to NAD. Thus, Fe seems to take both routes, depending on the conditions.
    The products of metal oxidation, the collidal hydroxydes, may be catalytically active; however, this possibility is rather unlikely, although it cannot be completely ruled out.
    I shall not comment other details that corroborate these conclusions.
    The paper is well written and structured, the experiments are conclusive, and the discussion is balanced and not too speculative. This paper is a mile stone in demonstrating the specific abiotic hydride transfer by metals to the universal hydrogen transfer metabolite NAD forming 1,4-NADH under hydrothermal vent conditions, as it occurs in biology by hydrogenases.
    I have no major questions or remarks. Minor:
    1. I feel the Abstract section is too "abstract", three quarters deal with serpentinization and other aspects, while the interesting results are condensed to a short statement. In the Introduction section, the knowledge of serpentinization is considered as general good. A few words extra would be welcome, especially for readers of The FEMS Journal.
    2. l. 195 ff. Please state how the metal to NAD ratio was varied.
    3. l. 266. Is the discussed exchange reaction plausible?
    4. l. 279. Could this be explained?
    5. Fig. 3. The results are indeed puzzling

    Decision letter by
    Cite this decision letter
    Reviewer report
    2021/12/03

    The authors tested the abiotic hydride transfer to NAD with elemental Fe, Co, and Ni (powdered metal), either from H2 or from H2O. They aimed at imitating hydrothermal vent conditions and the geological process of serpentinization.
    The calculated Nernst potential of the tested metals under the given conditions (Me/Me(OH)2 are -550 mV for Fe0, -407 mV for Co0, and -390 mV for Ni0. The hydrogen potential is near -510 mV, and the NADH potential at - 390 mV. This means that - based on thermodynamics - the metals possibly would be able to reduce NAD directly by water (thereby being oxidized to metal hydroxydes), in addition to catalyzing H2 oxidation and hydride transfer to NAD. The relative contribution of these two hydride sources was tested with D2O. The results are as follows:
    FTIR experiments showed that Fe oxidized most easily, as expected. The source of the hydride, i.e. either protons from water plus electrons from metal, or hydrides from hydrogen gas, was studied by proton-NMR. It confirmed that Fe reduces D2O and transfers D- to NAD; the results with Co were less clear and a matter of dispute. In contrast, Ni exclusively behaved as catalyst for hydride transfer from H2. In all cases 1,4-NADH was the main product.
    Fe behaves ambivalent. In the absence of hydrogen, it is oxidized and transfers hydride from water to NAD. In the presence of hydrogen gas, its oxidation is outcompeted, the hydride from hydrogen gas is transferred to NAD. Thus, Fe seems to take both routes, depending on the conditions.
    The products of metal oxidation, the collidal hydroxydes, may be catalytically active; however, this possibility is rather unlikely, although it cannot be completely ruled out.
    I shall not comment other details that corroborate these conclusions.
    The paper is well written and structured, the experiments are conclusive, and the discussion is balanced and not too speculative. This paper is a mile stone in demonstrating the specific abiotic hydride transfer by metals to the universal hydrogen transfer metabolite NAD forming 1,4-NADH under hydrothermal vent conditions, as it occurs in biology by hydrogenases.
    I have no major questions or remarks. Minor:
    1. I feel the Abstract section is too "abstract", three quarters deal with serpentinization and other aspects, while the interesting results are condensed to a short statement. In the Introduction section, the knowledge of serpentinization is considered as general good. A few words extra would be welcome, especially for readers of The FEMS Journal.
    2. l. 195 ff. Please state how the metal to NAD ratio was varied.
    3. l. 266. Is the discussed exchange reaction plausible?
    4. l. 279. Could this be explained?
    5. Fig. 3. The results are indeed puzzling

    Reviewed by
    Cite this review
    Reviewer report
    2021/11/29

    Henriques Pereira et al. describe an experimental study of the hydride-reduction of NAD+ by the metals Fe, Co and Ni in aqueous solutions containing H2; conditions aimed to mimic geochemical serpentizing systems. They show that all 3 metals are able to reduce NAD+ to NADH at 40 oC and suggest that metal-containing minerals may have been used by earlier life to hydride-reduce organic molecules prior to the emergence of hydrogenase enzymes.
    This is not my area of research, but I found the paper easy to read and engaging and I expect it may be of interest to FEBS J's readership.

    Minor points:

    How do Eq1 or Eq2 led to a change in pH? No protons are consumed during the reaction.

    Fig.3 One would expect the completely deuterated NADH to contain one each 1H and 2H at the NADH C4 position. Integration of the two CH4-H peaks (with normalisation to another proton peak) would help with interpretation of these data and allow quantification of amount of hydride originating from water cf. H2.

    pp.15 ‘The first cells or cell-like compartments such as LUCA6, were possibly still dependent upon metal hydrides from their geochemical surroundings...' This hypothesis relies on the organic hydride acceptor to be membrane permeable. Perhaps the authors should expand on this point, as these molecules probably require a transporter.

    Reviewed by
    Cite this review
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