Adaptation of Fe-S Cluster Assembly to Rising O2 Levels over Geological Time

Adaptation of Fe-S Cluster Assembly to Rising O2 Levels over Geological Time | 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 Adaptation of Fe-S Cluster Assembly to Rising O 2 Levels over Geological Time Hailiang Dong, Hongyu Chen, Franklin Outten, Li Huang, Zhenfeng Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7916008/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract One of the most important events in Earth history is the Great Oxidation Event (GOE). While O 2 killed most anaerobic microorganisms, some survived. Fe-S clusters are cofactors essential for cellular processes in all life forms, but how they adapt to rising O 2 remains unclear. Sulfur utilization factor (SUF) pathway is one of the most common Fe-S assembly pathways. We hypothesize that within the SUF pathway, SufE, as a sulfur-transfer partner of cysteine desulfurase SufS, maintains its functions under oxidative stress through molecular adaptation. Molecular clock dating showed SufE originated ~2.67 Ga (i.e., last common ancestor, LCA) and diversified considerably around the GOE (~2.14 Ga). The corresponding ancestral SufS was also reconstructed for these two times. Biochemical assays reveal that SufS LCA /SufE LCA is active at up to ~2% O 2 , higher than Archaean atmospheric O 2 , whereas SufS GOE /SufE GOE is active at up to ~10% O 2 , higher than the level during the GOE. These advanced evolutions may have provided resilience to redox fluctuations through Earth history. Growth experiments showed that overproduction of either SufE GOE or SufS GOE /SufE GOE in Escherichia coli mutants lacking SufE or SufSE better restores its growth than overproduction of their LCA counterparts, consistent with the in vitro results. Enzyme structure prediction revealed that such adaptation was achieved through replacement of a few amino acids in key catalytic sites and consequent conformational changes of key enzymes. Our results reveal the molecular mechanism of adaptation of Fe-S cluster assembly to rising O 2 and significantly contributes to the coevolution of the geosphere and biosphere. Biological sciences/Evolution Earth and environmental sciences/Biogeochemistry Biological sciences/Microbiology Figures Figure 1 Figure 2 Main One of the most important events in Earth history is the so-called Great Oxidation Event (GOE) 1,2 . It refers to a time period (between 2.46–2.06 Ga) when the Earth atmosphere and shallow sea experienced a rise in the free O 2 . During the latter half of GOE (i.e., Lomagundi-Jatuli Event, LJE, 2.22–2.06 Ga), atmospheric O 2 levels experienced a marked but temporary increase 3,4 . While accumulation of free O 2 killed the majority of anaerobic microorganisms, sometimes termed the first mass extinction event 5 , some survived through evolution to either tolerate or utilize O 2 6 . However, the molecular mechanisms underlying the evolution remain to be understood. Analysis of a biological process prevalent in extant life and yet ancient and sensitive to molecular oxygen may offer valuable clues. Iron-sulfur (Fe-S) clusters are such ancient cofactors. They are essential for a wide range of enzymes and metabolic processes, such as DNA repair and electron transfer 7–9 . Biosynthesis of Fe-S clusters requires multiprotein assembly systems that mobilize sulfur, iron, and electrons to construct clusters and insert them into target apoproteins 9 . In Escherichia coli , two major Fe-S assembly pathways exist: the housekeeping ISC system, functioning primarily under normal conditions, and the stress-responsive SUF system, which is induced under oxidative stress and iron limitation 10–13 . Comparative phylogenomic analysis indicates that the SUF pathway is one of the oldest Fe-S biogenesis machineries: minimal SUF-like systems are implicated in the last universal common ancestor and distributed among Bacteria and Archaea 14 . This broad distribution underscores the functional plasticity of SUF and suggests that this pathway played a central role in early microbial adaptation to fluctuating redox conditions. Notably, stress-inducible SUF systems (such as that in E. coli ) utilize SufE as the sulfur-transfer protein. Rate-smoothed phylogenies further indicate that the emergence of accessory cysteine desulfurase SufS and its sulfur-carrier partner SufE were triggered by oxygenation of the Earth 15 . However, the timing of their emergence remains uncertain and warrants further study. SufE is a small sulfur-transfer protein of the SUF system. In E. coli , SufE accepts a sulfur atom from the cysteine desulfurase SufS and then delivers it to the scaffold complex (SufBC 2 D) for Fe-S cluster assembly 16,17 . This transient SufS/SufE interaction is vital for efficient sulfur transfer 18 . SufE forms a hydrophobic pocket to protect cysteine-persulfide from oxygen in aqueous solution 19 . Indeed, SufS and SufE have been shown to be crucial for bacterial survival under oxidative stress conditions in E. coli 20 . However, how SufS/SufE has evolved in response to rising atmospheric O 2 over geological time remains unknown. We hypothesize that oxidative pressure favored genetic mutations in SufE and SufS to maintain Fe-S biogenesis in an increasingly oxidizing world. We focused on SufE because of its central role in sulfur transfer under oxidative stress and its small size, which would allow relatively rapid evolutionary adaptation 14 . We also examined SufS, as the SufS/SufE cooperating pair likely underpins the functionality of the Suf pathway under oxidative stress 20–22 . In this study, we investigated the O 2 -driven evolution of SufS/SufE. We performed phylogenetic analyses and molecular clock dating to predict the emergence and diversification of SufE in the context of the oxygenation history of Earth, and reconstructed ancestral sequences of SufE (and SufS) corresponding to two key time points, i.e., ~ 2.67 Ga, when SufE originated, and ~ 2.14 Ga (at the LJE). Our data reveal how O 2 has shaped the evolution of the proteins, and therefore provide significant insights into molecular events in the explosion of aerobic life through Earth history. SufE emergence before the GOE We constructed a maximum-likelihood phylogeny of SufE homologues from over 7,000 prokaryotic genomes (Extended Data Fig. 1). Among these basal candidate lineages, we chose Gammaproteobacteria clade II for molecular dating because (i) phylogenetic reconciliation of 120 single-copy core genes with sufE yielded fully congruent topologies, indicating strictly vertical inheritance with only one detectable horizontal gene transfer (HGT) event and (ii) the clade spans a well-resolved evolutionary gradient in heme-copper oxidase (HCO) types responsible for O 2 reduction to water. The basal lineages encode microaerobic C-type HCOs, whereas the more recently derived lineages possess fully aerobic A-type HCOs. This stepwise shift from microaerobic to aerobic respiration provides an ecological framework that directly links divergence times to progressive adaptation to increasing oxidative stress (Extended Data Figs. 1–2). Bayesian relaxed molecular clock analyses using the autocorrelation rates (AR) model placed the last common ancestor (LCA) of SufE (Fig. 1, hereafter named SufE LCA ), represented by the basal lineage of Gammaproteobacteria clade II near the root of the SufE phylogeny, at 2.67 Ga (95% Highest Posterior Density, HPD: 2.49–2.83 Ga), shortly after the inferred origin of oxygenic cyanobacteria (~ 2.73 Ga) (Figs. 1, Extended Data Fig. 3–4). This temporal proximity implies that SufE evolved in response to the “whiffs of O 2 ”, potentially preceding the global-scale GOE. This result suggests that the SufS/SufE system is an ancient invention, emerging between the appearance of oxygenic cyanobacteria and the advent of O 2 14 . Subsequent branching patterns indicate that major diversification or expansion of the SufE family occurred at the latter half of the GOE, e.g. LJE (Fig. 1, hereafter named SufE GOE ). Notably, the SufE GOE node represents the earliest SufE-carrying lineage isolated from terrestrial environments and was dated to 2.14 Ga (95% HPD: 1.98–2.29 Ga). This age coincides with the time of thickening of the continental crust 23 and rising O 2 levels, suggesting that SufE proteins were under selective pressure to accommodate elevated oxidative stress during the early colonization of land niches. We interpret this as evidence that the selective pressure from a rise in O₂ led to rapid diversification of SufE sequences and/or a selective sweep of O 2 -tolerant SufE variants around that period. Together, these results suggest that SufE underwent rapid evolutionary divergence under early oxidative stress, likely adapting to the sporadic O 2 “whiffs” produced by nascent cyanobacteria before the GOE 1 and to the rapid increase of O 2 during the LJE period. We set out to test if the changes in SufE sequence are bona fide molecular adaptations that resulted in improved SufE function under oxidative stress. Our approach involves ancestral sequence reconstruction coupled with the comparison of ancestral and existent proteins in biochemical and functional assays. In vitro response of SufSE activity to O 2 Two nodes in the SufE phylogenic tree, LCA and GOE SufE, were selected for detailed ancestral sequence reconstruction because they bracket the GOE, permitting a direct test of a functional shift in response to an increase in O 2 level. Three SufS/SufE pairs, corresponding to SufS LCA /SufE LCA , SufS GOE /SufE GOE and SufS modern /SufE modern ( E. coli K12), were expressed in E. coli and purified under anerobic conditions. The reconstructed and modern sequences were aligned (Extended Data Figs. 5–6). The core catalytic residues, such as C51 in SufE modern (corresponding to both C51 in SufE GOE and SufE LCA , Extended Data Fig. 5) and C364 in SufS modern (corresponding to C369 in SufS GOE and C368 in SufS LCA , Extended Data Fig. 6), have remained unchanged over time. However, there are mutations in the loop between β1 and β2 of SufE, sites of interaction between SufS and SufE, and the interface of the SufS homodimer (Extended Data Figs. 5–6). To determine the function of LCA, GOE and modern SufS/SufE pairs in response to rising O 2 , each pair was assayed for cysteine desulfurase activity by detecting sulfide production across an O 2 gradient (0–21%). All three variants of SufE greatly enhanced the low basal activity of SufS (data not shown), presumably by acting as a persulfide acceptor, thereby facilitating SufS turnover, consistent with previous studies for the modern pair 20 . Furthermore, under strictly anoxic conditions (0% O₂), all three SufS/SufE pairs were active, but the modern pair exhibited the highest catalytic efficiency (Fig. 2a). Both the modern E. coli and the GOE pairs showed a V max /K m ratio ~ 40–50% greater than that of the LCA pair. However, the three pairs exhibited markedly different patterns in response to O 2 . The activity of the LCA pair decreased precipitously at 2% O 2 (Fig. 2a, green curve), fell to less than half of its anoxic value at 5% O 2 , and essentially dropped to the baseline level of SufS at 10% O 2 . The SufS GOE /SufE GOE (Fig. 2a, blue curve) largely retained its anoxic activity at low O 2 levels (i.e., ~ 90% activity at 5% O 2 and ~ 70% at 10% O₂) but dropped to the baseline level of SufS at 15% O 2 . In stark contrast, the modern pair from E. coli K12 (Fig. 2a, red curve) retained its full activity up to ~ 5% O 2 and only dropped to ~ 80% at the modern O 2 level (21%). These results indicate a stepwise improvement in O 2 tolerance from the LCA to the GOE to the modern variants. We then determined if SufS or SufE was primarily responsible for the O 2 sensitivity by pairing the SufS modern with either the SufE LCA or the SufE GOE (Fig. 2b). Under anoxic condition, the SufS modern /SufE LCA combination increased the activity by ~ 100% relative to the SufS LCA /SufE LCA combination, yielding a catalytic efficiency indistinguishable from the SufS modern /SufE modern pair. Therefore, it is SufS that determines the maximal catalytic efficiency of the full SufS/SufE transpersulfuration reaction. As O 2 concentration increased, the activities of the three hybrid combinations show similar response patterns (Fig. 2b) to those of the three “age-match” combinations (Fig. 2a). It appears that, when SufS was kept unchanged (modern variant), different SufEs caused the difference in O 2 sensitivity among the three hybrid SufS/SufE pairs (Fig. 2b). To understand the structural basis of the difference in catalytic activity among the three SufS/SufE pairs, AlphaFold3 was used to predict the structures of SufS and SufE complexes based on a rigid docking model 18 . In the SufS LCA , a small and flexible histidine is present at position 349 (corresponding to 350 in SufS GOE and 345 in SufS modern , Extended Data Fig. 6) 24 . This residue, H349, fails to occupy the R121 cavity of SufE LCA (corresponding to R121 in SufE GOE and R119 in SufE LCA ) and allows R121 to stay at a blocking position (Fig. 2e). As a result, the distance between C368 of SufS LCA and C51 of SufE LCA (i.e., 15.723 Å) is sufficiently large to slow down the persulfide transfer from SufS to SufE, thus accounting for its low catalytical activity. In contrast, in SufS modern (also in SufS GOE ), a larger and more rigid tyrosine replaces histidine at position 345 (Extended Data Fig. 6) and occupies a cavity vacated by the C51 loop of SufE modern . Consequently, R119 in SufE modern is forced to move toward an outward position, allowing C51 of SufE modern to approach C364 of SufS modern for rapid persulfide transfer (Fig. 2f) 18 . Thus, the increased catalytical activity of the modern and GOE pairs, relative to the LCA pair (Fig. 2a), is likely triggered by the replacement of histidine in SufS LCA by tyrosine in SufS GOE and SufS modern to result in a more efficient interaction between the SufS and SufE catalytic sites. There are other residue-level substitutions along the β-latch/homodimer interface of SufS (i.e., K92 in SufS GOE versus R92 in SufS LCA and SufS modern variants, Q255 in SufS LCA versus E255 in SufS GOE and E250 in SufS modern variants), but they do not appear to compromise the monomer-monomer interaction of SufS (Extended Data Fig. 7) and the ability of SufS to interact with SufE. In addition to these substitutions on SufS, there are numerous differences among the three variants of SufE (Extended Data Fig. 5) that may jointly account for the measured differences in their catalytic activities among the three SufS/SufE pairs (Fig. 2a-b). For example, relative to the SufE modern , the SufE LCA and SufE GOE show insertions of two amino acid residues between β1 and β2, as well as numerous substitutions both between and within these β-strands (Extended Data Fig. 5). Although these sites are spatially distant from the SufS/SufE interface, they may perturb the overall conformation of SufE, thereby modulating its association with SufS and, in turn, the capacity of SufS/SufE pairs to withstand oxidative stress (Fig. 2). In vivo response of SufSE activity to O 2 To determine the response of SufS and SufE activity to rising O 2 levels in vivo, we analyzed the functionality of the SufS LCA /SufE LCA and SufS GOE /SufE GOE pairs in E. coli K12. First, we tested if these variants could rescue the synthetic lethality of strains lacking both the housekeeping Isc system and either SufE or both SufS/SufE. Normally, such mutants are inviable due to loss of Fe-S cluster-dependent isoprenoid biosynthesis 25 . Conditionally lethal Δ iscU-fdx Δ sufE::cm R and Δ iscU-fdx Δ sufSE::cm R mutant strains were constructed by inserting a non-native, hybrid mevalonate-dependent MVA system that does not require any Fe-S cluster enzymes for isoprenoid biosynthesis 26 . These strains absolutely rely on the non-native MVA system and addition of mevalonate to the growth media in order to restore isoprenoid biosynthesis. We then tested if introducing either ancient SufE alone (SufE LCA or SufE GOE ) or ancient SufS/SufE pair (SufS LCA /SufE LCA or SufS GOE /SufE GOE ) into a pBAD plasmid could allow the conditionally lethal strains to grow without mevalonate. We found that all SufE variants or SufS/SufE pairs rescued the lethality of the strains in the absence of mevalonate under atmospheric O 2 conditions (i.e., at the origin, Fig. 2c-d). We surmise that basal intracellular oxidative stress at atmospheric O 2 concentrations was probably not high enough to distinguish their ability in Fe-S cluster biogenesis, especially in the rich, glucose-supplemented media used for the experiment. To increase the oxidative stress, the Δ iscU-fdx Δ sufE::cm R strains transformed with the three variants of SufE on the pBAD plasmid were subjected to phenazine methosulfate (PMS). PMS generates intracellular superoxide radicals, imposing extra oxidative stress that damages Fe-S clusters and necessitates active repair/biogenesis systems 27 . Indeed, growth assays revealed clear differences in functional restoration of the three SufE variants under increasing PMS concentration (Fig. 2c). Across 0–30 µM PMS, all complemented strains exhibited a modest stimulatory response, with the maximum specific growth rate (µmax) increasing and peaking at 30 µM. From 30–60 µM, the µmax values declined modestly yet remained above that for the no-PMS control. Above 60 µM, the µmax values dropped sharply for all variants, and the resulting viability limits separated the three lineages: the SufE LCA ceased growth above 150 µM, the SufE GOE above 240 µM, and the E. coli K12 SufE modern above 300 µM. Thus, while the low dose stimulation is shared, the SufE modern extends the tolerable PMS window to a much higher level of PMS. These in vivo data mirror the in vitro biochemical assay results, showing that the evolutionary enhancements to SufE’s sequence translate into a tangible survival advantage under oxidative stress. Similar complementary experiments were performed for the Δ iscU-fdx Δ sufSE::cm R strain lacking both SufS and SufE. The normalized expression levels of SufS and SufE from LCA, GOE and modern strains were similar without PMS (Extended Data Fig. 8). Baseline growth in the absence of PMS showed that the SufS GOE /SufE GOE and SufS modern /SufE modern supported nearly identical maximum growth rates, both slightly lower (by ~ 0.1 h⁻¹) than in the Δ iscU-fdx Δ sufE::cm R strain complemented with SufE [~ 0.8 log(OD 450 ) h − 1 vs ~ 0.7, Fig. 2c-d]. In contrast, the SufS LCA /SufE LCA pair had a µmax value reduced by ~ 50% (Fig. 2d), matching the magnitude of its diminished catalytic efficiency in vitro (Fig. 2a). Unlike the results in the ∆ sufE strain, no hormesis was observed in the ∆ sufSE strain: the µmax value declined slightly over 0–30 µM PMS, then fell steeply from 30 to 240 µM (Fig. 2d). Consistently, the upper tolerance limits of PMS were markedly lower in the complemented ∆ sufSE strain compared to those in the complemented ∆ sufE strains, which ceased growth above 100 µM, 150 µM, and 240 µM PMS for LCA, GOE, and modern SufS/SufE pairs, respectively (Fig. 2d). Importantly, under higher PMS stress, the modern SufS/SufE consistently outperformed the GOE and LCA variants. Implications for life evolution through Earth oxygenation Our findings reveal a clear evolutionary trajectory in one of the most important biochemical pathways: SUF Fe-S biosynthesis system, driven by Earth’s rising atmospheric O 2 concentration. The SUF pathway, though present in early anaerobic life, had to be refined to remain functional as the atmospheric O 2 levels rose. By reconstructing ancient SufS and SufE proteins, we directly observed that the SufS LCA /SufE LCA and SufS GOE /SufE GOE complexes were poorly adapted to an O 2 -rich environment, while the modern complex was highly O 2 -tolerant. SufS determines the catalytic ceiling, but SufE governs how much oxidative load that the complex can withstand. Thus, neither component is subordinate: evolution jointly optimized SufS and SufE, likely by tuning their global geometries and coordinating a protected handoff of persulfide to SufBC 2 D scaffold. These results underscore the power of O 2 as an agent of natural selection. Certain housekeeping SUF systems (e.g., in Bacillus subtilis ) employ SufU as a SufE analog, serving as the cysteine desulfurase partner for sulfur transfer in those organisms 28 . A histidine 349 is critical for SufS LCA (corresponding to Y345 in SufS modern and Y350 in SufS GOE ) when it interacts with SufU which requires a Zn 2+ cofactor to coordinate interactions during persulfide transfer. Phylogenetically, SufU predates SufE (Extended Data Fig. 9). However, bioavailable Zn in Archaean oceans was extremely low 29 . As a result, SufE may have evolved as a zinc-independent ‘rescue’ module for the SUF pathway under anoxic condition. However, SufS/SufE pair faces a great challenge of Earth’s progressive oxygenation. Our molecular dating result indeed suggests that once localized O 2 appeared, molecular adaptation followed rapidly. The first detectable divergence within the SufE clade was only ~ 60 Myr after the median age of oxygenic cyanobacteria, which is a small age gap in the Archaean context. SufE emerged in lineages that likely shared ecological space with the earliest O 2 producers. Furthermore, the molecular adaptation was not only quick, but the oxidative tolerance of SufS and SufE actually evolved ahead of contemporaneous O 2 levels. The earliest-divergent SufE LCA already withstands ~ 2% O 2 , exceeding most estimates for ambient O 2 at this time (~ 0.1-~0.5%) 30,31 . Likewise, geochemical proxies indicate ~ 4% O 2 around the LJE 3 , but our in vitro biochemical assay showed that the SufE GOE already tolerates ~ 10% O 2 . These systematic overshoots of O 2 tolerance suggest selection for excess capacity as a safety margin in stress-response machineries, which possibly provides resilience to redox excursions and facilitates the progressive expansion of increasingly aerobic niches. A similar type of advanced preparedness in response to increasing stress is also shown in antibiotic stress-response 32 . In addition to oxidative tolerance, the increased catalytic efficiency of SufS/SufE from the LCA to GOE/modern variants may reflect selective pressure to increase rates of Fe-S cluster biogenesis in vivo. During aerobic growth, cells experience a constitutive level of Fe-S cluster damage in sensitive enzymes such as dehydratases, requiring a compensatory increased level of cluster biogenesis to maintain cell function 33 . In contrast, during anaerobic growth, Fe-S cluster turnover is much lower and the demand for cluster biogenesis is lower. From a broader perspective, the O 2 -driven evolution of SufE and SufS exemplifies how life’s molecular machinery has been molded by planetary change. The GOE forced the innovation of more efficient Fe-S cluster assembly tools, which in turn enabled organisms to exploit O 2 for metabolism while preserving their ancient biochemical capabilities. Without such adaptations, essential processes, including respiration, DNA repair, and metabolic pathways that depend on Fe-S proteins could have failed in an oxygenated world. One particular example of such adaptation is ancient nitrogenases, Fe-S proteins that are used for N 2 fixation. Molecular dating suggests that Mo-based nitrogenase originated in the anoxic mid-late Archaean age (3.1–2.7 Ga) 34 , but survived the GOE and retained the same basal structure and functions even in modern aerobic microorganisms 35 – 37 . Conformational change is one important mechanism against oxidative stress, where the Shethna (FeSII) protein binds to the nitrogenase complex to protect it from oxygen damage 38 , 39 . While the specifics may vary among different functional groups of organisms, our study reveals an important and possibly widespread defense mechanism against oxidative stress through the conformational change of the SufS/SufE interface. This work illustrates how geochemistry and biochemistry are intertwined: oxygenic photosynthesis leads to atmospheric oxygenation, and such a planetary-scale change drives molecular innovation, which in turn enables new biological capabilities. Our study therefore connects the evolution of a single protein complex to Earth’s largest evolutionary inflection point, even the Cambrian radiation, by way of the O 2 that links them. This integrative perspective from atmosphere to enzyme to evolution deepens our understanding of how life’s molecular machinery is molded by our changing planet and invites further investigations using ancestral protein reconstruction to explore other episodes where environmental change and biochemical evolution converged. Methods Molecular clock and Ancestral Sequence Reconstruction Multiple sequence alignment and phylogenetic analysis Four experimentally validated SufE/CsdE proteins from UniProt (downloaded 24 Oct 2022) with “UniProtKB reviewed” status (P76194, Q9EXP1, B5BA16, Q1C762, P0AGF2) were used as seeds for homology searches against the NCBI NR protein database (June 2022, 483,768,206 non‑redundant sequences) using DIAMOND blastp 40 with an E-value of < 1e-6 and a length filter of 90-170 amino acid residues, yielding 23,567 SufE‑like hits. Candidate sequences were verified by HMMER 41 hmmscan against Pfam 42 hidden Markov model (HMM) database, and validated sequences were dereplicated with CD‑HIT 43 (90% identity), grouped into 7,635 (NR) clusters. The longest sequence in each cluster was retained. The sequences were aligned with MAFFT 44 L‑INS‑i and trimmed using trimAl 45 with resoverlap 0.55 and seqoverlap 60. Maximum‑likelihood (ML) trees were inferred with FastTree 46 (for rapid exploration) and IQ‑TREE 2.2.1 47 (for focal subsets). Because of the lack of reliable outgroups for SufE, MAD 48 and MinVar 49 , outgroup‑independent methods were employed to allow identification of ancient lineages and candidate nodes for dating and ancestral sequence reconstruction. Visualization was performed with iTOL 50 , and sequences species annotations were fetched from NCBI identical protein group (ipg) database by Entrez 51 , using the most complete genome for each protein cluster using CheckM 52 completeness estimates. The putative oxygen‑respiration capacity of a species was inferred based on the presence of heme-copper oxidase (HCO) families A, B1, C, as detected with hmmscan (E-value < 1e-50), as described 53,54 . Gene-species tree reconciliation with curation of horizontal gene transfer (HGT) candidates To satisfy the vertical inheritance assumption for node dating and ancestral sequence reconstruction, SufE gene trees were reconciled with species trees, and those lineages which were likely recipients of HGT 55 were iteratively removed. For the focal lineage (301 proteins and 1,455 candidate genomes), open reading frames were predicted with Prokka 56 , and species phylogeny was built with GTDB‑Tk 57 bac120 single‑copy bacterial markers (hmmscan annotation, E-value < 1e-50). Each marker was aligned with MAFFT, trimmed with trimAl, and concatenated. The species tree was inferred under LG+C20+F+I+G in IQ‑TREE. Gene-species reconciliation was performed with GeneRax 58 under species‑tree‑aware ML, using SPR search and an unconstrained DTL model (radius 5). Leaf‑level HGT acceptors, acceptors at internal nodes transferring to leaves, and transfers between deep internal nodes of comparable depth were successively removed. After six iterations, transfers were reduced from 34 to 1, yielding a curated set of 131 vertically inherited SufE sequences and genomes for dating. Molecular clock analyses To anchor both deep Proterozoic and Phanerozoic timescales, the following two sets of species were used: (i) 21 oxygenic cyanobacteria and 3 melainabacteria (i.e., the cyanobacterial outgroup), and (ii) mitochondria‑proximal alphaproteobacteria and a set of mitochondrial markers (mito24) 59,60 . First, 10 alphaproteobacteria genomes were added close to mitochondria, built a bac120 species tree that included cyanobacteria and alphaproteobacteria, and then replaced the alpha clade with four alternative mitochondria-alphaproteobacteria topologies (TP1-TP4) reported in prior work 59 . The final pruned matrix contained 181 taxa (131 SufE genomes, 24 cyanobacteria/melainabacteria, and 26 mitochondria/alphaproteobacteria). The mito24 alignment contributed 6,749 concatenated amino‑acid positions after trimming. Eight calibrations spanning the root and key crown groups were used: origin of life, origin of oxygenic Cyanobacteria, crown Nostocales, crown Pleurocapsales, red algal Bangiophyceae and Florideophyceae, crown bryophytes, and crown eudicots. Root bounds explored broad windows reflecting early habitability constraints (upper bound 4.5 Ga; lower bounds 4.0, 3.5, or 3.0 Ga, sensu impact chronology and earliest microfossils). Cyanobacterial bounds bracketed literature estimates from geochemical proxies and biomarkers (upper 3.0 Ga 61 ; lower 2.32 or 2.50 Ga) 62 . Fossil minima for Nostocales (1.6 Ga) 63 and Pleurocapsales (1.7 Ga) 64 followed akinetes and microfossil occurrences. Eukaryotic calibrations followed recent syntheses for red algae, bryophytes and eudicots 59 . The root and cyanobacterial options with two eukaryote schemes (Euk1/Euk2) were combined to generate 12 calibration sets. These, along with four topologies for mitochondria (TP1-TP4) and two clock models (AR/IR; see below), produced 96 dating analyses (Supplementary Information Table S1). Bayesian relaxed‑clock dating was performed in PAML MCMCTree 4.10.7 65 using both auto‑correlated rates (AR) and independent rates (IR) relaxed-clock models. Before formal divergence time estimation, the gradient and Hessian matrices were obtained by using PAML CODEML to improve the accuracy of molecular clock results 66 . Each run involved a burn‑in of 2,000 iterations, sampling every 20 steps (20,000 posterior samples). Each configuration was run in duplicate with different random seeds. Convergence and performance were assessed by (i) mcmc3r diagnostics, (ii) infinite‑sites test, and (iii) stepping‑stone marginal likelihoods to compare topologies, calibrations, and clock models 67 . Ancestral protein reconstruction IQ-TREE (version 1.6) with the ‘-asr’ option was used to infer the amino acid sequences of ancestral SufE. The best-fit substitution model (LG+C20+I+G4) was chosen based on Akaike information criterion and Bayesian information criterion. The ancestral sequence reconstruction output provided the posterior probability for each amino acid at every site. Sites with a single amino acid having high posterior probability (> 0.30) were directly assigned that residue. For ambiguous sites with lower confidence, top three candidate residues were considered. If the top three residues exhibited similar properties, the highest-probability residue was chosen for that site. If candidates differed in property, the residue predicted to enhance protein solubility and allow the isoelectric point of the protein closer to that of E. coli SufE was selected. In cases where a gap (“-”) was the top inference for a site, top four candidates were considered. If none of the four candidate residues yielded a clear improvement in protein property or synthesis, the gap was retained 68 . ASR based on maximum likelihood (ML) method was also inferred by using PAML 65 and FastML 69 . The amino acid substitution model used in ML was LG. Representative reference SufS sequences were chosen from diverse taxa, including an extremophilic archaeon (D4gyV5, Haloferax volcanii ), a Gram-positive bacterium whose SufS interacts with SufU (O32164, Bacillus subtilis ), and E. coli K12, which encodes the canonical SufS (1C0N). SufS sequences were aligned as described above for SufE. A maximum likelihood tree of SufS genes was built from the 131 genomes used for SufE dating, using IQ-TREE, and ASR for SufS was performed in the same fashion as for SufE. Internal nodes corresponding to SufE LCA (2.67 Ga) and SufE GOE (2.17 Ga) were identified on the SufS tree, and the ancestral SufS sequences for these time points were inferred (designated as SufS LCA and SufS GOE , respectively). Ambiguous sites in the SufS reconstructions were resolved following the same criteria as those for SufE. AlphaFold3 protein structure prediction Reconstructed SufS and SufE variant structures were predicted by AlphaFold3 70 with the ratio of SufS to SufE equal to 2:1. The structure was visualized by ChimeraX. The PLP cofactor of SufS was matched by PDB 5XT6 SufS. In vitro experiments Protein preparation Genes encoding ancestral SufS/SufE proteins (LCA and GOE) were codon-optimized for E. coli and synthesized. The present-day sufS and sufE were PCR-amplified from the genomic DNA of E. coli MG1655. The resulting DNA fragments were cloned into pET-30a(+) via NdeI / XhoI to append a C-terminal His 6 tag. Since His 6 -SufS LCA construct was insoluble, SufS LCA was prepared by cloning the gene fragment into pMAL-c6T to generate an N-terminal MBP-TEV-SufS LCA fusion. All constructs were sequence-verified (primer lists and sequences in Supplementary Table S3). Plasmid constructs were transformed into E. coli BL21(DE3) pLysS. The strains were grown in LB at 37 °C to OD 600 ≈ 0.4, and protein synthesis was induced with the addition of 0.4 mM IPTG and subsequent incubation for 5 h at 37°C for SufE LCA , SufE GOE , SufE modern , SufS GOE , and SufS modern , and for overnight at 18 °C for MBP-SufS LCA . All of the following steps were performed in an anaerobic glove box (O 2 < 0.1%, Coy) at 4 °C. Cells were harvested, cell pellets were lysed in a buffer solution of 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 5 mM DTT and 1 mM phenylmethylsulfonyl fluoride (PMSF), and the lysates were removed (20,000 g, 30 min). His-tagged proteins were purified successively on a HisTrap column (Cytiva) with a 10-500 mM imidazole linear gradient, a HiTrap Q column (Cytiva) using a 50 mM-1 M NaCl linear gradient at a pH of ~2 units above the PI of the protein, and a Superdex 75 Increase 10/300 column (Cytiva) with an elution buffer of 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT. The MBP fusion protein was purified on an MBPTrap HP column (Cytiva) with maltose elution. The MBP tag was removed by overnight Tobacco Etch Virus (TEV) protease digestion at 4 °C, followed by subtractive Ni 2+ -affinity purification. Protein concentrations were determined by BCA assay against a BSA standard. Aliquots were flash-frozen and stored at -80 °C. Western blotting Proteins were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane for Western blotting, as previously described 71 , with an anti-His tag mouse monoclonal antibody (ThermoFisher Invitrogen) or anti-MBP monoclonal antibody (LabLead). An HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (ThermoFisher Invitrogen) was then used, and signals were detected using enhanced chemiluminescence (Thermo Scientific SuperSignal West Pico PLUS) and imaged (Tanon 1600). Cysteine desulfurase activity assay The cysteine desulfurase activity of the SufS/SufE system was measured using a methylene blue assay as described previously 72,73 . To determine the appropriate SufE concentration, the reaction mixture contained 0.5 μM SufS, a gradient of SufE (0, 0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10.0 μM ), and 0.5 mM L-cysteine in 25 mM Tris-HCl buffer (pH 7.4) with 150 mM NaCl. Using GraphPad Prism (v10.4) fit to the Michaelis-Menten equation to obtain the apparent K m of SufE. The formal reaction was conducted at a specified O 2 level in a modular atmosphere chamber, reaction conditions are 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 μM SufS, 2×K m concentrations of SufE, and L-cysteine 0-500 μM. SufS and SufE were pre-equilibrated separately in the chamber at the tested O 2 level for 30 min and then mixed for 15 min prior to the reaction. The reaction was initiated by adding L-cysteine. After incubation for 30 min at 27 o C, the reaction was quenched by adding 0.1 M NaOH. Next, DTT was added to 1 mM final concentration. Then 12.5 μL of 10% zinc acetate was added. Color development reagents, 25 μL of 20 mM DMPD and 25 μL of 30 mM FeCl 3 , were then introduced. The mixture was incubated for 30 min at room temperature in the dark and then shortly centrifuged. The absorbance of the supernatant at 670 nm was measured. One unit (1 mU) of cysteine desulfurase activity is defined as the formation of 1 μmol of S²⁻ per min under the assay conditions. In vivo experiments Strain construction To construct an E. coli strain for in vivo evaluation of the role of the reconstructed SufS/SufE proteins on oxidative stress response, we replaced a part of the isc operon ( iscUA-hscBA ) with a sequence encoding a ~4.2 kb heterologous mevalonate pathway 74 in E. coli strain MG1655 by using a high-efficiency, low-escape CRISPR/Cas9 genome editing method 75 with the designed guide sequences (Supplementary Information Table S3). The sgRNA protospacer was introduced by annealing oligonucleotides carrying ~20-bp vector overlaps and assembled into the linearized pTargetT backbone (SpeI-HF). A donor cassette (MVA) flanked by homology arms to the target locus (Δ iscU - hscA ) was assembled by overlap-extension PCR from genomic templates, followed by amplification with primers adding ~20-bp overlaps to the linearized pTargetT vector at 5’ end (Supplementary Information Table S3). pTargetT was linearized by restriction digestion and dephosphorylated (rSAP, NEB). The donor amplicon was inserted by Gibson assembly. All constructs were sequence-verified. E. coli MG1655 cells carrying pCas (Cas9/λ-Red) 76 were prepared at 30 °C. λ-Red was induced with 0.2% L-arabinose, and pTargetT-donor-sgRNA was then introduced into the cells by electroporation. Transformants were selected on LB agar with kanamycin (50 µg mL⁻¹) and streptomycin (50 µg mL⁻¹) in the presence of L-arabinose and/or MVA as indicated. Colonies were screened by PCR (Supplementary Information Table S3), and positive clones were confirmed by Sanger sequencing. pTargetT was cured by repeated passage in medium without streptomycin. pCas was subsequently cured by growth at 42 °C in the absence of antibiotics. The resulting strain was denoted E. coli MG1655 Δ iscU - hscA::MVA + (CHY97). To ensure that CHY97 closely matched the genetic background of other strains used for phenotype testing (WO539/WO541 from the W. Outten lab, see below), P1 phage transduction was used to move the MVA cassette from WO539/541 into the strain. In the WO539/541 strains, the PBAD-MVA-kan R cassette was integrated at the lac operon locus under the control of a BAD promoter and included a kanamycin resistance marker for selection 26 . P1 phage prepared on donor strain WO541 was transduced into CHY97, selecting for kan R colonies. This yielded strain CHY98 (genotype MG1655 Δ iscUAhscBA::MVA + + lac::[MVA+, kan R ] ), which carries the arabinose-inducible MVA pathway integrated at the lac locus, identical to WO539/541. CHY98 was used as a positive control strain in growth assays, as it retains the native suf operon and has the MVA pathway just like the WO539/541 mutants, allowing us to distinguish effects of the complementation constructs from any unintended effects of the MVA system or antibiotic markers. The donor and base strains used in this study: E. coli WO539 (MG1655 Δ iscU-fdx Δ sufSE::cm R MVA + -kan R ) and WO541 (MG1655 Δ iscU-fdx Δ sufE::cm R MVA + -kan R ) were constructed by multiple P1 transductions of mutations from previously constructed strains. Briefly, iscUA-hscBA-fdx was replaced with kan R from pKD4 and then the kan R was removed with plasmid pCP20 expressing flippase (FLP) recombinase 77 . The MVA + - kan R locus was then P1 transduced into the ∆ iscU - fdx background. Finally, the sufE gene or sufSE genes were replaced with cm R from pKD3 and then P1 transduced into the ∆ iscU-fdx MVA + - kan R background. The final transductants were selected on and maintained in “5-component SB medium” (see below) to ensure that they had the mevalonate supplement for initial recovery. This Super Broth (SB) was a rich medium (32 g L -1 tryptone, 20 g L -1 yeast extract and 5 g L -1 NaCl), supplemented with 0.2% (w/v) L-arabinose, 0.4% (w/v) D-glucose, 50 mg L -1 chloramphenicol, 50 mg L -1 kanamycin and 300 μM mevalonolactone (MVA, CAS 674-26-0). Complementation assays To complement the SufE/SufSE deletions in vivo, a series of arabinose-inducible plasmids carrying various combinations of present-day or ancestral sufE and sufS genes were constructed. For the single-gene constructs (pBAD/His B-SufE series), the coding sequences for SufE LCA , SufE GOE , and SufE modern were PCR-amplified (Supplementary Information Table S3) from their respective pET 30a(+) templates and cloned into pBAD/His B. This placed the sufE gene under the arabinose promoter, with an N-terminal His 6 -tag (from the vector) on the expressed SufE protein. For the sufS/sufE constructs (pBAD/His B-SufSE series), two genes were amplified in tandem, including their native intergenic spacer. In the case of the sufS modern and sufE modern , they were amplified directly from E. coli MG1655 genomic DNA using primer pair pBAD-SufSE-F and HindIII-His-SufE-R (Supplementary Information Table S3). For the sufS GOE /sufE GOE and sufS LCA /sufE LCA constructs, the sufS and sufE fragments were assembled by overlap extension PCR to include the native intergenic spacer (Supplementary Information Table S3). The two PCR fragments (ancestral sufS and s ufE ) were then joined by overlap PCR to create a full-length sufSE di-cistron fragment with spacer and tags. This fragment was digested and cloned into pBAD/His B. Similarly, for SufS LCA /SufE LCA adding MBP at N terminal, a bicistronic insert was generated where sufS LCA included the coding sequence for an MBP tag fused at its N-terminus (carried over from the pMAL-SufS LCA template, Supplementary Information Table S3). Thus, this construct tests the effect of co-expressing an MBP-tagged SufS LCA with SufE LCA in vivo. All pBAD constructs were verified by restriction mapping and sequencing. The resulting complementation plasmids were each introduced into the appropriate mutant background. Successful transformants were selected on 5-component SB with 50 mg L -1 ampicillin (to maintain the pBAD plasmid, referred to as “6-component SB), and verified. Glycerol stocks were made for each complementation strain. Growth assays under oxidative stress The growth phenotypes of the complemented strains under various oxidative stress conditions were assessed. The phenazine methosulfate (PMS) could generate oxidative stress inside cells. Concentration ranges for these stressors were determined based on literature and our pilot experiments: PMS was used at 0, 30, 60, 90, 150, 240, 300, and 450 μM 27 . For each strain, growth curves were obtained using a 96-well microplate format (200 μL culture per well, in at least three replicates). Prior to the experiment, all strains were revived from glycerol stocks on LB agar and then pre-cultured in a “6-component SB medium” (see above) to ensure they had the mevalonate supplement for initial recovery. After overnight growth in 6 component SB (37 °C, shaking), cultures were diluted 0.5% into the same medium but without mevalonate in order to test if the pBAD plasmids restored Fe-S dependent (MVA independent) isoprenoid biosynthesis. This medium still had ampicillin. We chose a 0.5% inoculum to minimize carryover of MVA from the starter culture. At this low inoculation, the control strain with empty plasmid (which cannot synthesize isoprenoids without MVA) did not grow for at least 48 h, confirming effective depletion of intracellular MVA with no carryover from the starter culture. Growth assays were performed in a FLUOstar Omega microplate reader (BMG Labtech) at 37 °C with continuous orbital shaking (200 rpm). The optical density was recorded every 5 min. Due to PMS’s color change upon oxidation (the medium gradually turned green, and even cell growth itself can alter PMS’s absorbance), a detailed spectral analysis (scanning 450-800 nm) showed that PMS causes elevated absorbance across a broad range (480-750 nm) and that high cell density plus PMS can increase OD readings in a wavelength-dependent manner. However, above 750 nm, the OD signal from cells alone started to diminish (due to scattering properties) and at wavelengths below 480 nm, the artifact due to PMS oxidation was minimal. OD 450 nm for PMS experiments was applied, as 450 nm gave a reliable indication of cell growth while avoiding most interference from PMS oxidation 78 . In all experiments, the first few OD readings (prior to significant growth, e.g. the mean of 0-5 min readings) were used as a blank and subtracted from subsequent readings for each well, to normalize starting OD. Each growth curve was then analyzed to extract key parameters. The R package gcplyr (v1.X) was utilized for growth curve analysis 79 . The OD data (log-transformed OD values vs. time) were fit to a growth model to estimate: the lag time (adjustment phase duration), the maximum growth rate (slope during exponential phase), and the maximum cell density (ODₘₐₓ). The results were plotted using GraphPad Prism. Parallel Reaction Monitoring (PRM) Targeted proteomics Synthesis of SufS and SufE in complementation cells was confirmed by targeted quantitative proteomics using PRM mass spectrometry. Cultures of the WO539 + pBAD/His B-SufSE (ancestral sufSE) strains were grown to log phase (OD 600 ~0.5) and stationary phase (overnight culture) in 6 component SB medium (with Amp and MVA) under aerobic conditions. Cells from 50 mL of each culture were harvested by centrifugation (5,000 g, 10 min, 4 °C), washed with cold PBS, resuspended in 200 μL of urea lysis buffer (7 M urea, 2 M thiourea, 0.1% CHAPS, 1× protease inhibitor cocktail), and lysed by sonication on ice (15 min total sonication time, in pulses). Total protein concentration in each sample was determined by BCA assay using a standard protocol 80 . An aliquot (~50 μg protein) of each sample was subjected to digestion and subsequent mass spectrum (MS) by following a filter-aided sample preparation (FASP) protocol 81 . For mass spectrometry, dried peptides (~0.5 μg) were analyzed by LC-MS/MS on an EASY-nLC 1200 UHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific). Separation was achieved on a C18 reversed-phase column (150 μm × 150 mm, 1.9 μm) using a 60 min gradient (8-45% acetonitrile/0.1% formic acid) at 500 nL min -1 . The mass spectrometer was operated in positive ion mode with full MS scans acquired at 120,000 resolution (m/z 350-1500), followed by higher-energy collisional dissociation (HCD) with normalized collision energy 30%. MS2 spectra were acquired in the Orbitrap at 15,000 mass resolution, with dynamic exclusion (45 s). The DDA MS/MS data were searched using Proteome Discoverer (v2.3, Thermo) against the UniProt E. coli K-12 proteome reference (downloaded 2024-06-11) with the sequences of SufS GOE /SufE GOE and SufS LCA /SufE LCA proteins appended (total 4406 sequences). Trypsin (up to 2 missed cleavages allowed) and Carbamidomethyl (Cys) as fixed, and Oxidation (Met) and Acetyl (protein N-terminus) as variable modifications were employed. Precursor ion mass tolerance was 10 ppm, and fragment ion tolerance 0.02 Da. Peptide-spectrum matches were filtered to a false discovery rate (FDR) of < 1% using a target-decoy strategy. Using Skyline (MacCoss Lab, v4.X), 3 unique peptides for each target protein (SufS variants and SufE variants of interest) that were confidently identified in the DDA run were selected. The precursor m/z for each target peptide and its charge state, along with the retention time (RT) window (~5 min around the observed RT), were compiled for PRM. In total, 12 peptides covering all target proteins were monitored by unique peptides. PRM assays were performed by measuring these peptides and corresponding stable isotope-labeled standards (SIS). Quantification was achieved by calculating the ratio of protein peptides to SIS peptide peak areas. Data and Code availability All data generated or analysed during this study are included in this published article, its Supplementary Information files and https://github.com/eacochen/SufE. Methods Reference s Buchfink, B., Reuter, K. & Drost, H.-G. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nature Methods 18 , 366-368 (2021). https://doi.org:10.1038/s41592-021-01101-x Potter, S. C. et al. HMMER web server: 2018 update. Nucleic acids research 46 , W200-W204 (2018). Mistry, J. et al. Pfam: The protein families database in 2021. Nucleic acids research 49 , D412-D419 (2021). Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics (Oxford, England) 28 , 3150-3152 (2012). https://doi.org:10.1093/bioinformatics/bts565 Katoh, K. & Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30 , 772-780 (2013). https://doi.org:10.1093/molbev/mst010 Capella-Gutierrez, S., Silla-Martinez, J. M. & Gabaldon, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics (Oxford, England) 25 , 1972-1973 (2009). https://doi.org:10.1093/bioinformatics/btp348 Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments. PLOS ONE 5 , e9490 (2010). https://doi.org:10.1371/journal.pone.0009490 Minh, B. Q. et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Molecular Biology and Evolution 37 , 1530-1534 (2020). https://doi.org:10.1093/molbev/msaa015 Tria, F. D. K., Landan, G. & Dagan, T. Phylogenetic rooting using minimal ancestor deviation. Nature ecology & evolution 1 , 0193 (2017). Mai, U., Sayyari, E. & Mirarab, S. Minimum variance rooting of phylogenetic trees and implications for species tree reconstruction. PloS one 12 , e0182238 (2017). Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic acids research 52 , W78-W82 (2024). Maglott, D., Ostell, J., Pruitt, K. D. & Tatusova, T. Entrez Gene: gene-centered information at NCBI. Nucleic acids research 33 , D54-D58 (2005). Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. & Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome research 25 , 1043-1055 (2015). Murali, R., Hemp, J. & Gennis, R. B. Evolution of quinol oxidation within the heme‑copper oxidoreductase superfamily. Biochim Biophys Acta Bioenerg 1863 , 148907 (2022). https://doi.org:10.1016/j.bbabio.2022.148907 Murali, R. et al. Diversity and evolution of nitric oxide reduction in bacteria and archaea. Proc Natl Acad Sci U S A 121 , e2316422121 (2024). https://doi.org:10.1073/pnas.2316422121 Ravenhall, M., Škunca, N., Lassalle, F. & Dessimoz, C. Inferring horizontal gene transfer. PLoS computational biology 11 , e1004095 (2015). Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics (Oxford, England) 30 , 2068-2069 (2014). https://doi.org:10.1093/bioinformatics/btu153 Chaumeil, P.-A., Mussig, A. J., Hugenholtz, P. & Parks, D. H. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics (Oxford, England) 38 , 5315-5316 (2022). Morel, B., Kozlov, A. M., Stamatakis, A. & Szöllősi, G. J. GeneRax: A Tool for Species-Tree-Aware Maximum Likelihood-Based Gene Family Tree Inference under Gene Duplication, Transfer, and Loss. Mol Biol Evol 37 , 2763-2774 (2020). https://doi.org:10.1093/molbev/msaa141 Wang, S. & Luo, H. Dating Alphaproteobacteria evolution with eukaryotic fossils. Nature Communications 12 , 3324 (2021). https://doi.org:10.1038/s41467-021-23645-4 Liao, T., Wang, S., Zhang, H., Stüeken, E. E. & Luo, H. Dating Ammonia-Oxidizing Bacteria with Abundant Eukaryotic Fossils. Molecular Biology and Evolution 41 , msae096 (2024). https://doi.org:10.1093/molbev/msae096 Crowe, S. A. et al. Atmospheric oxygenation three billion years ago. Nature 501 , 535-538 (2013). Zhang, H., Sun, Y., Zeng, Q., Crowe, S. A. & Luo, H. Snowball Earth, population bottleneck and Prochlorococcus evolution. Proceedings of the Royal Society B 288 , 20211956 (2021). Golubic, S., Sergeev, V. N. & Knoll, A. H. Mesoproterozoic Archaeoellipsoides: akinetes of heterocystous cyanobacteria. Lethaia 28 , 285-298 (1995). Golubic, S. & Seong-Joo, L. Early cyanobacterial fossil record: preservation, palaeoenvironments and identification. European Journal of Phycology 34 , 339-348 (1999). Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Molecular biology and evolution 24 , 1586-1591 (2007). dos Reis, M., Álvarez-Carretero, S. & Yang, Z. (2017). Reis, M. D. et al. Using phylogenomic data to explore the effects of relaxed clocks and calibration strategies on divergence time estimation: primates as a test case. Systematic Biology 67 , 594-615 (2018). Garcia, A. K., Fer, E., Sephus, C. & Kacar, B. in Environmental Microbial Evolution: Methods and Protocols (ed Haiwei Luo) 267-281 (Springer US, 2022). Ashkenazy, H. et al. FastML: a web server for probabilistic reconstruction of ancestral sequences. Nucleic acids research 40 , W580-W584 (2012). Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630 , 493-500 (2024). https://doi.org:10.1038/s41586-024-07487-w Kurien, B. T. & Scofield, R. H. Western blotting. Methods 38 , 283-293 (2006). Addo, M. A., Edwards, A. M. & Dos Santos, P. C. Methods to Investigate the Kinetic Profile of Cysteine Desulfurases. Methods Mol Biol 2353 , 173-189 (2021). https://doi.org:10.1007/978-1-0716-1605-5_10 Siegel, L. M. A direct microdetermination for sulfide. Analytical biochemistry 11 , 126-132 (1965). Liu, N. et al. Lycopene production from glucose, fatty acid and waste cooking oil by metabolically engineered Escherichia coli. Biochemical Engineering Journal 155 , 107488 (2020). Li, Q. et al. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai) 53 , 620-627 (2021). https://doi.org:10.1093/abbs/gmab036 Jiang, Y. et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81 , 2506-2514 (2015). https://doi.org:10.1128/AEM.04023-14 Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 97 , 6640-6645 (2000). https://doi.org:doi:10.1073/pnas.120163297 Jensen, P. R. & Hammer, K. Minimal requirements for exponential growth of Lactococcus lactis. Applied and Environmental Microbiology 59 , 4363-4366 (1993). Blazanin, M. gcplyr: an R package for microbial growth curve data analysis. BMC bioinformatics 25 , 232 (2024). Sapan, C. V., Lundblad, R. L. & Price, N. C. Colorimetric protein assay techniques. Biotechnology and applied Biochemistry 29 , 99-108 (1999). Wiśniewski, J. R. in Microbial proteomics: methods and protocols 3-10 (Springer, 2018). Javaux, E. J. & Lepot, K. The Paleoproterozoic fossil record: implications for the evolution of the biosphere during Earth's middle-age. Earth-Science Reviews 176 , 68-86 (2018). Wang, S. & Luo, H. Dating the bacterial tree of life based on ancient symbiosis. Systematic Biology , syae071 (2025). https://doi.org:10.1093/sysbio/syae071 Hanson-Smith, V., Kolaczkowski, B. & Thornton, J. W. Robustness of ancestral sequence reconstruction to phylogenetic uncertainty. Molecular biology and evolution 27 , 1988-1999 (2010). Mihara, H. et al. Structure of External Aldimine of Escherichia coli CsdB, an IscS/Nifs Homolog: Implications for Its Specificity toward Selenocysteine1. The Journal of Biochemistry 131 , 679-685 (2002). https://doi.org:10.1093/oxfordjournals.jbchem.a003151 Declarations Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (42192500 and 42192503). This work was supported by the High-performance Computing Platform of China University of Geosciences-Beijing. This work was also supported by the Laboratory of Microbiology Resources and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences. A grant from the United States National Institutes of Health (GM112919) also supported F.W.O. Author contributions H.D. led the study. H.D., L.H., and F.W.O. designed the research. H.C. performed gene retrieval and curation, phylogenetic analyses, and ancestral sequence reconstruction. H.C., D.X., and Z.Z. conducted the biochemical experiments. H.C. and M.Z. carried out the gene knockouts, and H.C. and X.T. carried out the gene knock-ins. H.C. performed the physiological assays. H.C. wrote the manuscript, and all authors contributed to discussions, with substantial input from H.D., L.H., and F.W.O.. Competing interests. The authors declare no competing interests. Additional information. Supplementary information is available for this paper. Correspondence and requests for material s should be addressed to Hailiang Dong. References Lyons TW et al (2024) Co-evolution of early Earth environments and microbial life. Nat Rev Microbiol 22:572–586. https://doi.org:10.1038/s41579-024-01044-y Lyons TW, Reinhard CT, Planavsky NJ (2014) The rise of oxygen in Earth's early ocean and atmosphere. Nature 506:307–315. https://doi.org:10.1038/nature13068 Blättler CL et al (2018) Two-billion-year-old evaporites capture Earth’s great oxidation. Science 360:320–323. https://doi.org:doi:10.1126/science.aar2687 Hodgskiss MSW, Crockford PW, Turchyn AV (2023) Deconstructing the Lomagundi-Jatuli Carbon Isotope Excursion. Annu Rev Earth Planet Sci 51:301–330. https://doi.org:10.1146/annurev-earth-031621-071250 Dorado G et al (2010) Biological mass extinctions on planet Earth. Archaeobios 4:53–64 Davín AA et al (2025) A geological timescale for bacterial evolution and oxygen adaptation. Science 388:eadp1853 Weiss MC et al (2016) The physiology and habitat of the last universal common ancestor. Nat Microbiol 1:16116. https://doi.org:10.1038/nmicrobiol.2016.116 Fontecave M (2006) Iron-sulfur clusters: ever-expanding roles. Nat Chem Biol 2:171–174. https://doi.org:10.1038/nchembio0406-171 Braymer JJ, Freibert SA, Rakwalska-Bange M, Lill R (2021) Mechanistic concepts of iron-sulfur protein biogenesis in Biology. Biochim et Biophys Acta (BBA) - Mol Cell Res 1868:118863. https://doi.org:https://doi.org/10.1016/j.bbamcr.2020.118863 Roche B et al (2013) Reprint of: Iron/sulfur proteins biogenesis in prokaryotes: Formation, regulation and diversity. Biochim et Biophys Acta (BBA) - Bioenergetics 1827:923–937. https://doi.org/10.1016/j.bbabio.2013.05.001 . https://doi.org: Blahut M, Sanchez E, Fisher CE, Outten FW (2020) Fe-S cluster biogenesis by the bacterial Suf pathway. Biochim Biophys Acta Mol Cell Res 1867:118829. https://doi.org:10.1016/j.bbamcr.2020.118829 Outten FW, Djaman O, Storz G (2004) A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli. Mol Microbiol 52:861–872. https://doi.org:10.1111/j.1365-2958.2004.04025.x Ayala-Castro C, Saini A, Outten FW (2008) Fe-S cluster assembly pathways in bacteria. Microbiol Mol Biol Rev 72:110–125 table of contents. https://doi.org:10.1128/MMBR.00034-07 Garcia PS et al (2022) An early origin of iron-sulfur cluster biosynthesis machineries before Earth oxygenation. Nat Ecol Evol 6:1564–1572. https://doi.org:10.1038/s41559-022-01857-1 Boyd ES, Thomas KM, Dai Y, Boyd JM, Outten FW (2014) Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway. Biochemistry 53:5834–5847. https://doi.org:10.1021/bi500488r Loiseau L, Ollagnier-de-Choudens S, Nachin L, Fontecave M, Barras F (2003) Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase. J Biol Chem 278:38352–38359. https://doi.org:10.1074/jbc.M305953200 Outten FW, Wood MJ, Munoz FM, Storz G (2003) The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli. J Biol Chem 278:45713–45719. https://doi.org:10.1074/jbc.M308004200 Gogar RK et al (2024) The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis. J Biol Chem 300:107641. https://doi.org:10.1016/j.jbc.2024.107641 Goldsmith-Fischman S et al (2004) The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes. J Mol Biol 344:549–565. https://doi.org:10.1016/j.jmb.2004.08.074 Dai Y, Outten FW (2012) The E. coli SufS-SufE sulfur transfer system is more resistant to oxidative stress than IscS-IscU. FEBS Lett 586:4016–4022. https://doi.org:10.1016/j.febslet.2012.10.001 Layer G et al (2007) SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. J Biol Chem 282:13342–13350. https://doi.org:10.1074/jbc.M608555200 Outten FW (2015) Recent advances in the Suf Fe-S cluster biogenesis pathway: Beyond the Proteobacteria. Biochim Biophys Acta 1853:1464–1469. https://doi.org:10.1016/j.bbamcr.2014.11.001 Condie KC, Kröner A (2013) The building blocks of continental crust: Evidence for a major change in the tectonic setting of continental growth at the end of the Archean. Gondwana Res 23:394–402. https://doi.org:10.1016/j.gr.2011.09.011 Fujishiro T et al (2017) Zinc-Ligand Swapping Mediated Complex Formation and Sulfur Transfer between SufS and SufU for Iron–Sulfur Cluster Biogenesis in Bacillus subtilis. J Am Chem Soc 139:18464–18467. https://doi.org:10.1021/jacs.7b11307 Tanaka N et al (2016) Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron–sulfur clusters. Mol Microbiol 99:835–848 CAMPOS N et al (2001) Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. Biochem J 353:59–67 Gerstel A et al (2020) Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis. PLoS Genet 16:e1009198. https://doi.org:10.1371/journal.pgen.1009198 Elchennawi I, Carpentier P, Caux C, Ponge M (2023) Ollagnier de Choudens, S. Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex. Biomolecules 13:732 Robbins L et al (2013) Authigenic iron oxide proxies for marine zinc over geological time and implications for eukaryotic metallome evolution. Geobiology 11:295–306 Daines SJ, Lenton TM (2016) The effect of widespread early aerobic marine ecosystems on methane cycling and the Great Oxidation. Earth Planet Sci Lett 434:42–51. https://doi.org:10.1016/j.epsl.2015.11.021 Riding R, Fralick P, Liang L (2014) Identification of an Archean marine oxygen oasis. Precambrian Res 251 Baym M et al (2016) Spatiotemporal microbial evolution on antibiotic landscapes. Science 353:1147–1151. https://doi.org:doi 10.1126/science.aag0822 Khademian M, Imlay JA (2021) How microbes evolved to tolerate oxygen. Trends Microbiol 29:428–440 Parsons C, Stueken EE, Rosen CJ, Mateos K, Anderson RE (2021) Radiation of nitrogen-metabolizing enzymes across the tree of life tracks environmental transitions in Earth history. Geobiology 19:18–34. https://doi.org:10.1111/gbi.12419 Garcia AK et al (2023) Nitrogenase resurrection and the evolution of a singular enzymatic mechanism. Elife 12:e85003 Cuevas Zuviría B et al (2025) Structural evolution of nitrogenase over 3 billion years. eLife 14, RP105613 https://doi.org:10.7554/eLife.105613 Zhou X et al (2024) Bioavailability of molybdenite to support nitrogen fixation on early Earth by an anoxygenic phototroph. Earth Planet Sci Lett 647:119056. https://doi.org/10.1016/j.epsl.2024.119056 . https://doi.org: Franke P, Freiberger S, Zhang L, Einsle O (2025) Conformational protection of molybdenum nitrogenase by Shethna protein II. Nature 637:998–1004. https://doi.org:10.1038/s41586-024-08355-3 Narehood SM et al (2025) Structural basis for the conformational protection of nitrogenase from O2. Nature 637:991–997. https://doi.org:10.1038/s41586-024-08311-1 Additional Declarations There is NO Competing Interest. Supplementary Files SIGuide.docx SI Guide file SupplementaryInformationTable1.xlsx Supplementary Table 1 SupplementaryInformationTable2.xlsx Supplementary Table 2 SupplementaryInformationTable3.xlsx Supplementary Table 3 ExtendedDataFigures.docx Cite Share Download PDF Status: Posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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07:54:51","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182705,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/3781c1b7f886615efc93d284.html"},{"id":99765140,"identity":"df1d7ec5-a358-4ee5-a1e2-b0d636f49ef7","added_by":"auto","created_at":"2026-01-08 07:54:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":628778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of SufE evolution in the context of Earth’s oxygenation.\u003c/strong\u003e A time-calibrated phylogenetic tree of SufE proteins from diverse bacteria is shown with a geological timeline. Diamonds at key nodes indicate reconstructed ancestral sequences. The timeline below the tree illustrates atmospheric O₂ levels over Earth’s history (blue line), highlighting the Great Oxygenation Event (GOE, ~2.4-2.0\u0026nbsp;Ga) and the Neoproterozoic Oxygenation Event (NOE, ~0.6-0.8\u0026nbsp;Ga) with shaded regions\u003csup\u003e1\u003c/sup\u003e. The deepest branches of the SufE tree (“Last Common Ancestor (LCA) SufE”, the corresponding protein named SufE\u003csup\u003eLCA\u003c/sup\u003e hereafter) predate the GOE, suggesting SufE existed in anoxic environments. A major diversification of SufE (“GOE SufE”, the corresponding protein named SufE\u003csup\u003eGOE\u003c/sup\u003e hereafter) aligns with the GOE (grey shading). Calibration lineages are noted (\u003cem\u003ee.g.\u003c/em\u003e, mitochondria-derived sequences in orange, and cyanobacteria in green). Others are annotated with their habitat (\u003cem\u003ee.g.\u003c/em\u003e, marine or terrestrial). The dating genome information was stored at github.com/eacochen. Bootstrap support values for key branches are indicated by black circles, and all values are higher than 95%. The analysis suggests that O\u003csub\u003e2\u003c/sub\u003e availability influenced the evolutionary radiation of SufE.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/1e38da4ddd1cf4f2c7556b4b.png"},{"id":99765183,"identity":"3bad0690-d60d-49c8-ba0a-6ebb1e766849","added_by":"auto","created_at":"2026-01-08 07:54:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":352926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e tolerance of ancestral and modern SufS/SufE complexes.\u003c/strong\u003e \u003cstrong\u003e(a-b)\u003c/strong\u003e: Catalytic efficiency (V\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e) of the LCA (green), GOE (blue), and modern (red) SufS/SufE proteins as a function of O₂ concentration. Each point represents the mean (± s.d.) of three experiments and error bars are too small to be visible. \u003cstrong\u003e(a)\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eMatched SufS/SufE pairs. \u003cstrong\u003e(b)\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eE. coli\u003c/em\u003e K12 SufS\u003csup\u003emodern\u003c/sup\u003e paired with either SufE\u003csup\u003eLCA\u003c/sup\u003e, SufE\u003csup\u003eGOE\u003c/sup\u003e or SufE\u003csup\u003emodern\u003c/sup\u003e variants. The K\u003csub\u003em\u003c/sub\u003e of SufE was determined as follows: SufS concentration was fixed at 0.5\u0026nbsp;μM and L-cysteine at 500\u0026nbsp;μM, while SufE was varied over a range (\u003cem\u003ee.g.\u003c/em\u003e 0-50\u0026nbsp;μM). According to the method, the Kₘ values of SufE for each SufS/SufE pair are as follows: modern pair showed Kₘ ≈3.64\u0026nbsp;μM; GOE pair Kₘ ≈4.88\u0026nbsp;μM; LCA pair Kₘ ≈2.48\u0026nbsp;μM. \u003cstrong\u003e(c-d)\u003c/strong\u003e: The maximum growth rate (per hour) of \u003cem\u003eE. coli\u003c/em\u003e strains lacking either SufE (WO541, \u003cem\u003eE. coli\u003c/em\u003e MG1655 Δ\u003cem\u003eiscU-fdx \u003c/em\u003eΔ\u003cem\u003esufE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e\u003cem\u003e MVA\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-kan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e mutant) or both SufS and SufE (WO539, \u003cem\u003eE. coli\u003c/em\u003e MG1655 Δ\u003cem\u003eiscU-fdx \u003c/em\u003eΔ\u003cem\u003esufSE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e\u003cem\u003e MVA\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-kan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e mutant), but complemented with ancestral or modern variants carried by pBAD, in the presence of the superoxide-generating agent phenazine methosulfate (PMS) (0-450µM). \u003cstrong\u003e(c)\u003c/strong\u003e ∆\u003cem\u003esufE\u003c/em\u003e strain WO541 complemented with SufE\u003csup\u003emodern\u003c/sup\u003e (red), SufE\u003csup\u003eGOE\u003c/sup\u003e (blue), or SufE\u003csup\u003eLCA\u003c/sup\u003e (green). \u003cstrong\u003e(d)\u003c/strong\u003e ∆\u003cem\u003esufSE\u003c/em\u003e strain WO539 complemented with SufS\u003csup\u003emodern\u003c/sup\u003e/SufE\u003csup\u003emodern\u003c/sup\u003e (red), SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e (blue), or SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e (green). Data points are mean ± s.d. for biological replicates (n = 3). \u003cstrong\u003e(e-f): \u003c/strong\u003eHypothetical structural model for SufS/SufE. A schematic model illustrating the interaction between the SufS homodimer (red and blue) and SufE (green).\u003cstrong\u003e (e)\u003c/strong\u003e AlphaFold3 predicted structure. H349 is present in SufS\u003csup\u003eLCA\u003c/sup\u003e, which prevents the catalytic two cysteine residues between SufS and SufE from approaching each other (distance 15.723 Å), this accounting for its low catalytical efficiency of persulfide transfer. \u003cstrong\u003e(f)\u003c/strong\u003e “Close approach” phase shows how SufS\u003csup\u003emodern\u003c/sup\u003e interacts with SufE\u003csup\u003emodern \u003c/sup\u003eadapted from Gogar, et al. \u003csup\u003e18\u003c/sup\u003e. In this case, the presence of Y345 on SufS allows a close approach between the catalytic two cysteine residues to allow efficient transfer of persulfide (distance 5.123 Å).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/4570ae2b070b4da3a11016ee.png"},{"id":102746320,"identity":"5999bf90-d65e-4459-b590-c8b5d2dc3eb0","added_by":"auto","created_at":"2026-02-16 08:56:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1956868,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/2051554d-b3d3-4ee7-b232-a3ce344ab8ee.pdf"},{"id":99765173,"identity":"09425c55-6264-4cf0-bd74-6dcc3ef1e2fd","added_by":"auto","created_at":"2026-01-08 07:54:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17509,"visible":true,"origin":"","legend":"SI Guide file","description":"","filename":"SIGuide.docx","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/f7fb0ad013c511d83f6e7660.docx"},{"id":99765199,"identity":"d91a9893-98e7-4e9f-b033-3961ef409728","added_by":"auto","created_at":"2026-01-08 07:54:52","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9954,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"SupplementaryInformationTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/9d411032e4a9b90f15d2463d.xlsx"},{"id":99765145,"identity":"5376ad6d-4b9f-46c3-93e4-7a26e9aaab2b","added_by":"auto","created_at":"2026-01-08 07:54:42","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10068,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"SupplementaryInformationTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/6b6b3b6f6981801555775657.xlsx"},{"id":99799160,"identity":"624e8bd1-647e-4565-b2ff-279cdcc0ba9c","added_by":"auto","created_at":"2026-01-08 13:49:17","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11473,"visible":true,"origin":"","legend":"Supplementary Table 3","description":"","filename":"SupplementaryInformationTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/048a34b7f0e99934d980a80b.xlsx"},{"id":99765191,"identity":"faffff3b-62f9-45a8-9870-8474855704c1","added_by":"auto","created_at":"2026-01-08 07:54:51","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3731776,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7916008/v1/a5bc7c09274b8b8554c49b4f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eAdaptation of Fe-S Cluster Assembly to Rising O\u003csub\u003e2\u003c/sub\u003e Levels over Geological Time\u003c/p\u003e","fulltext":[{"header":"Main","content":"\u003cp\u003eOne of the most important events in Earth history is the so-called Great Oxidation Event (GOE) \u003csup\u003e1,2\u003c/sup\u003e. It refers to a time period (between 2.46–2.06 Ga) when the Earth atmosphere and shallow sea experienced a rise in the free O\u003csub\u003e2\u003c/sub\u003e. During the latter half of GOE (i.e., Lomagundi-Jatuli Event, LJE, 2.22–2.06 Ga), atmospheric O\u003csub\u003e2\u003c/sub\u003e levels experienced a marked but temporary increase\u003csup\u003e3,4\u003c/sup\u003e. While accumulation of free O\u003csub\u003e2\u003c/sub\u003e killed the majority of anaerobic microorganisms, sometimes termed the first mass extinction event\u003csup\u003e5\u003c/sup\u003e, some survived through evolution to either tolerate or utilize O\u003csub\u003e2\u003c/sub\u003e \u003csup\u003e6\u003c/sup\u003e. However, the molecular mechanisms underlying the evolution remain to be understood. Analysis of a biological process prevalent in extant life and yet ancient and sensitive to molecular oxygen may offer valuable clues.\u003c/p\u003e\n\u003cp\u003eIron-sulfur (Fe-S) clusters are such ancient cofactors. They are essential for a wide range of enzymes and metabolic processes, such as DNA repair and electron transfer\u003csup\u003e7–9\u003c/sup\u003e. Biosynthesis of Fe-S clusters requires multiprotein assembly systems that mobilize sulfur, iron, and electrons to construct clusters and insert them into target apoproteins\u003csup\u003e9\u003c/sup\u003e. In \u003cem\u003eEscherichia coli\u003c/em\u003e, two major Fe-S assembly pathways exist: the housekeeping ISC system, functioning primarily under normal conditions, and the stress-responsive SUF system, which is induced under oxidative stress and iron limitation\u003csup\u003e10–13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eComparative phylogenomic analysis indicates that the SUF pathway is one of the oldest Fe-S biogenesis machineries: minimal SUF-like systems are implicated in the last universal common ancestor and distributed among Bacteria and Archaea\u003csup\u003e14\u003c/sup\u003e. This broad distribution underscores the functional plasticity of SUF and suggests that this pathway played a central role in early microbial adaptation to fluctuating redox conditions. Notably, stress-inducible SUF systems (such as that in \u003cem\u003eE. coli\u003c/em\u003e) utilize SufE as the sulfur-transfer protein. Rate-smoothed phylogenies further indicate that the emergence of accessory cysteine desulfurase SufS and its sulfur-carrier partner SufE were triggered by oxygenation of the Earth\u003csup\u003e15\u003c/sup\u003e. However, the timing of their emergence remains uncertain and warrants further study.\u003c/p\u003e\n\u003cp\u003eSufE is a small sulfur-transfer protein of the SUF system. In \u003cem\u003eE. coli\u003c/em\u003e, SufE accepts a sulfur atom from the cysteine desulfurase SufS and then delivers it to the scaffold complex (SufBC\u003csub\u003e2\u003c/sub\u003eD) for Fe-S cluster assembly\u003csup\u003e16,17\u003c/sup\u003e. This transient SufS/SufE interaction is vital for efficient sulfur transfer\u003csup\u003e18\u003c/sup\u003e. SufE forms a hydrophobic pocket to protect cysteine-persulfide from oxygen in aqueous solution\u003csup\u003e19\u003c/sup\u003e. Indeed, SufS and SufE have been shown to be crucial for bacterial survival under oxidative stress conditions in \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e20\u003c/sup\u003e. However, how SufS/SufE has evolved in response to rising atmospheric O\u003csub\u003e2\u003c/sub\u003e over geological time remains unknown. We hypothesize that oxidative pressure favored genetic mutations in SufE and SufS to maintain Fe-S biogenesis in an increasingly oxidizing world. We focused on SufE because of its central role in sulfur transfer under oxidative stress and its small size, which would allow relatively rapid evolutionary adaptation\u003csup\u003e14\u003c/sup\u003e. We also examined SufS, as the SufS/SufE cooperating pair likely underpins the functionality of the Suf pathway under oxidative stress\u003csup\u003e20–22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated the O\u003csub\u003e2\u003c/sub\u003e-driven evolution of SufS/SufE. We performed phylogenetic analyses and molecular clock dating to predict the emergence and diversification of SufE in the context of the oxygenation history of Earth, and reconstructed ancestral sequences of SufE (and SufS) corresponding to two key time points, i.e., ~ 2.67 Ga, when SufE originated, and ~ 2.14 Ga (at the LJE). Our data reveal how O\u003csub\u003e2\u003c/sub\u003e has shaped the evolution of the proteins, and therefore provide significant insights into molecular events in the explosion of aerobic life through Earth history.\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eSufE emergence before the GOE\u003c/h2\u003e\n \u003cp\u003eWe constructed a maximum-likelihood phylogeny of SufE homologues from over 7,000 prokaryotic genomes (Extended Data Fig.\u0026nbsp;1). Among these basal candidate lineages, we chose Gammaproteobacteria clade II for molecular dating because (i) phylogenetic reconciliation of 120 single-copy core genes with \u003cem\u003esufE\u003c/em\u003e yielded fully congruent topologies, indicating strictly vertical inheritance with only one detectable horizontal gene transfer (HGT) event and (ii) the clade spans a well-resolved evolutionary gradient in heme-copper oxidase (HCO) types responsible for O\u003csub\u003e2\u003c/sub\u003e reduction to water. The basal lineages encode microaerobic C-type HCOs, whereas the more recently derived lineages possess fully aerobic A-type HCOs. This stepwise shift from microaerobic to aerobic respiration provides an ecological framework that directly links divergence times to progressive adaptation to increasing oxidative stress (Extended Data Figs.\u0026nbsp;1–2). Bayesian relaxed molecular clock analyses using the autocorrelation rates (AR) model placed the last common ancestor (LCA) of SufE (Fig.\u0026nbsp;1, hereafter named SufE\u003csup\u003eLCA\u003c/sup\u003e), represented by the basal lineage of Gammaproteobacteria clade II near the root of the SufE phylogeny, at 2.67 Ga (95% Highest Posterior Density, HPD: 2.49–2.83 Ga), shortly after the inferred origin of oxygenic cyanobacteria (~ 2.73 Ga) (Figs.\u0026nbsp;1, Extended Data Fig.\u0026nbsp;3–4). This temporal proximity implies that SufE evolved in response to the “whiffs of O\u003csub\u003e2\u003c/sub\u003e”, potentially preceding the global-scale GOE. This result suggests that the SufS/SufE system is an ancient invention, emerging between the appearance of oxygenic cyanobacteria and the advent of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eSubsequent branching patterns indicate that major diversification or expansion of the SufE family occurred at the latter half of the GOE, \u003cem\u003ee.g.\u003c/em\u003e LJE (Fig.\u0026nbsp;1, hereafter named SufE\u003csup\u003eGOE\u003c/sup\u003e). Notably, the SufE\u003csup\u003eGOE\u003c/sup\u003e node represents the earliest SufE-carrying lineage isolated from terrestrial environments and was dated to 2.14 Ga (95% HPD: 1.98–2.29 Ga). This age coincides with the time of thickening of the continental crust\u003csup\u003e23\u003c/sup\u003e and rising O\u003csub\u003e2\u003c/sub\u003e levels, suggesting that SufE proteins were under selective pressure to accommodate elevated oxidative stress during the early colonization of land niches. We interpret this as evidence that the selective pressure from a rise in O₂ led to rapid diversification of SufE sequences and/or a selective sweep of O\u003csub\u003e2\u003c/sub\u003e-tolerant SufE variants around that period.\u003c/p\u003e\n \u003cp\u003eTogether, these results suggest that SufE underwent rapid evolutionary divergence under early oxidative stress, likely adapting to the sporadic O\u003csub\u003e2\u003c/sub\u003e “whiffs” produced by nascent cyanobacteria before the GOE\u003csup\u003e1\u003c/sup\u003e and to the rapid increase of O\u003csub\u003e2\u003c/sub\u003e during the LJE period. We set out to test if the changes in SufE sequence are bona fide molecular adaptations that resulted in improved SufE function under oxidative stress. Our approach involves ancestral sequence reconstruction coupled with the comparison of ancestral and existent proteins in biochemical and functional assays.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIn vitro response of SufSE activity to O\u003csub\u003e2\u003c/sub\u003e\u003c/h3\u003e\n\u003cp\u003eTwo nodes in the SufE phylogenic tree, LCA and GOE SufE, were selected for detailed ancestral sequence reconstruction because they bracket the GOE, permitting a direct test of a functional shift in response to an increase in O\u003csub\u003e2\u003c/sub\u003e level. Three SufS/SufE pairs, corresponding to SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e, SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e and SufS\u003csup\u003emodern\u003c/sup\u003e/SufE\u003csup\u003emodern\u003c/sup\u003e (\u003cem\u003eE. coli\u003c/em\u003e K12), were expressed in \u003cem\u003eE. coli\u003c/em\u003e and purified under anerobic conditions. The reconstructed and modern sequences were aligned (Extended Data Figs.\u0026nbsp;5–6). The core catalytic residues, such as C51 in SufE\u003csup\u003emodern\u003c/sup\u003e (corresponding to both C51 in SufE\u003csup\u003eGOE\u003c/sup\u003e and SufE\u003csup\u003eLCA\u003c/sup\u003e, Extended Data Fig.\u0026nbsp;5) and C364 in SufS\u003csup\u003emodern\u003c/sup\u003e (corresponding to C369 in SufS\u003csup\u003eGOE\u003c/sup\u003e and C368 in SufS\u003csup\u003eLCA\u003c/sup\u003e, Extended Data Fig.\u0026nbsp;6), have remained unchanged over time. However, there are mutations in the loop between β1 and β2 of SufE, sites of interaction between SufS and SufE, and the interface of the SufS homodimer (Extended Data Figs.\u0026nbsp;5–6).\u003c/p\u003e\n\u003cp\u003eTo determine the function of LCA, GOE and modern SufS/SufE pairs in response to rising O\u003csub\u003e2\u003c/sub\u003e, each pair was assayed for cysteine desulfurase activity by detecting sulfide production across an O\u003csub\u003e2\u003c/sub\u003e gradient (0–21%). All three variants of SufE greatly enhanced the low basal activity of SufS (data not shown), presumably by acting as a persulfide acceptor, thereby facilitating SufS turnover, consistent with previous studies for the modern pair\u003csup\u003e20\u003c/sup\u003e. Furthermore, under strictly anoxic conditions (0% O₂), all three SufS/SufE pairs were active, but the modern pair exhibited the highest catalytic efficiency (Fig.\u0026nbsp;2a). Both the modern \u003cem\u003eE. coli\u003c/em\u003e and the GOE pairs showed a V\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e ratio ~ 40–50% greater than that of the LCA pair.\u003c/p\u003e\n\u003cp\u003eHowever, the three pairs exhibited markedly different patterns in response to O\u003csub\u003e2\u003c/sub\u003e. The activity of the LCA pair decreased precipitously at 2% O\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;2a, green curve), fell to less than half of its anoxic value at 5% O\u003csub\u003e2\u003c/sub\u003e, and essentially dropped to the baseline level of SufS at 10% O\u003csub\u003e2\u003c/sub\u003e. The SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e (Fig.\u0026nbsp;2a, blue curve) largely retained its anoxic activity at low O\u003csub\u003e2\u003c/sub\u003e levels (i.e., ~ 90% activity at 5% O\u003csub\u003e2\u003c/sub\u003e and ~ 70% at 10% O₂) but dropped to the baseline level of SufS at 15% O\u003csub\u003e2\u003c/sub\u003e. In stark contrast, the modern pair from \u003cem\u003eE. coli\u003c/em\u003e K12 (Fig.\u0026nbsp;2a, red curve) retained its full activity up to ~ 5% O\u003csub\u003e2\u003c/sub\u003e and only dropped to ~ 80% at the modern O\u003csub\u003e2\u003c/sub\u003e level (21%). These results indicate a stepwise improvement in O\u003csub\u003e2\u003c/sub\u003e tolerance from the LCA to the GOE to the modern variants.\u003c/p\u003e\n\u003cp\u003eWe then determined if SufS or SufE was primarily responsible for the O\u003csub\u003e2\u003c/sub\u003e sensitivity by pairing the SufS\u003csup\u003emodern\u003c/sup\u003e with either the SufE\u003csup\u003eLCA\u003c/sup\u003e or the SufE\u003csup\u003eGOE\u003c/sup\u003e (Fig.\u0026nbsp;2b). Under anoxic condition, the SufS\u003csup\u003emodern\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e combination increased the activity by ~ 100% relative to the SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e combination, yielding a catalytic efficiency indistinguishable from the SufS\u003csup\u003emodern\u003c/sup\u003e/SufE\u003csup\u003emodern\u003c/sup\u003e pair. Therefore, it is SufS that determines the maximal catalytic efficiency of the full SufS/SufE transpersulfuration reaction. As O\u003csub\u003e2\u003c/sub\u003e concentration increased, the activities of the three hybrid combinations show similar response patterns (Fig.\u0026nbsp;2b) to those of the three “age-match” combinations (Fig.\u0026nbsp;2a). It appears that, when SufS was kept unchanged (modern variant), different SufEs caused the difference in O\u003csub\u003e2\u003c/sub\u003e sensitivity among the three hybrid SufS/SufE pairs (Fig.\u0026nbsp;2b).\u003c/p\u003e\n\u003cp\u003eTo understand the structural basis of the difference in catalytic activity among the three SufS/SufE pairs, AlphaFold3 was used to predict the structures of SufS and SufE complexes based on a rigid docking model\u003csup\u003e18\u003c/sup\u003e. In the SufS\u003csup\u003eLCA\u003c/sup\u003e, a small and flexible histidine is present at position 349 (corresponding to 350 in SufS\u003csup\u003eGOE\u003c/sup\u003e and 345 in SufS\u003csup\u003emodern\u003c/sup\u003e, Extended Data Fig.\u0026nbsp;6) \u003csup\u003e24\u003c/sup\u003e. This residue, H349, fails to occupy the R121 cavity of SufE\u003csup\u003eLCA\u003c/sup\u003e (corresponding to R121 in SufE\u003csup\u003eGOE\u003c/sup\u003e and R119 in SufE\u003csup\u003eLCA\u003c/sup\u003e) and allows R121 to stay at a blocking position (Fig.\u0026nbsp;2e). As a result, the distance between C368 of SufS\u003csup\u003eLCA\u003c/sup\u003e and C51 of SufE\u003csup\u003eLCA\u003c/sup\u003e (i.e., 15.723 Å) is sufficiently large to slow down the persulfide transfer from SufS to SufE, thus accounting for its low catalytical activity. In contrast, in SufS\u003csup\u003emodern\u003c/sup\u003e (also in SufS\u003csup\u003eGOE\u003c/sup\u003e), a larger and more rigid tyrosine replaces histidine at position 345 (Extended Data Fig.\u0026nbsp;6) and occupies a cavity vacated by the C51 loop of SufE\u003csup\u003emodern\u003c/sup\u003e. Consequently, R119 in SufE\u003csup\u003emodern\u003c/sup\u003e is forced to move toward an outward position, allowing C51 of SufE\u003csup\u003emodern\u003c/sup\u003e to approach C364 of SufS\u003csup\u003emodern\u003c/sup\u003e for rapid persulfide transfer (Fig.\u0026nbsp;2f) \u003csup\u003e18\u003c/sup\u003e. Thus, the increased catalytical activity of the modern and GOE pairs, relative to the LCA pair (Fig.\u0026nbsp;2a), is likely triggered by the replacement of histidine in SufS\u003csup\u003eLCA\u003c/sup\u003e by tyrosine in SufS\u003csup\u003eGOE\u003c/sup\u003e and SufS\u003csup\u003emodern\u003c/sup\u003e to result in a more efficient interaction between the SufS and SufE catalytic sites. There are other residue-level substitutions along the β-latch/homodimer interface of SufS (i.e., K92 in SufS\u003csup\u003eGOE\u003c/sup\u003e versus R92 in SufS\u003csup\u003eLCA\u003c/sup\u003e and SufS\u003csup\u003emodern\u003c/sup\u003e variants, Q255 in SufS\u003csup\u003eLCA\u003c/sup\u003e versus E255 in SufS\u003csup\u003eGOE\u003c/sup\u003e and E250 in SufS\u003csup\u003emodern\u003c/sup\u003e variants), but they do not appear to compromise the monomer-monomer interaction of SufS (Extended Data Fig.\u0026nbsp;7) and the ability of SufS to interact with SufE.\u003c/p\u003e\n\u003cp\u003eIn addition to these substitutions on SufS, there are numerous differences among the three variants of SufE (Extended Data Fig.\u0026nbsp;5) that may jointly account for the measured differences in their catalytic activities among the three SufS/SufE pairs (Fig.\u0026nbsp;2a-b). For example, relative to the SufE\u003csup\u003emodern\u003c/sup\u003e, the SufE\u003csup\u003eLCA\u003c/sup\u003e and SufE\u003csup\u003eGOE\u003c/sup\u003e show insertions of two amino acid residues between β1 and β2, as well as numerous substitutions both between and within these β-strands (Extended Data Fig.\u0026nbsp;5). Although these sites are spatially distant from the SufS/SufE interface, they may perturb the overall conformation of SufE, thereby modulating its association with SufS and, in turn, the capacity of SufS/SufE pairs to withstand oxidative stress (Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003ch3\u003eIn vivo response of SufSE activity to O\u003csub\u003e2\u003c/sub\u003e\u003c/h3\u003e\n\u003cp\u003eTo determine the response of SufS and SufE activity to rising O\u003csub\u003e2\u003c/sub\u003e levels in vivo, we analyzed the functionality of the SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e and SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e pairs in \u003cem\u003eE. coli\u003c/em\u003e K12. First, we tested if these variants could rescue the synthetic lethality of strains lacking both the housekeeping Isc system and either SufE or both SufS/SufE. Normally, such mutants are inviable due to loss of Fe-S cluster-dependent isoprenoid biosynthesis\u003csup\u003e25\u003c/sup\u003e. Conditionally lethal Δ\u003cem\u003eiscU-fdx\u003c/em\u003e Δ\u003cem\u003esufE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e and Δ\u003cem\u003eiscU-fdx\u003c/em\u003e Δ\u003cem\u003esufSE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e mutant strains were constructed by inserting a non-native, hybrid mevalonate-dependent MVA system that does not require any Fe-S cluster enzymes for isoprenoid biosynthesis\u003csup\u003e26\u003c/sup\u003e. These strains absolutely rely on the non-native MVA system and addition of mevalonate to the growth media in order to restore isoprenoid biosynthesis. We then tested if introducing either ancient SufE alone (SufE\u003csup\u003eLCA\u003c/sup\u003e or SufE\u003csup\u003eGOE\u003c/sup\u003e) or ancient SufS/SufE pair (SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e or SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e) into a pBAD plasmid could allow the conditionally lethal strains to grow without mevalonate. We found that all SufE variants or SufS/SufE pairs rescued the lethality of the strains in the absence of mevalonate under atmospheric O\u003csub\u003e2\u003c/sub\u003e conditions (i.e., at the origin, Fig.\u0026nbsp;2c-d). We surmise that basal intracellular oxidative stress at atmospheric O\u003csub\u003e2\u003c/sub\u003e concentrations was probably not high enough to distinguish their ability in Fe-S cluster biogenesis, especially in the rich, glucose-supplemented media used for the experiment.\u003c/p\u003e\n\u003cp\u003eTo increase the oxidative stress, the Δ\u003cem\u003eiscU-fdx\u003c/em\u003e Δ\u003cem\u003esufE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e strains transformed with the three variants of SufE on the pBAD plasmid were subjected to phenazine methosulfate (PMS). PMS generates intracellular superoxide radicals, imposing extra oxidative stress that damages Fe-S clusters and necessitates active repair/biogenesis systems\u003csup\u003e27\u003c/sup\u003e. Indeed, growth assays revealed clear differences in functional restoration of the three SufE variants under increasing PMS concentration (Fig.\u0026nbsp;2c). Across 0–30 µM PMS, all complemented strains exhibited a modest stimulatory response, with the maximum specific growth rate (µmax) increasing and peaking at 30 µM. From 30–60 µM, the µmax values declined modestly yet remained above that for the no-PMS control. Above 60 µM, the µmax values dropped sharply for all variants, and the resulting viability limits separated the three lineages: the SufE\u003csup\u003eLCA\u003c/sup\u003e ceased growth above 150 µM, the SufE\u003csup\u003eGOE\u003c/sup\u003e above 240 µM, and the \u003cem\u003eE. coli\u003c/em\u003e K12 SufE\u003csup\u003emodern\u003c/sup\u003e above 300 µM. Thus, while the low dose stimulation is shared, the SufE\u003csup\u003emodern\u003c/sup\u003e extends the tolerable PMS window to a much higher level of PMS. These in vivo data mirror the in vitro biochemical assay results, showing that the evolutionary enhancements to SufE’s sequence translate into a tangible survival advantage under oxidative stress.\u003c/p\u003e\n\u003cp\u003eSimilar complementary experiments were performed for the Δ\u003cem\u003eiscU-fdx\u003c/em\u003e Δ\u003cem\u003esufSE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e strain lacking both SufS and SufE. The normalized expression levels of SufS and SufE from LCA, GOE and modern strains were similar without PMS (Extended Data Fig.\u0026nbsp;8). Baseline growth in the absence of PMS showed that the SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e and SufS\u003csup\u003emodern\u003c/sup\u003e/SufE\u003csup\u003emodern\u003c/sup\u003e supported nearly identical maximum growth rates, both slightly lower (by ~ 0.1 h⁻¹) than in the Δ\u003cem\u003eiscU-fdx\u003c/em\u003e Δ\u003cem\u003esufE::cm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e strain complemented with SufE [~ 0.8 log(OD\u003csub\u003e450\u003c/sub\u003e) h\u003csup\u003e− 1\u003c/sup\u003e vs ~ 0.7, Fig.\u0026nbsp;2c-d]. In contrast, the SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e pair had a µmax value reduced by ~ 50% (Fig.\u0026nbsp;2d), matching the magnitude of its diminished catalytic efficiency in vitro (Fig.\u0026nbsp;2a). Unlike the results in the ∆\u003cem\u003esufE\u003c/em\u003e strain, no hormesis was observed in the ∆\u003cem\u003esufSE\u003c/em\u003e strain: the µmax value declined slightly over 0–30 µM PMS, then fell steeply from 30 to 240 µM (Fig.\u0026nbsp;2d). Consistently, the upper tolerance limits of PMS were markedly lower in the complemented ∆\u003cem\u003esufSE\u003c/em\u003e strain compared to those in the complemented ∆\u003cem\u003esufE\u003c/em\u003e strains, which ceased growth above 100 µM, 150 µM, and 240 µM PMS for LCA, GOE, and modern SufS/SufE pairs, respectively (Fig.\u0026nbsp;2d). Importantly, under higher PMS stress, the modern SufS/SufE consistently outperformed the GOE and LCA variants.\u003c/p\u003e\n\u003ch3\u003eImplications for life evolution through Earth oxygenation\u003c/h3\u003e\n\u003cp\u003eOur findings reveal a clear evolutionary trajectory in one of the most important biochemical pathways: SUF Fe-S biosynthesis system, driven by Earth\u0026rsquo;s rising atmospheric O\u003csub\u003e2\u003c/sub\u003e concentration. The SUF pathway, though present in early anaerobic life, had to be refined to remain functional as the atmospheric O\u003csub\u003e2\u003c/sub\u003e levels rose. By reconstructing ancient SufS and SufE proteins, we directly observed that the SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e and SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e complexes were poorly adapted to an O\u003csub\u003e2\u003c/sub\u003e-rich environment, while the modern complex was highly O\u003csub\u003e2\u003c/sub\u003e-tolerant. SufS determines the catalytic ceiling, but SufE governs how much oxidative load that the complex can withstand. Thus, neither component is subordinate: evolution jointly optimized SufS and SufE, likely by tuning their global geometries and coordinating a protected handoff of persulfide to SufBC\u003csub\u003e2\u003c/sub\u003eD scaffold. These results underscore the power of O\u003csub\u003e2\u003c/sub\u003e as an agent of natural selection.\u003c/p\u003e \u003cp\u003eCertain housekeeping SUF systems (e.g., in \u003cem\u003eBacillus subtilis\u003c/em\u003e) employ SufU as a SufE analog, serving as the cysteine desulfurase partner for sulfur transfer in those organisms\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. A histidine 349 is critical for SufS\u003csup\u003eLCA\u003c/sup\u003e (corresponding to Y345 in SufS\u003csup\u003emodern\u003c/sup\u003e and Y350 in SufS\u003csup\u003eGOE\u003c/sup\u003e) when it interacts with SufU which requires a Zn\u003csup\u003e2+\u003c/sup\u003e cofactor to coordinate interactions during persulfide transfer. Phylogenetically, SufU predates SufE (Extended Data Fig.\u0026nbsp;9). However, bioavailable Zn in Archaean oceans was extremely low\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. As a result, SufE may have evolved as a zinc-independent \u0026lsquo;rescue\u0026rsquo; module for the SUF pathway under anoxic condition. However, SufS/SufE pair faces a great challenge of Earth\u0026rsquo;s progressive oxygenation.\u003c/p\u003e \u003cp\u003eOur molecular dating result indeed suggests that once localized O\u003csub\u003e2\u003c/sub\u003e appeared, molecular adaptation followed rapidly. The first detectable divergence within the SufE clade was only\u0026thinsp;~\u0026thinsp;60 Myr after the median age of oxygenic cyanobacteria, which is a small age gap in the Archaean context. SufE emerged in lineages that likely shared ecological space with the earliest O\u003csub\u003e2\u003c/sub\u003e producers. Furthermore, the molecular adaptation was not only quick, but the oxidative tolerance of SufS and SufE actually evolved ahead of contemporaneous O\u003csub\u003e2\u003c/sub\u003e levels. The earliest-divergent SufE\u003csup\u003eLCA\u003c/sup\u003e already withstands\u0026thinsp;~\u0026thinsp;2% O\u003csub\u003e2\u003c/sub\u003e, exceeding most estimates for ambient O\u003csub\u003e2\u003c/sub\u003e at this time (~\u0026thinsp;0.1-~0.5%)\u003csup\u003e30,31\u003c/sup\u003e. Likewise, geochemical proxies indicate\u0026thinsp;~\u0026thinsp;4% O\u003csub\u003e2\u003c/sub\u003e around the LJE\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, but our in vitro biochemical assay showed that the SufE\u003csup\u003eGOE\u003c/sup\u003e already tolerates\u0026thinsp;~\u0026thinsp;10% O\u003csub\u003e2\u003c/sub\u003e. These systematic overshoots of O\u003csub\u003e2\u003c/sub\u003e tolerance suggest selection for excess capacity as a safety margin in stress-response machineries, which possibly provides resilience to redox excursions and facilitates the progressive expansion of increasingly aerobic niches. A similar type of advanced preparedness in response to increasing stress is also shown in antibiotic stress-response\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to oxidative tolerance, the increased catalytic efficiency of SufS/SufE from the LCA to GOE/modern variants may reflect selective pressure to increase rates of Fe-S cluster biogenesis in vivo. During aerobic growth, cells experience a constitutive level of Fe-S cluster damage in sensitive enzymes such as dehydratases, requiring a compensatory increased level of cluster biogenesis to maintain cell function\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In contrast, during anaerobic growth, Fe-S cluster turnover is much lower and the demand for cluster biogenesis is lower.\u003c/p\u003e \u003cp\u003eFrom a broader perspective, the O\u003csub\u003e2\u003c/sub\u003e-driven evolution of SufE and SufS exemplifies how life\u0026rsquo;s molecular machinery has been molded by planetary change. The GOE forced the innovation of more efficient Fe-S cluster assembly tools, which in turn enabled organisms to exploit O\u003csub\u003e2\u003c/sub\u003e for metabolism while preserving their ancient biochemical capabilities. Without such adaptations, essential processes, including respiration, DNA repair, and metabolic pathways that depend on Fe-S proteins could have failed in an oxygenated world. One particular example of such adaptation is ancient nitrogenases, Fe-S proteins that are used for N\u003csub\u003e2\u003c/sub\u003e fixation. Molecular dating suggests that Mo-based nitrogenase originated in the anoxic mid-late Archaean age (3.1\u0026ndash;2.7 Ga) \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, but survived the GOE and retained the same basal structure and functions even in modern aerobic microorganisms\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Conformational change is one important mechanism against oxidative stress, where the Shethna (FeSII) protein binds to the nitrogenase complex to protect it from oxygen damage\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While the specifics may vary among different functional groups of organisms, our study reveals an important and possibly widespread defense mechanism against oxidative stress through the conformational change of the SufS/SufE interface.\u003c/p\u003e \u003cp\u003eThis work illustrates how geochemistry and biochemistry are intertwined: oxygenic photosynthesis leads to atmospheric oxygenation, and such a planetary-scale change drives molecular innovation, which in turn enables new biological capabilities. Our study therefore connects the evolution of a single protein complex to Earth\u0026rsquo;s largest evolutionary inflection point, even the Cambrian radiation, by way of the O\u003csub\u003e2\u003c/sub\u003e that links them. This integrative perspective from atmosphere to enzyme to evolution deepens our understanding of how life\u0026rsquo;s molecular machinery is molded by our changing planet and invites further investigations using ancestral protein reconstruction to explore other episodes where environmental change and biochemical evolution converged.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMolecular clock and Ancestral Sequence Reconstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMultiple sequence alignment and phylogenetic analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFour experimentally validated SufE/CsdE proteins from UniProt (downloaded 24 Oct 2022) with\u0026nbsp;\u0026ldquo;UniProtKB reviewed\u0026rdquo; status\u0026nbsp;(P76194, Q9EXP1,\u0026nbsp;B5BA16,\u0026nbsp;Q1C762,\u0026nbsp;P0AGF2)\u0026nbsp;were used as seeds for homology searches\u0026nbsp;against the NCBI NR protein database (June 2022, 483,768,206 non‑redundant sequences) using DIAMOND blastp\u003csup\u003e40\u003c/sup\u003e with an E-value of \u0026lt; 1e-6 and a length filter of 90-170 amino acid residues, yielding 23,567 SufE‑like hits. Candidate sequences were verified by HMMER\u003csup\u003e41\u003c/sup\u003e hmmscan against Pfam\u003csup\u003e42\u003c/sup\u003e hidden Markov model (HMM) database, and validated sequences were dereplicated with CD‑HIT\u003csup\u003e43\u003c/sup\u003e (90% identity), grouped into 7,635 (NR) clusters. The longest sequence in each cluster was retained. The sequences were aligned with MAFFT\u003csup\u003e44\u003c/sup\u003e L‑INS‑i and trimmed using trimAl\u003csup\u003e45\u003c/sup\u003e with resoverlap 0.55 and seqoverlap 60. Maximum‑likelihood (ML) trees were inferred with FastTree\u003csup\u003e46\u003c/sup\u003e (for rapid exploration) and IQ‑TREE 2.2.1\u003csup\u003e47\u003c/sup\u003e (for focal subsets). Because of the lack of reliable outgroups for SufE, MAD\u003csup\u003e48\u003c/sup\u003e and MinVar\u003csup\u003e49\u003c/sup\u003e, outgroup‑independent methods were employed to allow identification of ancient lineages and candidate nodes for dating and ancestral sequence reconstruction. Visualization was performed with iTOL\u003csup\u003e50\u003c/sup\u003e, and sequences species annotations were fetched from NCBI identical protein group (ipg) database by Entrez\u003csup\u003e51\u003c/sup\u003e, using the most complete genome for each protein cluster using CheckM\u003csup\u003e52\u003c/sup\u003e completeness estimates. The putative oxygen‑respiration capacity of a species was inferred based on the presence of heme-copper oxidase (HCO) families A, B1, C, as detected with hmmscan (E-value \u0026lt; 1e-50), as described\u003csup\u003e53,54\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGene-species tree reconciliation with curation of horizontal gene transfer (HGT) candidates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo satisfy the vertical inheritance assumption for node dating and ancestral sequence reconstruction, SufE gene trees were reconciled with species trees, and those lineages which were likely recipients of HGT\u003csup\u003e55\u003c/sup\u003e were iteratively removed. For the focal lineage (301 proteins and 1,455 candidate genomes), open reading frames were predicted with Prokka\u003csup\u003e56\u003c/sup\u003e, and species phylogeny was built with GTDB‑Tk\u003csup\u003e57\u003c/sup\u003e bac120 single‑copy bacterial markers (hmmscan annotation, E-value \u0026lt; 1e-50). Each marker was aligned with MAFFT, trimmed with trimAl, and concatenated. The species tree was inferred under LG+C20+F+I+G in IQ‑TREE. Gene-species reconciliation was performed with GeneRax\u003csup\u003e58\u003c/sup\u003e under species‑tree‑aware ML, using SPR search and an unconstrained DTL model (radius 5). Leaf‑level HGT acceptors, acceptors at internal nodes transferring to leaves, and transfers between deep internal nodes of comparable depth were successively removed. After six iterations, transfers were reduced from 34 to 1, yielding a curated set of 131 vertically inherited SufE sequences and genomes for dating.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMolecular clock analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo anchor both deep Proterozoic and Phanerozoic timescales, the following two sets of species were used: (i) 21 oxygenic cyanobacteria and 3 melainabacteria (i.e., the cyanobacterial outgroup), and (ii) mitochondria‑proximal alphaproteobacteria and a set of mitochondrial markers (mito24)\u003csup\u003e\u0026nbsp;59,60\u003c/sup\u003e. First, 10 alphaproteobacteria genomes were added close to mitochondria, built a bac120 species tree that included cyanobacteria and alphaproteobacteria, and then replaced the alpha clade with four alternative mitochondria-alphaproteobacteria topologies (TP1-TP4) reported in prior work\u003csup\u003e59\u003c/sup\u003e. The final pruned matrix contained 181 taxa (131 SufE genomes, 24 cyanobacteria/melainabacteria, and 26 mitochondria/alphaproteobacteria). The mito24 alignment contributed 6,749 concatenated amino‑acid positions after trimming.\u003c/p\u003e\n\u003cp\u003eEight calibrations spanning the root and key crown groups were used: origin of life, origin of oxygenic Cyanobacteria, crown Nostocales, crown Pleurocapsales, red algal Bangiophyceae and Florideophyceae, crown bryophytes, and crown eudicots. Root bounds explored broad windows reflecting early habitability constraints (upper bound 4.5\u0026nbsp;Ga; lower bounds 4.0, 3.5, or 3.0\u0026nbsp;Ga, sensu impact chronology and earliest microfossils). Cyanobacterial bounds bracketed literature estimates from geochemical proxies and biomarkers (upper 3.0\u0026nbsp;Ga\u003csup\u003e61\u003c/sup\u003e; lower 2.32 or 2.50\u0026nbsp;Ga)\u003csup\u003e\u0026nbsp;62\u003c/sup\u003e. Fossil minima for Nostocales (1.6\u0026nbsp;Ga)\u003csup\u003e\u0026nbsp;63\u003c/sup\u003e and Pleurocapsales (1.7 Ga)\u003csup\u003e\u0026nbsp;64\u003c/sup\u003e followed akinetes and microfossil occurrences. Eukaryotic calibrations followed recent syntheses for red algae, bryophytes and eudicots\u003csup\u003e59\u003c/sup\u003e. The root and cyanobacterial options with two eukaryote schemes (Euk1/Euk2) were combined to generate 12 calibration sets. These, along with four topologies for mitochondria (TP1-TP4) and two clock models (AR/IR; see below), produced 96 dating analyses (Supplementary Information Table S1).\u003c/p\u003e\n\u003cp\u003eBayesian relaxed‑clock dating was performed in PAML MCMCTree\u0026nbsp;4.10.7\u003csup\u003e65\u003c/sup\u003e using both auto‑correlated rates (AR) and independent rates (IR) relaxed-clock models. Before formal divergence time estimation, the gradient and Hessian matrices were obtained by using PAML CODEML to improve the accuracy of molecular clock results\u003csup\u003e66\u003c/sup\u003e. Each run involved a burn‑in of 2,000 iterations, sampling every 20 steps (20,000 posterior samples). Each configuration was run in duplicate with different random seeds. Convergence and performance were assessed by (i) mcmc3r diagnostics, (ii) infinite‑sites test, and (iii) stepping‑stone marginal likelihoods to compare topologies, calibrations, and clock models\u003csup\u003e67\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAncestral protein reconstruction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIQ-TREE (version 1.6) with the \u0026lsquo;-asr\u0026rsquo; option was used to infer the amino acid sequences of ancestral SufE. The best-fit substitution model (LG+C20+I+G4) was chosen based on Akaike information criterion and Bayesian information criterion. The ancestral sequence reconstruction output provided the posterior probability for each amino acid at every site. Sites with a single amino acid having high posterior probability (\u0026gt; 0.30) were directly assigned that residue. For ambiguous sites with lower confidence, top three candidate residues were considered. If the top three residues exhibited similar properties, the highest-probability residue was chosen for that site. If candidates differed in property, the residue predicted to enhance protein solubility and allow the isoelectric point of the protein closer to that of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eSufE was selected. In cases where a gap (\u0026ldquo;-\u0026rdquo;) was the top inference for a site, top four candidates were considered. If none of the four candidate residues yielded a clear improvement in protein property or synthesis, the gap was retained\u003csup\u003e68\u003c/sup\u003e. ASR based on maximum likelihood (ML) method was also inferred by using PAML\u003csup\u003e65\u003c/sup\u003e and FastML\u003csup\u003e69\u003c/sup\u003e. The amino acid substitution model used in ML was LG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRepresentative reference SufS sequences were chosen from diverse taxa, including an extremophilic archaeon (D4gyV5, \u003cem\u003eHaloferax volcanii\u003c/em\u003e), a Gram-positive bacterium whose SufS interacts with SufU (O32164, \u003cem\u003eBacillus subtilis\u003c/em\u003e), and \u003cem\u003eE. coli\u003c/em\u003e K12, which encodes the canonical SufS (1C0N). SufS sequences were aligned as described above for SufE. A maximum likelihood tree of SufS genes was built from the 131 genomes used for SufE dating, using IQ-TREE, and ASR for SufS was performed in the same fashion as for SufE. Internal nodes corresponding to SufE\u003csup\u003eLCA\u003c/sup\u003e (2.67 Ga) and SufE\u003csup\u003eGOE\u003c/sup\u003e (2.17 Ga) were identified on the SufS tree, and the ancestral SufS sequences for these time points were inferred (designated as SufS\u003csup\u003eLCA\u003c/sup\u003e and SufS\u003csup\u003eGOE\u003c/sup\u003e, respectively). Ambiguous sites in the SufS reconstructions were resolved following the same criteria as those for SufE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlphaFold3 protein structure prediction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eReconstructed SufS and SufE variant structures were predicted by AlphaFold3\u003csup\u003e70\u003c/sup\u003e with the ratio of SufS to SufE equal to 2:1. The structure was visualized by ChimeraX. The PLP cofactor of SufS was matched by PDB 5XT6 SufS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProtein preparation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenes encoding ancestral SufS/SufE proteins (LCA and GOE) were codon-optimized for \u003cem\u003eE. coli\u003c/em\u003e and synthesized. The present-day \u003cem\u003esufS\u003c/em\u003e and \u003cem\u003esufE\u003c/em\u003e were PCR-amplified from the genomic DNA of \u003cem\u003eE. coli\u003c/em\u003e MG1655. The resulting DNA fragments were cloned into pET-30a(+) via \u003cem\u003eNdeI\u003c/em\u003e/\u003cem\u003eXhoI\u003c/em\u003e to append a C-terminal His\u003csub\u003e6\u003c/sub\u003e tag. Since His\u003csub\u003e6\u003c/sub\u003e-SufS\u003csup\u003eLCA\u003c/sup\u003e construct was insoluble, SufS\u003csup\u003eLCA\u003c/sup\u003e was prepared by cloning the gene fragment into pMAL-c6T to generate an N-terminal MBP-TEV-SufS\u003csup\u003eLCA\u003c/sup\u003e fusion. All constructs were sequence-verified (primer lists and sequences in Supplementary Table S3).\u003c/p\u003e\n\u003cp\u003ePlasmid constructs were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) pLysS. The strains were grown in LB at 37 \u0026deg;C to OD\u003csub\u003e600\u003c/sub\u003e \u0026asymp; 0.4, and protein synthesis was induced with the addition of 0.4 mM IPTG and subsequent incubation for 5 h at 37\u0026deg;C for SufE\u003csup\u003eLCA\u003c/sup\u003e, SufE\u003csup\u003eGOE\u003c/sup\u003e, SufE\u003csup\u003emodern\u003c/sup\u003e, SufS\u003csup\u003eGOE\u003c/sup\u003e, and SufS\u003csup\u003emodern\u003c/sup\u003e, and for overnight at 18 \u0026deg;C for MBP-SufS\u003csup\u003eLCA\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAll of the following steps were performed in an anaerobic glove box (O\u003csub\u003e2\u003c/sub\u003e \u0026lt; 0.1%, Coy) at 4 \u0026deg;C. Cells were harvested, cell pellets were lysed in a buffer solution of 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 5 mM DTT and 1 mM phenylmethylsulfonyl fluoride (PMSF), and the lysates were removed (20,000 g, 30 min). His-tagged proteins were purified successively on a HisTrap column (Cytiva) with a 10-500 mM imidazole linear gradient, a HiTrap Q column (Cytiva) using a 50 mM-1 M NaCl linear gradient at a pH of ~2 units above the PI of the protein, and a Superdex 75 Increase 10/300 column (Cytiva) with an elution buffer of 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT. The MBP fusion protein was purified on an MBPTrap HP column (Cytiva) with maltose elution. The MBP tag was removed by overnight Tobacco Etch Virus (TEV) protease digestion at 4 \u0026deg;C, followed by subtractive Ni\u003csup\u003e2+\u003c/sup\u003e-affinity purification. Protein concentrations were determined by BCA assay against a BSA standard. Aliquots were flash-frozen and stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern blotting\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProteins were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane for Western blotting, as previously described\u003csup\u003e71\u003c/sup\u003e, with an anti-His tag mouse monoclonal antibody (ThermoFisher Invitrogen) or anti-MBP monoclonal antibody (LabLead). An HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (ThermoFisher Invitrogen) was then used, and signals were detected using enhanced chemiluminescence (Thermo Scientific SuperSignal West Pico PLUS) and imaged (Tanon 1600).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCysteine desulfurase activity assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cysteine desulfurase activity of the SufS/SufE system was measured using a methylene blue assay as described previously\u003csup\u003e72,73\u003c/sup\u003e. To determine the appropriate SufE concentration, the reaction mixture contained 0.5 \u0026mu;M SufS, a gradient of SufE (0, 0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10.0 \u0026mu;M ), and 0.5 mM L-cysteine in 25 mM Tris-HCl buffer (pH 7.4) with 150 mM NaCl. Using GraphPad Prism (v10.4) fit to the Michaelis-Menten equation to obtain the apparent K\u003csub\u003em\u003c/sub\u003e of SufE. The formal reaction was conducted at a specified O\u003csub\u003e2\u003c/sub\u003e level in a modular atmosphere chamber,\u0026nbsp;reaction conditions are 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 \u0026mu;M SufS, 2\u0026times;K\u003csub\u003em\u003c/sub\u003e concentrations of SufE, and L-cysteine 0-500 \u0026mu;M. SufS and SufE were pre-equilibrated separately in the chamber at the tested O\u003csub\u003e2\u003c/sub\u003e level for 30 min and then mixed for 15 min prior to the reaction. The reaction was initiated by adding L-cysteine. After incubation for 30 min at 27\u003csup\u003eo\u003c/sup\u003eC, the reaction was quenched by adding 0.1 M NaOH. Next, DTT was added to 1\u0026nbsp;mM final concentration. Then 12.5 \u0026mu;L of 10% zinc acetate was added. Color development reagents, 25\u0026nbsp;\u0026mu;L of 20\u0026nbsp;mM DMPD and 25\u0026nbsp;\u0026mu;L of 30\u0026nbsp;mM FeCl\u003csub\u003e3\u003c/sub\u003e, were then introduced. The mixture was incubated for 30\u0026nbsp;min at room temperature in the dark and then shortly centrifuged. The absorbance of the supernatant at 670\u0026nbsp;nm was measured. One unit (1 mU) of cysteine desulfurase activity is defined as the formation of 1 \u0026mu;mol of S\u0026sup2;⁻ per min under the assay conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStrain construction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo construct an \u003cem\u003eE. coli\u003c/em\u003e strain for in vivo evaluation of the role of the reconstructed SufS/SufE proteins on oxidative stress response, we replaced a part of the \u003cem\u003eisc\u003c/em\u003e operon (\u003cem\u003eiscUA-hscBA\u003c/em\u003e) with a sequence encoding a ~4.2 kb heterologous mevalonate pathway\u003csup\u003e74\u003c/sup\u003e in \u003cem\u003eE. coli\u003c/em\u003e strain MG1655 by using a high-efficiency, low-escape CRISPR/Cas9 genome editing method\u003csup\u003e75\u003c/sup\u003e with the designed guide sequences (Supplementary Information Table S3). The sgRNA protospacer was introduced by annealing oligonucleotides carrying ~20-bp vector overlaps and assembled into the linearized pTargetT backbone (SpeI-HF). A donor cassette (MVA) flanked by homology arms to the target locus (\u0026Delta;\u003cem\u003eiscU\u003c/em\u003e-\u003cem\u003ehscA\u003c/em\u003e) was assembled by overlap-extension PCR from genomic templates, followed by amplification with primers adding ~20-bp overlaps to the linearized pTargetT vector at 5\u0026rsquo; end (Supplementary Information Table S3). pTargetT was linearized by restriction digestion and dephosphorylated (rSAP, NEB). The donor amplicon was inserted by Gibson assembly. All constructs were sequence-verified.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e MG1655 cells carrying pCas (Cas9/\u0026lambda;-Red) \u003csup\u003e76\u003c/sup\u003ewere prepared at 30 \u0026deg;C. \u0026lambda;-Red was induced with 0.2% L-arabinose, and pTargetT-donor-sgRNA was then introduced into the cells by electroporation. Transformants were selected on LB agar with kanamycin (50 \u0026micro;g mL⁻\u0026sup1;) and streptomycin (50 \u0026micro;g mL⁻\u0026sup1;) in the presence of L-arabinose and/or MVA as indicated. Colonies were screened by PCR (Supplementary Information Table S3), and positive clones were confirmed by Sanger sequencing. pTargetT was cured by repeated passage in medium without streptomycin. pCas was subsequently cured by growth at 42 \u0026deg;C in the absence of antibiotics. The resulting strain was denoted \u003cem\u003eE. coli\u003c/em\u003e MG1655 \u0026Delta;\u003cem\u003eiscU\u003c/em\u003e-\u003cem\u003ehscA::MVA\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e (CHY97).\u003c/p\u003e\n\u003cp\u003eTo ensure that CHY97 closely matched the genetic background of other strains used for phenotype testing (WO539/WO541 from the W. Outten lab, see below), P1 phage transduction was used to move the MVA cassette from WO539/541 into the strain. In the WO539/541 strains, the \u003cem\u003ePBAD-MVA-kan\u003csup\u003eR\u003c/sup\u003e\u003c/em\u003e cassette was integrated at the \u003cem\u003elac\u003c/em\u003e operon locus under the control of a BAD promoter and included a kanamycin resistance marker for selection\u003csup\u003e26\u003c/sup\u003e. P1 phage prepared on donor strain WO541 was transduced into CHY97, selecting for \u003cem\u003ekan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e colonies. This yielded strain CHY98 (genotype MG1655\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003eiscUAhscBA::MVA\u003csup\u003e+\u003c/sup\u003e + lac::[MVA+, kan\u003csup\u003eR\u003c/sup\u003e]\u003c/em\u003e), which carries the arabinose-inducible MVA pathway integrated at the \u003cem\u003elac\u0026nbsp;\u003c/em\u003elocus, identical to WO539/541. CHY98 was used as a positive control strain in growth assays, as it retains the native \u003cem\u003esuf\u003c/em\u003e operon and has the MVA pathway just like the WO539/541 mutants, allowing us to distinguish effects of the complementation constructs from any unintended effects of the MVA system or antibiotic markers.\u003c/p\u003e\n\u003cp\u003eThe donor and base strains used in this study: \u003cem\u003eE. coli\u003c/em\u003e WO539 (MG1655 \u0026Delta;\u003cem\u003eiscU-fdx\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003esufSE::cm\u003csup\u003eR\u003c/sup\u003e MVA\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e-kan\u003csup\u003eR\u003c/sup\u003e\u003c/em\u003e) and WO541 (MG1655 \u0026Delta;\u003cem\u003eiscU-fdx\u0026nbsp;\u003c/em\u003e\u0026Delta;\u003cem\u003esufE::cm\u003csup\u003eR\u003c/sup\u003e MVA\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e-kan\u003csup\u003eR\u003c/sup\u003e\u003c/em\u003e) were constructed by multiple P1 transductions of mutations from previously constructed strains. Briefly, \u003cem\u003eiscUA-hscBA-fdx\u003c/em\u003e was replaced with \u003cem\u003ekan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e from pKD4 and then the \u003cem\u003ekan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e was removed with plasmid pCP20 expressing flippase (FLP) recombinase\u003csup\u003e77\u003c/sup\u003e. The \u003cem\u003eMVA\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e-\u003cem\u003ekan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e locus was then P1 transduced into the ∆\u003cem\u003eiscU\u003c/em\u003e-\u003cem\u003efdx\u003c/em\u003e background. Finally, the \u003cem\u003esufE\u003c/em\u003e gene or \u003cem\u003esufSE\u0026nbsp;\u003c/em\u003egenes were replaced with \u003cem\u003ecm\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e from pKD3 and then P1 transduced into the ∆\u003cem\u003eiscU-fdx MVA\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e-\u003cem\u003ekan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e background. The final transductants were selected on and maintained in \u0026ldquo;5-component SB medium\u0026rdquo; (see below) to ensure that they had the mevalonate supplement for initial recovery. This Super Broth (SB) was a rich medium (32 g L\u003csup\u003e-1\u003c/sup\u003e tryptone, 20 g L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eyeast extract and 5 g L\u003csup\u003e-1\u003c/sup\u003e NaCl), supplemented with 0.2% (w/v) L-arabinose, 0.4% (w/v) D-glucose, 50 mg L\u003csup\u003e-1\u003c/sup\u003e chloramphenicol, 50 mg L\u003csup\u003e-1\u003c/sup\u003e kanamycin and 300 \u0026mu;M mevalonolactone (MVA, CAS 674-26-0).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComplementation assays\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo complement the SufE/SufSE deletions in vivo, a series of arabinose-inducible plasmids carrying various combinations of present-day or ancestral \u003cem\u003esufE\u003c/em\u003e and \u003cem\u003esufS\u003c/em\u003e genes were constructed. For the single-gene constructs (pBAD/His B-SufE series), the coding sequences for SufE\u003csup\u003eLCA\u003c/sup\u003e, SufE\u003csup\u003eGOE\u003c/sup\u003e, and SufE\u003csup\u003emodern\u003c/sup\u003e were PCR-amplified (Supplementary Information Table S3) from their respective pET 30a(+) templates and cloned into pBAD/His B. This placed the \u003cem\u003esufE\u003c/em\u003e gene under the arabinose promoter, with an N-terminal His\u003csub\u003e6\u003c/sub\u003e-tag (from the vector) on the expressed SufE protein. For the \u003cem\u003esufS/sufE\u003c/em\u003e constructs (pBAD/His B-SufSE series), two genes were amplified in tandem, including their native intergenic spacer. In the case of the \u003cem\u003esufS\u003csup\u003emodern\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003esufE\u003csup\u003emodern\u003c/sup\u003e\u003c/em\u003e, they were amplified directly from \u003cem\u003eE. coli\u003c/em\u003e MG1655 genomic DNA using primer pair pBAD-SufSE-F and HindIII-His-SufE-R (Supplementary Information Table S3). For the \u003cem\u003esufS\u003csup\u003eGOE\u003c/sup\u003e/sufE\u003csup\u003eGOE\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003esufS\u003csup\u003eLCA\u003c/sup\u003e/sufE\u003csup\u003eLCA\u003c/sup\u003e\u003c/em\u003e constructs, the \u003cem\u003esufS\u003c/em\u003e and \u003cem\u003esufE\u003c/em\u003e fragments were assembled by overlap extension PCR to include the native intergenic spacer (Supplementary Information Table S3). The two PCR fragments (ancestral \u003cem\u003esufS\u003c/em\u003e and s\u003cem\u003eufE\u003c/em\u003e) were then joined by overlap PCR to create a full-length \u003cem\u003esufSE\u003c/em\u003e di-cistron fragment with spacer and tags. This fragment was digested and cloned into pBAD/His B. Similarly, for SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e adding MBP at N terminal, a bicistronic insert was generated where \u003cem\u003esufS\u003csup\u003eLCA\u003c/sup\u003e\u003c/em\u003e included the coding sequence for an MBP tag fused at its N-terminus (carried over from the pMAL-SufS\u003csup\u003eLCA\u003c/sup\u003e template, Supplementary Information Table S3). Thus, this construct tests the effect of co-expressing an MBP-tagged SufS\u003csup\u003eLCA\u003c/sup\u003e with SufE\u003csup\u003eLCA\u003c/sup\u003e in vivo. All pBAD constructs were verified by restriction mapping and sequencing. The resulting complementation plasmids were each introduced into the appropriate mutant background. Successful transformants were selected on 5-component SB with 50 mg L\u003csup\u003e-1\u003c/sup\u003e ampicillin (to maintain the pBAD plasmid, referred to as \u0026ldquo;6-component SB), and verified. Glycerol stocks were made for each complementation strain.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGrowth assays under oxidative stress\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe growth phenotypes of the complemented strains under various oxidative stress conditions were assessed. The phenazine methosulfate (PMS) could generate oxidative stress inside cells. Concentration ranges for these stressors were determined based on literature and our pilot experiments: PMS was used at 0, 30, 60, 90, 150, 240, 300, and 450\u0026nbsp;\u0026mu;M\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor each strain, growth curves were obtained using a 96-well microplate format (200 \u0026mu;L culture per well, in at least three replicates). Prior to the experiment, all strains were revived from glycerol stocks on LB agar and then pre-cultured in a \u0026ldquo;6-component SB medium\u0026rdquo; (see above) to ensure they had the mevalonate supplement for initial recovery. After overnight growth in 6 component SB (37 \u0026deg;C, shaking), cultures were diluted 0.5% into the same medium but without mevalonate in order to test if the pBAD plasmids restored Fe-S dependent (MVA independent) isoprenoid biosynthesis. This medium still had ampicillin. We chose a 0.5% inoculum to minimize carryover of MVA from the starter culture. At this low inoculation, the control strain with empty plasmid (which cannot synthesize isoprenoids without MVA) did not grow for at least 48\u0026nbsp;h, confirming effective depletion of intracellular MVA with no carryover from the starter culture.\u003c/p\u003e\n\u003cp\u003eGrowth assays were performed in a FLUOstar Omega microplate reader (BMG Labtech) at 37\u0026nbsp;\u0026deg;C with continuous orbital shaking (200\u0026nbsp;rpm). The optical density was recorded every 5\u0026nbsp;min. Due to PMS\u0026rsquo;s color change upon oxidation (the medium gradually turned green, and even cell growth itself can alter PMS\u0026rsquo;s absorbance), a detailed spectral analysis (scanning 450-800 nm) showed that PMS causes elevated absorbance across a broad range (480-750 nm) and that high cell density plus PMS can increase OD readings in a wavelength-dependent manner. However, above 750 nm, the OD signal from cells alone started to diminish (due to scattering properties) and at wavelengths below 480 nm, the artifact due to PMS oxidation was minimal. OD\u003csub\u003e450\u003c/sub\u003e nm for PMS experiments was applied, as 450 nm gave a reliable indication of cell growth while avoiding most interference from PMS oxidation\u003csup\u003e78\u003c/sup\u003e. In all experiments, the first few OD readings (prior to significant growth, \u003cem\u003ee.g.\u003c/em\u003e the mean of 0-5 min readings) were used as a blank and subtracted from subsequent readings for each well, to normalize starting OD.\u003c/p\u003e\n\u003cp\u003eEach growth curve was then analyzed to extract key parameters. The R package gcplyr (v1.X) was utilized for growth curve analysis\u003csup\u003e79\u003c/sup\u003e. The OD data (log-transformed OD values vs. time) were fit to a growth model to estimate: the lag time (adjustment phase duration), the maximum growth rate (slope during exponential phase), and the maximum cell density (ODₘₐₓ). The results were plotted using GraphPad Prism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParallel Reaction Monitoring (PRM) Targeted proteomics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSynthesis of SufS and SufE in complementation cells was confirmed by targeted quantitative proteomics using PRM mass spectrometry. Cultures of the WO539 + pBAD/His B-SufSE (ancestral sufSE) strains were grown to log phase (OD\u003csub\u003e600\u003c/sub\u003e ~0.5) and stationary phase (overnight culture) in 6 component SB medium (with Amp and MVA) under aerobic conditions. Cells from 50 mL of each culture were harvested by centrifugation (5,000 g, 10 min, 4 \u0026deg;C), washed with cold PBS, resuspended in 200 \u0026mu;L of urea lysis buffer (7 M urea, 2 M thiourea, 0.1% CHAPS, 1\u0026times; protease inhibitor cocktail), and lysed by sonication on ice (15 min total sonication time, in pulses). Total protein concentration in each sample was determined by BCA assay using a standard protocol\u003csup\u003e80\u003c/sup\u003e. An aliquot (~50\u0026nbsp;\u0026mu;g protein) of each sample was subjected to digestion and subsequent mass spectrum (MS) by following a filter-aided sample preparation (FASP) protocol\u003csup\u003e81\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor mass spectrometry, dried peptides (~0.5\u0026nbsp;\u0026mu;g) were analyzed by LC-MS/MS on an EASY-nLC 1200 UHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific). Separation was achieved on a C18 reversed-phase column (150 \u0026mu;m\u0026nbsp;\u0026times;\u0026nbsp;150\u0026nbsp;mm, 1.9\u0026nbsp;\u0026mu;m) using a 60 min gradient (8-45% acetonitrile/0.1% formic acid) at 500 nL min\u003csup\u003e-1\u003c/sup\u003e. The mass spectrometer was operated in positive ion mode with full MS scans acquired at 120,000 resolution (m/z 350-1500), followed by higher-energy collisional dissociation (HCD) with normalized collision energy 30%. MS2 spectra were acquired in the Orbitrap at 15,000 mass resolution, with dynamic exclusion (45 s). The DDA MS/MS data were searched using Proteome Discoverer (v2.3, Thermo) against the UniProt \u003cem\u003eE. coli\u003c/em\u003e K-12 proteome reference (downloaded 2024-06-11) with the sequences of SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e and SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e proteins appended (total 4406 sequences). Trypsin (up to 2 missed cleavages allowed) and Carbamidomethyl (Cys) as fixed, and Oxidation (Met) and Acetyl (protein N-terminus) as variable modifications were employed. Precursor ion mass tolerance was 10 ppm, and fragment ion tolerance 0.02 Da. Peptide-spectrum matches were filtered to a false discovery rate (FDR) of \u0026lt; 1% using a target-decoy strategy.\u003c/p\u003e\n\u003cp\u003eUsing Skyline (MacCoss Lab, v4.X), 3 unique peptides for each target protein (SufS variants and SufE variants of interest) that were confidently identified in the DDA run were selected. The precursor m/z for each target peptide and its charge state, along with the retention time (RT) window (~5 min around the observed RT), were compiled for PRM. In total, 12 peptides covering all target proteins were monitored by unique peptides.\u0026nbsp;PRM assays were performed by measuring these peptides and corresponding stable isotope-labeled standards (SIS). Quantification was achieved by calculating the ratio of protein peptides to SIS peptide peak areas.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData and Code availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article, its Supplementary Information files and https://github.com/eacochen/SufE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"40\"\u003e\n \u003cli\u003eBuchfink, B., Reuter, K. \u0026amp; Drost, H.-G. Sensitive protein alignments at tree-of-life scale using DIAMOND. \u003cem\u003eNature Methods\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 366-368 (2021). https://doi.org:10.1038/s41592-021-01101-x\u003c/li\u003e\n \u003cli\u003ePotter, S. C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e HMMER web server: 2018 update. \u003cem\u003eNucleic acids research\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, W200-W204 (2018).\u003c/li\u003e\n \u003cli\u003eMistry, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Pfam: The protein families database in 2021. \u003cem\u003eNucleic acids research\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, D412-D419 (2021).\u003c/li\u003e\n \u003cli\u003eFu, L., Niu, B., Zhu, Z., Wu, S. \u0026amp; Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 3150-3152 (2012). https://doi.org:10.1093/bioinformatics/bts565\u003c/li\u003e\n \u003cli\u003eKatoh, K. \u0026amp; Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 772-780 (2013). https://doi.org:10.1093/molbev/mst010\u003c/li\u003e\n \u003cli\u003eCapella-Gutierrez, S., Silla-Martinez, J. M. \u0026amp; Gabaldon, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1972-1973 (2009). https://doi.org:10.1093/bioinformatics/btp348\u003c/li\u003e\n \u003cli\u003ePrice, M. N., Dehal, P. S. \u0026amp; Arkin, A. P. FastTree 2 \u0026ndash; Approximately Maximum-Likelihood Trees for Large Alignments. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e9490 (2010). https://doi.org:10.1371/journal.pone.0009490\u003c/li\u003e\n \u003cli\u003eMinh, B. Q.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 1530-1534 (2020). https://doi.org:10.1093/molbev/msaa015\u003c/li\u003e\n \u003cli\u003eTria, F. D. K., Landan, G. \u0026amp; Dagan, T. Phylogenetic rooting using minimal ancestor deviation. \u003cem\u003eNature ecology \u0026amp; evolution\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 0193 (2017).\u003c/li\u003e\n \u003cli\u003eMai, U., Sayyari, E. \u0026amp; Mirarab, S. Minimum variance rooting of phylogenetic trees and implications for species tree reconstruction. \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0182238 (2017).\u003c/li\u003e\n \u003cli\u003eLetunic, I. \u0026amp; Bork, P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. \u003cem\u003eNucleic acids research\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, W78-W82 (2024).\u003c/li\u003e\n \u003cli\u003eMaglott, D., Ostell, J., Pruitt, K. D. \u0026amp; Tatusova, T. Entrez Gene: gene-centered information at NCBI. \u003cem\u003eNucleic acids research\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, D54-D58 (2005).\u003c/li\u003e\n \u003cli\u003eParks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. \u0026amp; Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. \u003cem\u003eGenome research\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1043-1055 (2015).\u003c/li\u003e\n \u003cli\u003eMurali, R., Hemp, J. \u0026amp; Gennis, R. B. Evolution of quinol oxidation within the heme‑copper oxidoreductase superfamily. \u003cem\u003eBiochim Biophys Acta Bioenerg\u003c/em\u003e \u003cstrong\u003e1863\u003c/strong\u003e, 148907 (2022). https://doi.org:10.1016/j.bbabio.2022.148907\u003c/li\u003e\n \u003cli\u003eMurali, R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Diversity and evolution of nitric oxide reduction in bacteria and archaea. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, e2316422121 (2024). https://doi.org:10.1073/pnas.2316422121\u003c/li\u003e\n \u003cli\u003eRavenhall, M., \u0026Scaron;kunca, N., Lassalle, F. \u0026amp; Dessimoz, C. Inferring horizontal gene transfer. \u003cem\u003ePLoS computational biology\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e1004095 (2015).\u003c/li\u003e\n \u003cli\u003eSeemann, T. Prokka: rapid prokaryotic genome annotation. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 2068-2069 (2014). https://doi.org:10.1093/bioinformatics/btu153\u003c/li\u003e\n \u003cli\u003eChaumeil, P.-A., Mussig, A. J., Hugenholtz, P. \u0026amp; Parks, D. H. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 5315-5316 (2022).\u003c/li\u003e\n \u003cli\u003eMorel, B., Kozlov, A. M., Stamatakis, A. \u0026amp; Sz\u0026ouml;llősi, G. J. GeneRax: A Tool for Species-Tree-Aware Maximum Likelihood-Based Gene Family Tree Inference under Gene Duplication, Transfer, and Loss. \u003cem\u003eMol Biol Evol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 2763-2774 (2020). https://doi.org:10.1093/molbev/msaa141\u003c/li\u003e\n \u003cli\u003eWang, S. \u0026amp; Luo, H. Dating Alphaproteobacteria evolution with eukaryotic fossils. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 3324 (2021). https://doi.org:10.1038/s41467-021-23645-4\u003c/li\u003e\n \u003cli\u003eLiao, T., Wang, S., Zhang, H., Stüeken, E. E. \u0026amp; Luo, H. Dating Ammonia-Oxidizing Bacteria with Abundant Eukaryotic Fossils. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, msae096 (2024). https://doi.org:10.1093/molbev/msae096\u003c/li\u003e\n \u003cli\u003eCrowe, S. A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Atmospheric oxygenation three billion years ago. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e501\u003c/strong\u003e, 535-538 (2013).\u003c/li\u003e\n \u003cli\u003eZhang, H., Sun, Y., Zeng, Q., Crowe, S. A. \u0026amp; Luo, H. Snowball Earth, population bottleneck and Prochlorococcus evolution. \u003cem\u003eProceedings of the Royal Society B\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 20211956 (2021).\u003c/li\u003e\n \u003cli\u003eGolubic, S., Sergeev, V. N. \u0026amp; Knoll, A. H. Mesoproterozoic Archaeoellipsoides: akinetes of heterocystous cyanobacteria. \u003cem\u003eLethaia\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 285-298 (1995).\u003c/li\u003e\n \u003cli\u003eGolubic, S. \u0026amp; Seong-Joo, L. Early cyanobacterial fossil record: preservation, palaeoenvironments and identification. \u003cem\u003eEuropean Journal of Phycology\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 339-348 (1999).\u003c/li\u003e\n \u003cli\u003eYang, Z. PAML 4: phylogenetic analysis by maximum likelihood. \u003cem\u003eMolecular biology and evolution\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1586-1591 (2007).\u003c/li\u003e\n \u003cli\u003edos Reis, M., \u0026Aacute;lvarez-Carretero, S. \u0026amp; Yang, Z. (2017).\u003c/li\u003e\n \u003cli\u003eReis, M. D.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Using phylogenomic data to explore the effects of relaxed clocks and calibration strategies on divergence time estimation: primates as a test case. \u003cem\u003eSystematic Biology\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 594-615 (2018).\u003c/li\u003e\n \u003cli\u003eGarcia, A. K., Fer, E., Sephus, C. \u0026amp; Kacar, B. in \u003cem\u003eEnvironmental Microbial Evolution: Methods and Protocols\u003c/em\u003e (ed Haiwei Luo) 267-281 (Springer US, 2022).\u003c/li\u003e\n \u003cli\u003eAshkenazy, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e FastML: a web server for probabilistic reconstruction of ancestral sequences. \u003cem\u003eNucleic acids research\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, W580-W584 (2012).\u003c/li\u003e\n \u003cli\u003eAbramson, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Accurate structure prediction of biomolecular interactions with AlphaFold 3. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e630\u003c/strong\u003e, 493-500 (2024). https://doi.org:10.1038/s41586-024-07487-w\u003c/li\u003e\n \u003cli\u003eKurien, B. T. \u0026amp; Scofield, R. H. Western blotting. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 283-293 (2006).\u003c/li\u003e\n \u003cli\u003eAddo, M. A., Edwards, A. M. \u0026amp; Dos Santos, P. C. Methods to Investigate the Kinetic Profile of Cysteine Desulfurases. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e2353\u003c/strong\u003e, 173-189 (2021). https://doi.org:10.1007/978-1-0716-1605-5_10\u003c/li\u003e\n \u003cli\u003eSiegel, L. M. A direct microdetermination for sulfide. \u003cem\u003eAnalytical biochemistry\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 126-132 (1965).\u003c/li\u003e\n \u003cli\u003eLiu, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Lycopene production from glucose, fatty acid and waste cooking oil by metabolically engineered Escherichia coli. \u003cem\u003eBiochemical Engineering Journal\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e, 107488 (2020).\u003c/li\u003e\n \u003cli\u003eLi, Q.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. \u003cem\u003eActa Biochim Biophys Sin (Shanghai)\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 620-627 (2021). https://doi.org:10.1093/abbs/gmab036\u003c/li\u003e\n \u003cli\u003eJiang, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 2506-2514 (2015). https://doi.org:10.1128/AEM.04023-14\u003c/li\u003e\n \u003cli\u003eDatsenko, K. A. \u0026amp; Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 6640-6645 (2000). https://doi.org:doi:10.1073/pnas.120163297\u003c/li\u003e\n \u003cli\u003eJensen, P. R. \u0026amp; Hammer, K. Minimal requirements for exponential growth of Lactococcus lactis. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 4363-4366 (1993).\u003c/li\u003e\n \u003cli\u003eBlazanin, M. gcplyr: an R package for microbial growth curve data analysis. \u003cem\u003eBMC bioinformatics\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 232 (2024).\u003c/li\u003e\n \u003cli\u003eSapan, C. V., Lundblad, R. L. \u0026amp; Price, N. C. Colorimetric protein assay techniques. \u003cem\u003eBiotechnology and applied Biochemistry\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 99-108 (1999).\u003c/li\u003e\n \u003cli\u003eWiśniewski, J. R. in \u003cem\u003eMicrobial proteomics: methods and protocols\u003c/em\u003e 3-10 (Springer, 2018).\u003c/li\u003e\n \u003cli\u003eJavaux, E. J. \u0026amp; Lepot, K. The Paleoproterozoic fossil record: implications for the evolution of the biosphere during Earth\u0026apos;s middle-age. \u003cem\u003eEarth-Science Reviews\u003c/em\u003e \u003cstrong\u003e176\u003c/strong\u003e, 68-86 (2018).\u003c/li\u003e\n \u003cli\u003eWang, S. \u0026amp; Luo, H. Dating the bacterial tree of life based on ancient symbiosis. \u003cem\u003eSystematic Biology\u003c/em\u003e, syae071 (2025). https://doi.org:10.1093/sysbio/syae071\u003c/li\u003e\n \u003cli\u003eHanson-Smith, V., Kolaczkowski, B. \u0026amp; Thornton, J. W. Robustness of ancestral sequence reconstruction to phylogenetic uncertainty. \u003cem\u003eMolecular biology and evolution\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1988-1999 (2010).\u003c/li\u003e\n \u003cli\u003eMihara, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structure of External Aldimine of Escherichia coli CsdB, an IscS/Nifs Homolog: Implications for Its Specificity toward Selenocysteine1. \u003cem\u003eThe Journal of Biochemistry\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 679-685 (2002). https://doi.org:10.1093/oxfordjournals.jbchem.a003151\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (42192500 and 42192503). This work was supported by the High-performance Computing Platform of China University of Geosciences-Beijing. This work was also supported by the Laboratory of Microbiology Resources and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences. A grant from the United States National Institutes of Health (GM112919) also supported F.W.O.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.D. led the study. H.D., L.H., and F.W.O. designed the research. H.C. performed gene retrieval and curation, phylogenetic analyses, and ancestral sequence reconstruction. H.C., D.X., and Z.Z. conducted the biochemical experiments. H.C. and M.Z. carried out the gene knockouts, and H.C. and X.T. carried out the gene knock-ins. H.C. performed the physiological assays. H.C. wrote the manuscript, and all authors contributed to discussions, with substantial input from H.D., L.H., and F.W.O..\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAdditional information.\u003c/strong\u003e Supplementary information is available for this paper. Correspondence and requests for material\u003cstrong\u003es\u0026nbsp;\u003c/strong\u003eshould be addressed to Hailiang Dong.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLyons TW et al (2024) Co-evolution of early Earth environments and microbial life. Nat Rev Microbiol 22:572\u0026ndash;586. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41579-024-01044-y\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41579-024-01044-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLyons TW, Reinhard CT, Planavsky NJ (2014) The rise of oxygen in Earth's early ocean and atmosphere. Nature 506:307\u0026ndash;315. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/nature13068\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/nature13068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBl\u0026auml;ttler CL et al (2018) Two-billion-year-old evaporites capture Earth\u0026rsquo;s great oxidation. Science 360:320\u0026ndash;323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:doi:10.1126/science.aar2687\u003c/span\u003e\u003cspan address=\"https://doi.org:doi:10.1126/science.aar2687\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHodgskiss MSW, Crockford PW, Turchyn AV (2023) Deconstructing the Lomagundi-Jatuli Carbon Isotope Excursion. Annu Rev Earth Planet Sci 51:301\u0026ndash;330. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1146/annurev-earth-031621-071250\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1146/annurev-earth-031621-071250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorado G et al (2010) Biological mass extinctions on planet Earth. Archaeobios 4:53\u0026ndash;64\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDav\u0026iacute;n AA et al (2025) A geological timescale for bacterial evolution and oxygen adaptation. Science 388:eadp1853\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiss MC et al (2016) The physiology and habitat of the last universal common ancestor. Nat Microbiol 1:16116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/nmicrobiol.2016.116\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/nmicrobiol.2016.116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFontecave M (2006) Iron-sulfur clusters: ever-expanding roles. Nat Chem Biol 2:171\u0026ndash;174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/nchembio0406-171\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/nchembio0406-171\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraymer JJ, Freibert SA, Rakwalska-Bange M, Lill R (2021) Mechanistic concepts of iron-sulfur protein biogenesis in Biology. Biochim et Biophys Acta (BBA) - Mol Cell Res 1868:118863. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:https://doi.org/10.1016/j.bbamcr.2020.118863\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.bbamcr.2020.118863\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoche B et al (2013) Reprint of: Iron/sulfur proteins biogenesis in prokaryotes: Formation, regulation and diversity. Biochim et Biophys Acta (BBA) - Bioenergetics 1827:923\u0026ndash;937. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbabio.2013.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.bbabio.2013.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlahut M, Sanchez E, Fisher CE, Outten FW (2020) Fe-S cluster biogenesis by the bacterial Suf pathway. Biochim Biophys Acta Mol Cell Res 1867:118829. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.bbamcr.2020.118829\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.bbamcr.2020.118829\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOutten FW, Djaman O, Storz G (2004) A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli. Mol Microbiol 52:861\u0026ndash;872. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1111/j.1365-2958.2004.04025.x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1111/j.1365-2958.2004.04025.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAyala-Castro C, Saini A, Outten FW (2008) Fe-S cluster assembly pathways in bacteria. Microbiol Mol Biol Rev 72:110\u0026ndash;125 table of contents. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1128/MMBR.00034-07\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1128/MMBR.00034-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia PS et al (2022) An early origin of iron-sulfur cluster biosynthesis machineries before Earth oxygenation. Nat Ecol Evol 6:1564\u0026ndash;1572. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41559-022-01857-1\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41559-022-01857-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyd ES, Thomas KM, Dai Y, Boyd JM, Outten FW (2014) Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway. Biochemistry 53:5834\u0026ndash;5847. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1021/bi500488r\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1021/bi500488r\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoiseau L, Ollagnier-de-Choudens S, Nachin L, Fontecave M, Barras F (2003) Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase. J Biol Chem 278:38352\u0026ndash;38359. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1074/jbc.M305953200\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1074/jbc.M305953200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOutten FW, Wood MJ, Munoz FM, Storz G (2003) The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli. J Biol Chem 278:45713\u0026ndash;45719. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1074/jbc.M308004200\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1074/jbc.M308004200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGogar RK et al (2024) The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis. J Biol Chem 300:107641. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.jbc.2024.107641\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.jbc.2024.107641\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldsmith-Fischman S et al (2004) The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes. J Mol Biol 344:549\u0026ndash;565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.jmb.2004.08.074\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.jmb.2004.08.074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai Y, Outten FW (2012) The E. coli SufS-SufE sulfur transfer system is more resistant to oxidative stress than IscS-IscU. FEBS Lett 586:4016\u0026ndash;4022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.febslet.2012.10.001\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.febslet.2012.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLayer G et al (2007) SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. J Biol Chem 282:13342\u0026ndash;13350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1074/jbc.M608555200\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1074/jbc.M608555200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOutten FW (2015) Recent advances in the Suf Fe-S cluster biogenesis pathway: Beyond the Proteobacteria. Biochim Biophys Acta 1853:1464\u0026ndash;1469. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.bbamcr.2014.11.001\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.bbamcr.2014.11.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCondie KC, Kr\u0026ouml;ner A (2013) The building blocks of continental crust: Evidence for a major change in the tectonic setting of continental growth at the end of the Archean. Gondwana Res 23:394\u0026ndash;402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.gr.2011.09.011\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.gr.2011.09.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujishiro T et al (2017) Zinc-Ligand Swapping Mediated Complex Formation and Sulfur Transfer between SufS and SufU for Iron\u0026ndash;Sulfur Cluster Biogenesis in Bacillus subtilis. J Am Chem Soc 139:18464\u0026ndash;18467. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1021/jacs.7b11307\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1021/jacs.7b11307\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka N et al (2016) Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron\u0026ndash;sulfur clusters. Mol Microbiol 99:835\u0026ndash;848\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCAMPOS N et al (2001) Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. Biochem J 353:59\u0026ndash;67\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerstel A et al (2020) Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis. PLoS Genet 16:e1009198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1371/journal.pgen.1009198\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1371/journal.pgen.1009198\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElchennawi I, Carpentier P, Caux C, Ponge M (2023) Ollagnier de Choudens, S. Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex. Biomolecules 13:732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobbins L et al (2013) Authigenic iron oxide proxies for marine zinc over geological time and implications for eukaryotic metallome evolution. Geobiology 11:295\u0026ndash;306\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaines SJ, Lenton TM (2016) The effect of widespread early aerobic marine ecosystems on methane cycling and the Great Oxidation. Earth Planet Sci Lett 434:42\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.epsl.2015.11.021\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.epsl.2015.11.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiding R, Fralick P, Liang L (2014) Identification of an Archean marine oxygen oasis. Precambrian Res 251\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaym M et al (2016) Spatiotemporal microbial evolution on antibiotic landscapes. Science 353:1147\u0026ndash;1151. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:doi\u003c/span\u003e\u003cspan address=\"https://doi.org:doi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.aag0822\u003c/span\u003e\u003cspan address=\"10.1126/science.aag0822\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhademian M, Imlay JA (2021) How microbes evolved to tolerate oxygen. Trends Microbiol 29:428\u0026ndash;440\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParsons C, Stueken EE, Rosen CJ, Mateos K, Anderson RE (2021) Radiation of nitrogen-metabolizing enzymes across the tree of life tracks environmental transitions in Earth history. Geobiology 19:18\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1111/gbi.12419\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1111/gbi.12419\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia AK et al (2023) Nitrogenase resurrection and the evolution of a singular enzymatic mechanism. Elife 12:e85003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuevas Zuvir\u0026iacute;a B et al (2025) Structural evolution of nitrogenase over 3 billion years. \u003cem\u003eeLife\u003c/em\u003e 14, RP105613 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.7554/eLife.105613\u003c/span\u003e\u003cspan address=\"https://doi.org:10.7554/eLife.105613\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X et al (2024) Bioavailability of molybdenite to support nitrogen fixation on early Earth by an anoxygenic phototroph. Earth Planet Sci Lett 647:119056. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.epsl.2024.119056\u003c/span\u003e\u003cspan address=\"10.1016/j.epsl.2024.119056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranke P, Freiberger S, Zhang L, Einsle O (2025) Conformational protection of molybdenum nitrogenase by Shethna protein II. Nature 637:998\u0026ndash;1004. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41586-024-08355-3\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41586-024-08355-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNarehood SM et al (2025) Structural basis for the conformational protection of nitrogenase from O2. Nature 637:991\u0026ndash;997. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41586-024-08311-1\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41586-024-08311-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7916008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7916008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"One of the most important events in Earth history is the Great Oxidation Event (GOE). While O\u003csub\u003e2\u003c/sub\u003e killed most anaerobic microorganisms, some survived. Fe-S clusters are cofactors essential for cellular processes in all life forms, but how they adapt to rising O\u003csub\u003e2\u003c/sub\u003e remains unclear. Sulfur utilization factor (SUF) pathway is one of the most common Fe-S assembly pathways. We hypothesize that within the SUF pathway, SufE, as a sulfur-transfer partner of cysteine desulfurase SufS, maintains its functions under oxidative stress through molecular adaptation. Molecular clock dating showed SufE originated ~2.67 Ga (i.e., last common ancestor, LCA) and diversified considerably around the GOE (~2.14 Ga). The corresponding ancestral SufS was also reconstructed for these two times. Biochemical assays reveal that SufS\u003csup\u003eLCA\u003c/sup\u003e/SufE\u003csup\u003eLCA\u003c/sup\u003e is active at up to ~2% O\u003csub\u003e2\u003c/sub\u003e, higher than Archaean atmospheric O\u003csub\u003e2\u003c/sub\u003e, whereas SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e is active at up to ~10% O\u003csub\u003e2\u003c/sub\u003e, higher than the level during the GOE. These advanced evolutions may have provided resilience to redox fluctuations through Earth history. Growth experiments showed that overproduction of either SufE\u003csup\u003eGOE\u003c/sup\u003e or SufS\u003csup\u003eGOE\u003c/sup\u003e/SufE\u003csup\u003eGOE\u003c/sup\u003e in \u003ci\u003eEscherichia coli\u003c/i\u003e mutants lacking SufE or SufSE better restores its growth than overproduction of their LCA counterparts, consistent with the in vitro results. Enzyme structure prediction revealed that such adaptation was achieved through replacement of a few amino acids in key catalytic sites and consequent conformational changes of key enzymes. Our results reveal the molecular mechanism of adaptation of Fe-S cluster assembly to rising O\u003csub\u003e2\u003c/sub\u003e and significantly contributes to the coevolution of the geosphere and biosphere.","manuscriptTitle":"Adaptation of Fe-S Cluster Assembly to Rising O2 Levels over Geological Time","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 07:53:13","doi":"10.21203/rs.3.rs-7916008/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e8f4e815-3ec8-4960-a5c7-28007a4be915","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60794025,"name":"Biological sciences/Evolution"},{"id":60794026,"name":"Earth and environmental sciences/Biogeochemistry"},{"id":60794027,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-02-11T17:55:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 07:53:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7916008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7916008","identity":"rs-7916008","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}