Resurrected nitrogenases recapitulate canonical N-isotope biosignatures over two billion years

Domain: neuroscience
Published: 2026-01-22
Editorial outcome: minor_revision

Abstract
Nitrogen isotope fractionation (ε15N) in sedimentary rocks has provided evidence for biological nitrogen fixation, and thus primary productivity, on the early Earth. However, the extent to which molecular evolution has influenced the isotopic signatures of nitrogenase, the enzyme that catalyzes the conversion of atmospheric nitrogen (N₂) to bioavailable ammonia, remains unresolved. Here, we reconstruct and experimentally characterize a library of synthetic ancestral nitrogenase genes, spanning over 2 billion years of evolutionary history. We assess the resulting ε15N values under controlled laboratory conditions. All engineered strains exhibit ε15N values within a narrow range comparable to that of modern microbes, suggesting that molybdenum (Mo)-dependent nitrogenase has been largely invariant throughout evolutionary time since the origins of this pathway. The results of this study support the early origin of molybdenum nitrogenase and the resilience of nitrogen-isotope biosignatures in ancient rocks, while also demonstrating their potential as powerful tools in the search for life beyond Earth.

Introduction
The metabolic and physiological characteristics of Earth’s earliest life remain largely obscure, with only the broad outlines discernible for the first 2 billion years of evolution1–7. In the absence of diagnostic fossils, stable isotopes of carbon and sulfur have served as key geochemical proxies for ancient biological activity2,8–11. Nitrogen, owing to its centrality in biochemistry, is an appealing candidate for early life reconstructions. However, the complexity and diversity of its metabolic pathways have hindered the interpretation of its record. Uniquely, nitrogen fixation of N into bioavailable N is catalyzed by a single enzyme, nitrogenase. This constraint raises the possibility that the evolutionary history of this single enzyme could inform the interpretation of nitrogen isotopes across deep time.

The oldest geochemical evidence for biological nitrogen fixation comes from nitrogen isotopic compositions of ~3.2 Ga Archean sedimentary rocks, with δ15N values ranging from +0.7‰ to −2.8‰12. These values closely resemble those of the biomass of modern organisms using Mo-dependent nitrogenase, which typically fall between +1‰ and −3‰12–16. By contrast, modern organisms expressing alternative vanadium (V)- and iron (Fe)-based nitrogenase isozymes exhibit more negative biomass fractionations (−6‰ to −8‰)13. Similar isotopic patterns in ~2.9 Ga rocks have been interpreted as evidence for the primordial nitrogen cycle driven by Mo-nitrogenase17. Additionally, nitrogen isotope signatures from transient atmospheric oxygenation episodes (“whiffs”) at ~2.66 Ga and ~2.5 Ga suggest that Mo-nitrogenase remained the dominant nitrogen-fixing enzyme before the onset of the Great Oxidation Event (GOE)9,18 at approximately 2.45 Ga, after which time the atmosphere became permanently oxygenated19. Although post-GOE oxidative processes increasingly overprint nitrogen fixation signals in younger rocks, Mesoproterozoic offshore marine sediments deposited under low-oxygen conditions still preserve δ15N values consistent with Mo-nitrogenase activity18,20,21. Even during Phanerozoic ocean anoxic events, such as those in the late Devonian, Jurassic, and Cretaceous, marine sedimentary δ15N values often return to the canonical Mo-nitrogenase range22. Collectively, these records depend on a critical assumption: that nitrogenase-mediated fractionation has remained stable over billions of years, despite profound environmental, ecological, and evolutionary shifts.

Isotopic fractionation during biological nitrogen fixation varies across modern diazotrophs depending on species16, nitrogenase isozyme13, and environmental conditions14,16,23,24. Importantly, all extant nitrogenases are the product of billions of years of molecular evolution, and are adapted to an Earth with an oxygenated atmosphere and an ecologically diverse biosphere25. These factors can impact the isotopic fractionation effect generated from nitrogenase activity by modifying the mechanistic aspects of catalysis. Therefore, it remains uncertain whether the isotopic fractionation signatures observed in modern biology can be reliably extrapolated to interpret nitrogen isotope signatures in the ancient sedimentary record.

We constructed a library of ancient nitrogenases, spanning over 2 billion years, and engineered a model diazotroph, Azotobacter vinelandii (A. vinelandii), to express these ancient variants in vivo. Our work separates itself from prior efforts by beginning with fully synthetic ancient DNA rather than modifying extant genes. We introduce a synthetic-biology framework that reconstructs nitrogenase from first principles, establishing a molecular platform for probing ancient biosignatures. We measured the nitrogen isotope fractionation in the cell biomass of engineered A. vinelandii strains, with all fixed nitrogen coming from the activity of the synthetic ancient nitrogenase. This system enabled a direct test of the evolutionary stability of nitrogen isotope fractionation across deep time, using controlled expression of biochemically functional, phylogenetically inferred ancient enzymes. The resulting isotopic signatures were then evaluated within the context of the sedimentary nitrogen isotopic record and long-term changes in Earth’s biogeochemistry.

Results
Reconstruction of nitrogenase ancestors
Our study builds on a nitrogenase phylogeny and an ancestral nitrogenase protein sequence database (Fig. 1, “Methods”). The phylogeny broadly samples known nitrogenase sequences and host taxonomic diversity, grouping homologs into three major clades: Groups I to III (Fig. 1a). The Nif–III clade also contains two subclades corresponding to the alternative nitrogenases, Vnf and Anf, which incorporate VFe- and FeFe-cofactors, respectively, instead of the canonical FeMo-cofactor found in most nitrogenases26. This overall phylogenetic topology is largely consistent with prior reconstructions of nitrogenase evolution27–29.

The Group-I clade is distinguished by its association with oxygen-tolerant hosts. In our dataset, approximately 62.5% of Nif-I nitrogenases are either found in, or predicted to occur in, aerobic or facultatively anaerobic prokaryotes, compared to only 6.5% in Group-II and 0% in Group-III (see “Methods”). This strong association between most Group-I nitrogenases and host oxygen tolerance suggests that the diversification of major Group-I lineages likely followed the initial rise of atmospheric oxygen (the GOE) and the resulting emergence of aerobic metabolism5,30. Notably, the Nif-I clade also contains a monophyletic cluster of nitrogenases hosted by cyanobacteria, consistent with abundant cyanobacterial microfossils preserved in Proterozoic strata31,32. Both observations make the Group-I clade particularly well suited for correlating nitrogenase evolution with the sedimentary rock record.

To experimentally reconstruct ancient nitrogenase isotopic signatures, we engineered four ancient nitrogenases representing a temporal series from the present into the deep past (~700 Ma to 2.3 Ga) in A. vinelandii (Supplemental Table S1). These variants (Anc1 to Anc4), reconstructed from phylogenetic nodes along the oxygen-tolerant Group-I lineage, were individually introduced into a nitrogenase-deficient background to replace one or more native nif genes (Supplemental Table S1). This inferred evolutionary context places the reconstructed ancestors after the GOE and captures a period of permanent but varying atmospheric oxygenation.

Notably, Anc3 and Anc4 precede the diversification of heterocystous cyanobacteria, which form specialized nitrogen-fixing cells33. Phylogenetic analysis places Anc3 prior to the emergence of a monophyletic clade that includes nitrogenase homologs from heterocyst-forming Nostocales, which compartmentalize nitrogen fixation34. The presence of heterocystous cyanobacteria in the Proterozoic fossil record32,35 constraints the minimum ages of Anc3 and Anc4 to approximately 700 Ma to 2.3 Ga (Fig. 1).

Functional characteristics of ancestral nitrogenases
The catalytic function of the ancestral nitrogenases was evaluated by their ability to support diazotrophic growth and catalyze acetylene reduction in vivo (Fig. 2e, f) (“Methods”). Relative to the wild-type, all ancestral strains showed lower rates of ethylene production, a standard proxy for N₂ reduction36, reflecting measurable differences in catalytic performance across evolutionary time (Fig. 2f).

Purified ancestral nitrogenases exhibited lower in vitro specific activities than their WT counterparts across all tested substrates (Fig. 2g). Despite this, the engineered strains maintained nitrogen-fixation efficiencies comparable to the WT37. Immunodetection showed that nitrogenase protein expressions are generally comparable to the WT (Extended Data Fig. 3), and a prior transcriptomic analysis of Anc2 revealed no significant upregulation of nif genes38. These results suggest that differences arise primarily from intrinsic functional properties rather than changes in expression.

Nitrogen isotope fractionation: Archean to now
Batch cultures of A. vinelandii strains expressing ancestral nitrogenases were grown under diazotrophic conditions, and cell biomass was harvested for nitrogen isotopic analysis. The isotopic fractionation associated with nitrogen fixation (ε fixed nitrogen; ε¹⁵N) was calculated relative to atmospheric nitrogen (0‰). All ancestral strains exhibited ε¹⁵N values ranging from −0.9 to −2.9‰ (Fig. 3), and notably, all overlap with the range displayed by the WT. Anc1 and Anc3 were statistically similar to WT, while Anc2 and Anc4 differed significantly from WT and each other, but there was no discernible trend between fractionation values and increasing ancestral enzyme age (Fig. 3b). The isotopic fractionations (ε15N) associated with the ancestral nitrogenases fall within the extremities of the range reported for modern diazotrophs (+3.7 to −5.86‰) and align with average values observed in extant bacteria (−1.38‰) and archaea (−4.13‰)16 (Fig. 4). Among the variants, Anc3, the second oldest enzyme, exhibited the broadest range in ε¹⁵N values and the most negative average fractionation.

We further assessed whether nitrogen isotopic fractionation changed as a function of diazotrophic growth phase (Fig. 3c, d). Previous work with WT bacteria has shown up to 2‰ variation in isotopic values throughout growth14. In contrast, we observed no significant trend in ε15N values throughout early log phase for WT, Anc1, or Anc2, whereas biomass from Anc3 and Anc4 became slightly more 15N-depleted (Pearson coefficients = −0.75 and −0.66, respectively). Most strains exhibited <1‰ variation in biomass ε15N values, with all strains remaining within the ≤2‰ range reported previously14. At late log to stationary phases (i.e., OD600 of 8–25), biomass 15N increased slightly (i.e., became less 15N depleted) for WT, Anc1, and Anc2 (Pearson coefficients = 0.98, 0.67, and 0.96, respectively), while Anc3 and Anc4 showed weak depletion (Pearson coefficients = −0.23 and −0.4, respectively). Overall, isotopic variation across all growth phases was not a primary control on nitrogen isotopic signatures.

Methods
Ancestral sequence reconstruction
Phylogenetic analysis and ancestral sequence reconstruction of nitrogenase proteins were performed previously37. Briefly, nitrogenase protein sequences were collected by BLASTp search48 against the NCBI non-redundant protein database (accessed August 2020) and curated manually, yielding 385 sets of H-, D-, and K-subunit sequences. Sequences for each subunit were aligned separately by MAFFT v.7.45049. For ancestor Anc2, protein sequence alignments were trimmed using trimAl v.1.250. Evolutionary model selection (LG+G+F) was performed by ModelFinder51 implemented in IQ-TREE v.1.6.1252. Tree reconstruction (using the trimmed alignment) and ancestral sequence reconstruction (using the untrimmed alignment) were performed by RAxML v.8.2.1053 using the best-fit LG+G+F model. For ancestors Anc1, Anc3, and Anc4, an updated tree was built using an untrimmed alignment and RAxML. Ancestral sequence reconstruction was instead performed by PAML v.4.9j54 using the LG+G+model. Oxygen metabolism was calculated via the Oxyphen package55,56.

Sequence and structural comparison
The DDKK complex structures for the WT Nif from A. vinelandii and the selected Group I ancestors were extracted from the Nitrogenase database (https://nsdb.bact.wisc.edu/)57 (Extended Data Fig. 1). To assess the structural variation, we calculated Root Mean Squared Deviation (RMSD) and sequence identity for each ancestor with respect to the WT complex using US-align58 (Fig. 2c).

Sequence embeddings were calculated for the different NifD variant sequences at each node generated during ancestral sequence reconstruction using a 5-dimensional physicochemical representation for each amino acid residue58. Dimensionality reduction and clustering visualization were performed using t-distributed stochastic neighbor embedding (t-SNE) from the Scikit-learn implementation59 (Extended Data Fig. 1c). Mean posterior probability and SH-like aLRT for each residue and node of the ancestral sequences were calculated (Supplemental Table S3).

Strain engineering and functional assessment
Ancestral nitrogenase sequences were obtained from a previously constructed phylogeny37 (sequences and primers available in Supplemental Tables S1, 2). Strains Anc1-3 were constructed in previous studies (Anc1-237 and Anc360). Anc4 was constructed following these previously established methods. Briefly, ancestral sequences are integrated into a plasmid via Gibson assembly, and this plasmid is then transformed into a Δnif strain. Transformants were screened for nitrogen fixation rescue and loss of kanamycin resistance on solid Burk’s (“B”) medium. The growth rate of each strain in nitrogen-fixing conditions was quantified using previously reported methods61. In brief, liquid B medium was inoculated from a 24-h seed culture to an optical density at 600 nm of 0.05. Three biological replicates of each strain were tested. The cultures were grown in a 96-well, flat-bottom plate (Greiner Bio-One) with a Breathe-Easy adhesive seal (Diversified Biotech). The plate was incubated at 30°C with a 200-rpm double orbital agitation inside a SPECTROstar Nano Microplate Reader (BMG Labtech, Ortenberg, Germany). An optical density reading at 600 nm was taken every 30 min for over 75 h. Doubling time and data visualization were conducted using the R package Growthcurver62.

Acetylene reduction assay was conducted following previous methods37. Biological replicates of each culture were grown overnight and then used to inoculate 100 mL of Burk’s medium with ammonium acetate (“BN media”) to an OD600 of 0.01. Cultures were then grown for 16 h, centrifuged down to a cell pellet at 4255 x g for 10 min, and then resuspended in 100 mL of B medium. The cultures were shaken at 30°C and agitated at 300 rpm for 4 h, at which time a rubber septum was placed over each flask. 25 mL of headspace air was removed and replaced with 25 mL of acetylene. Headspace measurements were taken 20, 40, and 60 min after acetylene incubation. Ethylene production was quantified via Nexis GC-2030 gas chromatograph (Shimadzu). Cell pellets were collected after the final sampling by centrifuging 24 mL of cultures at 4255 x g for 10 min, washing with 3 mL of phosphate buffer, and pelleting once more as above. Cell pellets were stored at –80°C. The total protein of each pellet was quantified using a Quick Start Bradford Protein Assay kit (Bio-Rad) and CLARIOstar Plus plate reader (BMG Labtech). The total protein of each pellet was used to normalize the acetylene reduction rates.

Growth and sample collection
BN medium was inoculated and grown at 30°C and agitated at 250–300 rpm. Biological replicates (n = 6 to 9) were prepared for each strain. After 20–24 h, the optical density at 600 nm (OD600) of each culture was measured. These cultures were then used to inoculate 500 mL of Burk’s media without any nitrogen (“B media”) in 1L Erlenmeyer flasks to an OD600 of 0.01. The cultures were placed in a shaker at 30°C and agitated at 250–300 rpm. The OD600 of the cultures was monitored until the cultures reached log or stationary growth.

To collect samples for isotope analysis, 50 mL of culture was taken from each flask and centrifuged for 10 min at 4255 x g. The pellet was resuspended and washed with 50 mL 1x PO4, centrifuging as before. The resulting cell pellets were resuspended and washed again with 1 mL 1x PO4 in a benchtop microcentrifuge (2400 x g for 10 min). The supernatant was removed, and the pellets were dried in an oven at 50°C for 48 h. Laboratory air samples were also collected for analysis.

Isotopic analyses
All analyses of nitrogen isotopic composition were carried out at the University of Washington Isolab facilities. The bulk nitrogen isotopic analyses were analyzed using an elemental analyzer (Costech ECS 4010) coupled to a continuous-flow isotope-ratio mass spectrometer (CF-IRMS; Thermo Finnigan MAT 253).

Approximately 0.500–2.000 mg of freeze-dried A. vinelandii biomass powders were weighed into 3.5 mm x 5 mm tin capsules (Costech) and flash-combusted at 1000°C under an oxygen-rich atmosphere. The resulting N2 and CO2 gases were purified with chromium oxide at 1000°C to convert trace CO gases to CO2, and silvered cobalt oxide at 1000°C to capture sulfur gases and halogens. Then, another purification step was performed with copper at 650°C to convert trace NOx gases to N2. Traces of H2O were scrubbed with magnesium perchlorate. Subsequently, N2 and CO2 gases were separated chromatographically in a GC column and analyzed for their isotopic composition.

An aliquot of the lab air (2 mL) was analyzed via a Sercon CryoPrep gas concentration system interfaced to a Sercon 20–20 isotope-ratio mass spectrometer at the University of California, Davis Stable Isotope Facility. The measured lab air value was 0.3±0.05‰. The isotope fractionation values associated with nitrogen fixation, calculated using this value for air, are given in Extended Data Fig. 4.

Isotope and abundance measurements were calibrated using three in-house standards (two glutamic acids, GA1 and GA2, and dried salmon, SA). In-house standards were calibrated against the international reference materials USGS40 and USGS4163 (Supplemental Tables S4, S5). Isotope measurements were reported in standard delta notation relative to Air N2:

δ15N = [(15N/14N)sample / (15N/14N)standard - 1] x 1000 (1)

Calculations and statistics
ε15N of the biomass was calculated using the following equation:

ε(fixed nitrogen=N2) = [(1000+δ15N sample) / (1000+δ15N N2) - 1] x 1000 (2)

Where δ15N for the biomass samples is atmospheric N2 (0‰) (Supplemental Table S6).

Statistical significance was calculated using one-way ANOVA, Pearson correlation coefficient, and Tukey’s Honest Significant Difference tests. A significance level of 0.05 was used for all analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper describes measuring isotopic fractionation of WT and ancestrally reconstructed nitrogenases. The reconstructed\nnitrogenases have similar δ15N values to modern nitrogenases, which helps to constrain values over geological timescales.\nThe engineering of nitrogenases and measurements of activity are not trivial, and the results are important and interesting.\nThe work is of interest to those interested in the early Earth, and also are of interest to those making engineered\nnitrogenases – a wide range! The data broadly support the conclusions, subject to caveats below. Methods seem\nappropriate and reproducible. I support publication, subject to some minor points.\nI am not too surprised by the results, as I would predict that the core active site residues around the FeMoCo cluster are\nconserved amongst all these nitrogenases, and so differential isotope effects should be small. It would be interesting to add\nthe VFe and FeFe nitrogenases to Ext Fig 1d to highlight the differences that presumably do lead to larger isotope effects. It\nwould also be interesting to add a sequence logo for the MoFe sequences used in the reconstruction. Do the authors have\nany explanation/rationalization for why the alternative nitrogenases have a more negative δ15N? The assumption of stable\nnitrogenase-mediated fractionation also presumably has the parallel assumption of stable enzyme-mediated nitrogen\ncycling, such as denitrification?\nMinor points:\np. 3 line 72 “nearly 3 billion years”. I think this is stretching the interpretation of the Anc4 time.\np.4 line 119. 2.3-0.7 Ga – I think this is meant to be 2.3 - 2.7 Ga? Or 2.3 ± 0.7 Ga?\np 5. Figure 1b. I don’t see the “respective oxygen tolerances” on this figure. Is this the node colors? Please clarify. Also, I\nthink the “oxygen tolerance” is really of the parental organisms. Diazotrophs have a much wider range of O2 tolerance (~0%\nto >40% O2 atmosphere) than the nitrogenase enzyme itself. Please clarify.\np.6 line 132. Does lower ethylene production and specific activity reflect catalytic performance over evolutionary time, or\nreduced holoenzyme assembly due to incompatibility between reconstructed sequences and the modern A. vinelandii\nnitrogenase assembly machinery? Perhaps hedge this statement more.\nAn interesting control here might have been a modern WT nitrogenase from another organism, such as a cyanobacterium.\nFig 2c x-axis “indentity”\np.7 line 147 expression → expressions\nExt Fig 1d. I like this sequence alignment (discussed above) – it would be good to put it into the main text, perhaps in Fig 1\nor 2?\np. 12 Is there any estimate of the expected error in ancestrally reconstructed sequences? For example identity between the\nreconstruction and the true ancestral sequence? Please include some statement on the accuracy of the Anc{1,2,3,4}\nsequences.", "reviewer_2": "Nature Comms manuscript review\nRucker et al. Present a compelling and important study in the evolution of nitrogenase activity. I find the work fascinating and\nrelevant to the field, and I think it is scope is appropriate for Nature Communications. My concerns / comments on the study\nare centred primarily on the rigour and confidence in the ancestral sequence reconstruction / phylogenetic analysis that\nunderpins the results and (more importantly) how they are interpreted in terms of ancient biogeographic trends.\nMajor comments:\nThe study would benefit from additional phylogenetic analysis. Ancestral sequence reconstruction is uniquely positioned to\nstudy bio geochemical trends. However, as a method, ASR is probabilistic and sensitive to biases and model assumptions.\nThe authors should report bootstrap values at key nodes in the phylogenetic tree and perform multiple and independent tree\nsearches with tree topology testing (such as the approximately unbiased test). This will provide more compelling evidence\nthat the tree topology that the authors report (and build their results from) is a statistically reliable and stable representation of\nnitrogenase evolution.\nSimilarly, at a minimum the posterior probabilities of each ancestral enzyme should be reported and commented on. If\nposterior probabilities are poor, it is important that the authors consider, discuss and confirm how ambiguity in the\nreconstruction may affect the conclusions they draw, including experimental characterisation of “AltAll” reconstructions\n(https://pmc.ncbi.nlm.nih.gov/articles/PMC6095102/). This will provide stronger evidence that the conclusions the authors\nmake on the emergence nitrogenase catalytic properties are not the consequences of sampling error introduced during ASR.\nA potentially interesting point that the authors overlook in their discussion is how prehistoric global temperature may\ninfluence the catalytic behaviour of their ancestral proteins (e.g.,\nhttps://www.biorxiv.org/content/10.1101/2025.03.23.644840v2.abstract). For example, the generally slower nitrogenase\nactivity they report may be a consequence of thermophilicity, as global temperatures and climates are likely to have been\nharsher and hotter during the timeframes the authors are studying. While I do not expect that it is feasible to test the ancestral\nproteins outside of mesophilic experimental conditions, how the environment they would have in is likely to influenced their\ncatalytic properties is an important discussion point (both in terms of thermophilicity and gas substrate solubility).", "reviewer_3": "Foreword:\nI am a stable isotope biogeochemist, working on Precambrian biogeochemical cycles. Please keep in mind while reading\nmy review that I am not qualified to evaluate the innovative nature and soundness of the biological methods (in rough terms\nreconstructing ancient nitrogenase, introducing it in organisms and growing them). I hope that another reviewer is able to\nevaluate these aspects.\nAccepting that the biological methods are correct, my opinion is that this paper pertains to a topic absolutely worthy of\npublication in XXX. I have not heard of anything like this done in the case of nitrogenase and the implications for the\nunderstanding the history of primary productivity through time are very important. I would recommend publication after my\nthree comments are taken into account.\nIn short, the isotope fractionation associated with present day nitrogenase enzymes has been used to infer their operation in\nthe past assuming actualism, which implies not only 1) that this enzyme has been fractionating the N-isotopes in the same\nway since their appearance but also 2) that no other enzymatic pathway, now unknown or even lost, was doing the same\nthing in the past. This paper tests the hypothesis 1 by reconstructing ancient nitrogenase, introducing them in modern\norganisms, themselves grown, harvested and analysed for their isotope composition.\nHowever, the hypothesis 2 remains impossible to test today (unless my knowledge in biology is too limited), and this should\nnot be omitted from the introduction, the discussion, conclusions and hence abstract. This in important because the findings\nof this paper are indeed compatible with the interpretation that the nitrogenase was there very early in the Earth history but it\nis not a demonstration for it. It might also be possible that other enzyme were able to fix N2 (comment 1).\nAs a specialist of the measurement of nitrogen stable isotope I do not have comment to make about the measurement of the\nbiomass, but it seems to me that the measurement of the laboratory air d15N needs more work (comment 2). The isotope\nvalue of the laboratory air reported in the paper is weirdly at +1,5‰. Air is the international standard and its d15N should\ntherefore be at 0‰. This has been established by Mariotti since the 80 (Mariotti, A. Natural 15N abundance measurements\nand atmospheric nitrogen standard calibration. Nature 311, 251–252, 1984). Changing it to +1,5‰ given the 80% of N2 in air\nseems to me to be mission impossible. I have often measured air in my lab to validate off-line extraction protocols and it was\nsystematically around 0‰ although with significant scatter owing the presence of Argon that produced interferences in the\nmass spectrometer.\nA value of +1,5‰ cannot be published as it is without questioning it/discussing it even if it had been doubled checked in\nanother lab (which should be done here). It would be far more appropriate to use the value of 0‰ which would increase the\nvalues of the fractionation factor by 25% relative to the values determined in the manuscript. While this would not change the\nconclusions much, it might have strong implications for future work using these numbers.\nAnd finally, my comment 3 derives directly from comment 1: most of the rock record presents d15N values much higher than\n-3‰. These values imply either that other processes of the biogeochemical N-cycle were occurring that increased the d15N\nrecorded in the sediments (as discussed in the manuscript), or that an unknown and possibly extinct enzymatic pathways\nwere fixing N2, with a fractionation factor compatible with the more positive values. This possibility would deserve to be\nmentioned or dismissed by discussing it in the paper.\nFigure 3 d : I do not understand what the linear regression are for. I wonder if the differences in isotope values could be due\nto analytical artefacts. This could be discussed.\nFigure 4 : the word archaea is missing in the last sentence of its legend.\nExtended figure 3: the references for bacteria and archaea fractionation factors should be provided in the legend.\nhoping this helps\nMagali Ader"}, "author_response": "We greatly appreciate the reviewers’ time and effort in evaluating our manuscript. We\nfound the feedback to be both insightful and constructive and we thank them for\nrecognizing the significance of this work and the effort invested in it.\nBelow, we provide detailed responses to each of the reviewer’s comments and\nsuggestions. We have addressed all reviewer comments and performed all requested\nrevisions. We would like to note that per reviewer suggestions, we have added additional\nfigures and tables to the extended and supplemental data files, as a result, some figure\nand table numbers have been updated relative to those cited in the original comments.\nResponse to Reviewer #1:\nThis paper describes measuring isotopic fractionation of WT and ancestrally\nreconstructed nitrogenases. The reconstructed nitrogenases have similar δ15N values to\nmodern nitrogenases, which helps to constrain values over geological timescales. The\nengineering of nitrogenases and measurements of activity are not trivial, and the results\nare important and interesting. The work is of interest to those interested in the early\nEarth, and also are of interest to those making engineered nitrogenases – a wide range!\nThe data broadly support the conclusions, subject to caveats below. Methods seem\nappropriate and reproducible. I support publication, subject to some minor points.\nI am not too surprised by the results, as I would predict that the core active site residues\naround the FeMoCo cluster are conserved amongst all these nitrogenases, and so\ndifferential isotope effects should be small. It would be interesting to add the VFe and\nFeFe nitrogenases to Ext Fig 1d to highlight the differences that presumably do lead to\nlarger isotope effects. It would also be interesting to add a sequence logo for the MoFe\nsequences used in the reconstruction.\nThank you, we have now added the VFe and FeFe nitrogenase active site residues to the\nalignment and have included it in Figure 2. We also appreciate the suggestion for adding\nsequence logos for the sequences used in this paper, which has been added as Extended\nFigure 2.\nDo the authors have any explanation/rationalization for why the alternative nitrogenases\nhave a more negative δ15N? The assumption of stable nitrogenase-mediated\nfractionation also presumably has the parallel assumption of stable enzyme-mediated\nnitrogen cycling, such as denitrification?\nThe reviewer raises a very good point. The precise fractionation step(s) in nitrogen fixation\nremain somewhat enigmatic. Recent work (Han et al., 2025 PNAS Nexus) suggests that\ndiffusion into the substrate channel results in the kinetic isotope effect (KIE) observed for\nMo-nitrogenase. It is unclear how such diffusion may differ in the alternative nitrogenases,\nresulting in their more negative δ15N. An extension of the Han et al. study to the\nalternatives may be insightful but is beyond the scope of this work. We hypothesize that\nthe increased reversibility of the alternative nitrogenases (i.e., the formation of H rather\n2\nthan NH as a result of a reversible E4 state in the enzymatic pathway) (Lukoyanov et al.,\n4\nδ15N observed for the alternative. While directly testing this hypothesis is beyond the\nscope of the present study, it offers a plausible mechanistic framework for future\ninvestigation.\nMinor points:\n p. 3 line 72 “nearly 3 billion years”. I think this is stretching the interpretation of\nthe Anc4 time.\nThank you, we have updated this text to “spanning over 2 billion years”.\n p.4 line 119. 2.3-0.7 Ga – I think this is meant to be 2.3 - 2.7 Ga? Or 2.3 ± 0.7\nGa?\nThe correct range is indeed 0.7 Ga to 2.3 Ga, and we have updated the text to\n“(~700 Ma to 2.3 Ga)” for improved clarity.\n p 5. Figure 1b. I don’t see the “respective oxygen tolerances” on this figure. Is\nthis the node colors? Please clarify. Also, I think the “oxygen tolerance” is really\nof the parental organisms. Diazotrophs have a much wider range of O2 tolerance\n(~0% to >40% O2 atmosphere) than the nitrogenase enzyme itself. Please\nclarify.\nThank you for this observation. You are correct that the respective oxygen\ntolerances are not depicted in this figure. We have revised the figure legend to\nremove this reference and clarified that the node colors represent taxonomic\ngroupings. A sentence has been added to clarify this.\n p.6 line 132. Does lower ethylene production and specific activity reflect catalytic\nperformance over evolutionary time, or reduced holoenzyme assembly due to\nincompatibility between reconstructed sequences and the modern A. vinelandii\nnitrogenase assembly machinery? Perhaps hedge this statement more. An\ninteresting control here might have been a modern WT nitrogenase from another\norganism, such as a cyanobacterium.\nThank you for this question. The observed decreases in specific activity and\nsubstrate reduction likely reflect reduced holoenzyme assembly, as previously\nreported (Harris et al., 2024 mBio). Importantly, the catalytic efficiencies of the\nancestral nitrogenases, as defined by the H to N ratio, have been shown to be\n2 2\nthe same as WT (~2, Garcia et al., 2023 eLife) suggesting that the fundamental\ncatalytic mechanisms remain conserved.\nWe agree that comparing activity to other modern nitrogenases would be an\ninformative direction. In ongoing work, we are constructing a library of\nnitrogenase variants, including strains expressing modern nitrogenase\nsequences from other taxa for a separate study. As these values are prepared for\npublication, we can share that insertion of the nitrogenase sequence from\nRhodoferax antarcticus, a purple nonsulfur bacteria, results in a log2 fold change\nof -0.66 nmol Ethylene per hour, which is similar to the fold change reported for\nour ancestral strains in this work.\n Fig 2c x-axis “indentity”\nThank you, this has been fixed.\n p.7 line 147 expression → expressions\nThank you, this has been fixed.\n Ext Fig 1d. I like this sequence alignment (discussed above) – it would be good\nto put it into the main text, perhaps in Fig 1 or 2?\nThank you, this has now been added to Figure 2.\n p. 12 Is there any estimate of the expected error in ancestrally reconstructed\nsequences? ;For example identity between the reconstruction and the true\nancestral sequence? Please include some statement on the accuracy of the\nAnc{1,2,3,4} sequences.\nThank you for this suggestion. Accordingly, we have included a table of the mean\nposterior probability and branch support values for each ancestral sequence has\nbeen added to the supplemental data (Table S3).\nResponse to Reviewer #2:\nRucker et al. Present a compelling and important study in the evolution of nitrogenase\nactivity. I find the work fascinating and relevant to the field, and I think it is scope is\nappropriate for Nature Communications. My concerns / comments on the study are\ncentred primarily on the rigour and confidence in the ancestral sequence reconstruction\n/ phylogenetic analysis that underpins the results and (more importantly) how they are\ninterpreted in terms of ancient biogeographic trends.\nMajor comments:\nThe study would benefit from additional phylogenetic analysis. Ancestral sequence\nreconstruction is uniquely positioned to study biogeochemical trends. However, as a\nmethod, ASR is probabilistic and sensitive to biases and model assumptions. The\nauthors should report bootstrap values at key nodes in the phylogenetic tree and\nperform multiple and independent tree searches with tree topology testing (such as the\napproximately unbiased test). This will provide more compelling evidence that the tree\ntopology that the authors report (and build their results from) is a statistically reliable\nand stable representation of nitrogenase evolution.\nThank you for these suggestions. As requested, we have now added branch support\nvalues (Shimodaira–Hasegawa approximate likelihood ratio test, or SH-like aLRT) to the\nsupplemental data (Table S3). The tree topology for this work is further supported by\nseveral previously published phylogenies (Garcia et al., 2020, Geobiology; Garcia et al.,\n2023 eLife; Cuevas-Zuviría et al., 2024, Molecular Biology and Evolution), and all\nrelevant information used in this study is now publicly available via GitHub.\nSimilarly, at a minimum the posterior probabilities of each ancestral enzyme should be\nreported and commented on. If posterior probabilities are poor, it is important that the\nauthors consider, discuss and confirm how ambiguity in the reconstruction may affect\nthe conclusions they draw, including experimental characterisation of “AltAll”\nreconstructions (https://pmc.ncbi.nlm.nih.gov/articles/PMC6095102/). This will provide\nstronger evidence that the conclusions the authors make on the emergence nitrogenase\ncatalytic properties are not the consequences of sampling error introduced during ASR.\nThank you for this helpful comment. We have added the mean posterior probabilities for\neach ancestor to the supplemental data (Table S3). The posterior probabilities were\nconsistently high (0.94-0.98) indicating that the reconstructed ancestral sequences are\nwell supported. Given this strong support and the previously characterized kinetic\nproperties of these ancestors, we believe that the analyses presented, including those\nshown in Extended Data Fig.1, provide a robust assessment of the reconstructions. We\nfurther expanded and clarified these references throughout the text to better orient the\nreader to relevant supporting work.\nA potentially interesting point that the authors overlook in their discussion is how\nprehistoric global temperature may influence the catalytic behaviour of their ancestral\nproteins (e.g., https://www.biorxiv.org/content/10.1101/2025.03.23.644840v2.abstract).\nFor example, the generally slower nitrogenase activity they report may be a\nconsequence of thermophilicity, as global temperatures and climates are likely to have\nbeen harsher and hotter during the timeframes the authors are studying. While I do not\nexpect that it is feasible to test the ancestral proteins outside of mesophilic experimental\nconditions, how the environment they would have in is likely to influenced their\ncatalytic properties is an important discussion point (both in terms of thermophilicity and gas\nsubstrate solubility).\nThank you for this suggestion. We have added this point to Discussion to highlight this\nidea and note it as an important direction for future work.\nResponse to Reviewer #3:\nForeword:\nI am a stable isotope biogeochemist, working on Precambrian biogeochemical cycles.\nPlease keep in mind while reading my review that I am not qualified to evaluate the\ninnovative nature and soundness of the biological methods (in rough terms\nreconstructing ancient nitrogenase, introducing it in organisms and growing them). I\nhope that another reviewer is able to evaluate these aspects.\nAccepting that the biological methods are correct, my opinion is that this paper pertains\nto a topic absolutely worthy of publication in XXX. I have not heard of anything like this\ndone in the case of nitrogenase and the implications for the understanding the history of\nprimary productivity through time are very important. I would recommend publication\nafter my three comments are taken into account.\nIn short, the isotope fractionation associated with present day nitrogenase enzymes has\nbeen used to infer their operation in the past assuming actualism, which implies not only\n1) that this enzyme has been fractionating the N-isotopes in the same way since their\nappearance but also 2) that no other enzymatic pathway, now unknown or even lost,\nwas doing the same thing in the past. This paper tests the hypothesis 1 by\nreconstructing ancient nitrogenase, introducing them in modern organisms, themselves\ngrown, harvested and analysed for their isotope composition.\nHowever, the hypothesis 2 remains impossible to test today (unless my knowledge in\nbiology is too limited), and this should not be omitted from the introduction, the\ndiscussion, conclusions and hence abstract. This in important because the findings of\nthis paper are indeed compatible with the interpretation that the nitrogenase was there\nvery early in the Earth history but it is not a demonstration for it. It might also be\npossible that other enzyme were able to fix N2 (comment 1).\nThank you for this thoughtful comment. While it is indeed possible that an alternative\nenzyme could have catalyzed nitrogen fixation prior to the emergence of the canonical\nnitrogenase, all current phylogenetic and biochemical evidence supports the most\nparsimonious conclusion that nitrogen fixation evolved only once, via the nitrogenase\nenzyme. We have nevertheless added a paragraph in the Discussion to acknowledge this\npossibility and to emphasize that our findings do not exclude other potential ancestral\nextinct pathways. We also note that future synthetic biology approaches could, in\nprinciple, explore alternative catalytic solutions to nitrogen fixation, which would represent\nan exciting direction for future research.\nAs a specialist of the measurement of nitrogen stable isotope I do not have comment to\nmake about the measurement of the biomass, but it seems to me that the measurement\nof the laboratory air d15N needs more work (comment 2). The isotope value of the\nlaboratory air reported in the paper is weirdly at +1,5‰. Air is the international standard\nand its d15N should therefore be at 0‰. This has been established by Mariotti since the\n80 (Mariotti, A. Natural 15N abundance measurements and atmospheric nitrogen\nstandard calibration. Nature 311, 251–252, 1984). Changing it to +1,5‰ given the 80%\nof N2 in air seems to me to be mission impossible. I have often measured air in my lab\nto validate off-line extraction protocols and it was systematically around 0‰ although\nwith significant scatter owing the presence of Argon that produced interferences in the\nmass spectrometer.\nA value of +1,5‰ cannot be published as it is without questioning it/discussing it even if\nit had been doubled checked in another lab (which should be done here). It would be far\nmore appropriate to use the value of 0‰ which would increase the values of the\nfractionation factor by 25% relative to the values determined in the manuscript. While this\nwould not change the conclusions much, it might have strong implications for future work\nusing these numbers.\nThank you, we appreciate this comment and agree with your reasoning. We have\nrecalculated the fractionation using the standard air value of 0‰, as well as updated the\ndata and figures (Fig. 3 and 4; Extended Data Fig. 5) in the manuscript accordingly. We\nhave also measured a sample of lab air at the Stable Isotope Facility at UC-Davis and\nnow included a figure in the extended data (Extended Data Figure 4) that shows\nfractionation using the UC-Davis lab air measurement. We have included a description of\nUC-Davis methods in the methods section as well.\nAnd finally, my comment 3 derives directly from comment 1: most of the rock record\npresents d15N values much higher than -3‰. These values imply either that other\nprocesses of the biogeochemical N-cycle were occurring that increased the d15N\nrecorded in the sediments (as discussed in the manuscript), or that an unknown and\npossibly extinct enzymatic pathways were fixing N2, with a fractionation factor\ncompatible with the more positive values. This possibility would deserve to be\nmentioned or dismissed by discussing it in the paper.\nThank you for this comment; we have mentioned this possibility in the discussion. Values\nhigher than -3‰ in the rock record are likely associated with abiotic processes, such as\nNH volatilization or NH + oxidation, that occurred with the expansion and evolution of the\n3 4\nN-cycle (Thomazo et al., 2011 Geobiology; Stüeken et al., 2015 Earth Planet Sci Lett;\nVelazquez et al., 2025 Geobiology). Since the exact cause(s) of high δ15N in the rock\nrecord are still unresolved, we have kept mention of these oxidation processes as is.\nFigure comments\n Figure 3 d : I do not understand what the linear regression are for. I wonder if the\ndifferences in isotope values could be due to analytical artefacts. This could be\ndiscussed.\nThank you for this comment. The linear regression line is included to visualize\npotential correlations between OD and δ15N values. The data were not\ntransformed in any way with the addition of this trend line, and the R2 values are\nconsistent with calculated Pearson correlation coefficients as well. We have now\nclarified this in the figure caption. We also note that the small variations observed\nare within analytical uncertainty. Similar data presentation approaches have been\nused in previously published work (e.g., Zerkle et al., 2008 Journal of\nGeophysical Research: Biogeosciences).\n Figure 4 : the word archaea is missing in the last sentence of its legend.\nThank you, this has been added.\n Extended figure 3: the references for bacteria and archaea fractionation factors\nshould be provided in the legend.\nThank you, we have added the appropriate references. Please note that this\nfigure is now Extended Figure 5."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have satisfactorily addressed all of my comments and concerns and present an important, original and\ninteresting study at an (under-explored) interface between biogeochemistry and ancestral sequence reconstruction.\nIt is my opinion that manuscript is acceptable for publication in Nature Communications in its current state.", "reviewer_3": "My comments have ben taken into account and I suggest minor clarifications and corrections.\nThe sentence added in the method for the measurement of d15N in air :\n\"An aliquot of the lab air (2 mL) was analyzed via a Sercon CryoPrep gas concentration system interfaced to a Sercon 20-20\nisotope-ratio mass spectrometer at the University of California, Davis Stable Isotope Facility (Extended Data Fig. 4).\"\nshould be modified to include the value that has been measured (value + number of replicates + precision...). Or this value\nshould be given in a result section somewhere or in the section when it is explained that the value chosen for N2 was 0‰.\nIn this sentence the words \"Extended Data Fig. 4\" should be replaced by : \"the isotope fractionation associated with nitrogen\nfixation calculated using this value for air are given in the extended Data Fig. 4\"\nThe extended data table S5 does not seem to have been adjusted. The fractionation factors are much too high (probably\ncalculated using the 1,5‰ value for air), and the reference (n°41) for modern fixation is not right.\nTable S4, S5 and S6 : Only one figure after the coma should be given on delta epsilon values and two on standard\ndeviations since the later are usually not better than 0,1‰.\ntable S6 : the measured values must be in d15N as it was initially. Espilon is not measured, it is calculated from the\nmeasured values d15N using a d15N value for air, and is an interpretation."}, "author_response": "Response to reviewer comments:\nR3: My comments have ben taken into account and I suggest minor clarifications and corrections.\nThe sentence added in the method for the measurement of d15N in air :\n\"An aliquot of the lab air (2 mL) was analyzed via a Sercon CryoPrep gas concentration system interfaced to a Sercon 20-20\nisotope-ratio mass spectrometer at the University of California, Davis Stable Isotope Facility (Extended Data Fig. 4).\" should be\nmodified to include the value that has been measured (value + number of replicates + precision...). Or this value should be given in\na result section somewhere or in the section when it is explained that the value chosen for N2 was 0‰.\nIn this sentence the words \"Extended Data Fig. 4\" should be replaced by : \"the isotope fractionation associated with nitrogen fixation\ncalculated using this value for air are given in the extended Data Fig. 4\"\nThank you. We have added the value to this portion of the methods section and modified the sentence to include the reference to\nextended data figure 4.\nThe extended data table S5 does not seem to have been adjusted. The fractionation factors are much too high (probably calculated\nusing the 1,5‰ value for air), and the reference (n°41) for modern fixation is not right.\nWe believe the reviewer is referring to extended data figure S5, which we have now updated to reflect the adjusted fractionation\nfactors. We have also updated the citation.\nTable S4, S5 and S6 : Only one figure after the coma should be given on delta epsilon values and two on standard deviations since\nthe later are usually not better than 0,1‰.\nThank you, these values have been updated accordingly.\ntable S6 : the measured values must be in d15N as it was initially. Espilon is not measured, it is calculated from the measured\nvalues d15N using a d15N value for air, and is an interpretation.\nWe have reverted the table legend back to δ15N."}]

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