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Molecular oxygen is a stable diradical. All O 2 -dependent enzymes employ a radical mechanism. Generated by cyanobacteria, O 2 started accumulating on Earth 2.4 billion years ago. Its evolutionary impact is traditionally sought in respiration and energy yield. We mapped 365 O 2 -dependent enzymatic reactions of prokaryotes to phylogenies for the corresponding 792 protein families. The main physiological adaptations imparted by O 2 -dependent enzymes were not energy conservation, but novel organic substrate oxidations and O 2 -dependent, hence O 2 -tolerant, alternative pathways for O 2 -inhibited reactions. Oxygen-dependent enzymes evolved in ancestrally anaerobic pathways for essential cofactor biosynthesis including NAD + , pyridoxal, thiamine, ubiquinone, cobalamin, heme, and chlorophyll. These innovations allowed prokaryotes to synthesize essential cofactors in O 2 -containing environments, a prerequisite for the later emergence of aerobic respiratory chains., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Prokaryotic genomes constantly undergo gene flux via lateral gene transfer, generating a pangenome structure consisting of a conserved core genome surrounded by a more variable accessory genome shell. Over time, flux generates change in genome content. Here, we measure and compare the rate of genome flux for 5655 prokaryotic genomes as a function of amino acid sequence divergence in 36 universally distributed proteins of the informational core (IC). We find a clock of gene content change. The long-term average rate of gene content flux is remarkably constant across all higher prokaryotic taxa sampled, whereby the size of the accessory genome-the proportion of the genome harboring gene content difference for genome pairs-varies across taxa. The proportion of species-level accessory genes per genome, varies from 0% (Chlamydia) to 30%-33% (Alphaproteobacteria, Gammaproteobacteria, and Clostridia). A clock-like rate of gene content change across all prokaryotic taxa sampled suggest that pangenome structure is a general feature of prokaryotic genomes and that it has been in existence since the divergence of bacteria and archaea., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The Moon-forming impact vaporized part of Earth's mantle, and turned the rest into a magma ocean, from which carbon dioxide degassed into the atmosphere, where it stayed until water rained out to form the oceans. The rain dissolved CO 2 and made it available to react with transition metal catalysts in the Earth's crust so as to ultimately generate the organic compounds that form the backbone of microbial metabolism. The Moon-forming impact was key in building a planet with the capacity to generate life in that it converted carbon on Earth into a homogeneous and accessible substrate for organic synthesis. Today all ecosystems, without exception, depend upon primary producers, organisms that fix CO 2 . According to theories of autotrophic origin, it has always been that way, because autotrophic theories posit that the first forms of life generated all the molecules needed to build a cell from CO 2 , forging a direct line of continuity between Earth's initial CO 2 -rich atmosphere and the first microorganisms. By modern accounts these were chemolithoautotrophic archaea and bacteria that initially colonized the crust and still inhabit that environment today., (© 2023 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Serpentinization in hydrothermal vents is central to some autotrophic theories for the origin of life because it generates compartments, reductants, catalysts and gradients. During the process of serpentinization, water circulates through hydrothermal systems in the crust where it oxidizes Fe (II) in ultramafic minerals to generate Fe (III) minerals and H 2 . Molecular hydrogen can, in turn, serve as a freely diffusible source of electrons for the reduction of CO 2 to organic compounds, provided that suitable catalysts are present. Using catalysts that are naturally synthesized in hydrothermal vents during serpentinization H 2 reduces CO 2 to formate, acetate, pyruvate, and methane. These compounds represent the backbone of microbial carbon and energy metabolism in acetogens and methanogens, strictly anaerobic chemolithoautotrophs that use the acetyl-CoA pathway of CO 2 fixation and that inhabit serpentinizing environments today. Serpentinization generates reduced carbon, nitrogen and - as newer findings suggest - reduced phosphorous compounds that were likely conducive to the origins process. In addition, it gives rise to inorganic microcompartments and proton gradients of the right polarity and of sufficient magnitude to support chemiosmotic ATP synthesis by the rotor-stator ATP synthase. This would help to explain why the principle of chemiosmotic energy harnessing is more conserved (older) than the machinery to generate ion gradients via pumping coupled to exergonic chemical reactions, which in the case of acetogens and methanogens involve H 2 -dependent CO 2 reduction. Serpentinizing systems exist in terrestrial and deep ocean environments. On the early Earth they were probably more abundant than today. There is evidence that serpentinization once occurred on Mars and is likely still occurring on Saturn's icy moon Enceladus, providing a perspective on serpentinization as a source of reductants, catalysts and chemical disequilibrium for life on other worlds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schwander, Brabender, Mrnjavac, Wimmer, Preiner and Martin.)

Though all theories for the origin of life require a source of energy to promote primordial chemical reactions, the nature of energy that drove the emergence of metabolism at origins is still debated. We reasoned that evidence for the nature of energy at origins should be preserved in the biochemical reactions of life itself, whereby changes in free energy, Δ G , which determine whether a reaction can go forward or not, should help specify the source. By calculating values of Δ G across the conserved and universal core of 402 individual reactions that synthesize amino acids, nucleotides and cofactors from H 2 , CO 2 , NH 3 , H 2 S and phosphate in modern cells, we find that 95-97% of these reactions are exergonic (Δ G ≤ 0 kJ⋅mol -1 ) at pH 7-10 and 80-100°C under nonequilibrium conditions with H 2 replacing biochemical reductants. While 23% of the core's reactions involve ATP hydrolysis, 77% are ATP-independent, thermodynamically driven by Δ G of reactions involving carbon bonds. We identified 174 reactions that are exergonic by -20 to -300 kJ⋅mol -1 at pH 9 and 80°C and that fall into ten reaction types: six pterin dependent alkyl or acyl transfers, ten S-adenosylmethionine dependent alkyl transfers, four acyl phosphate hydrolyses, 14 thioester hydrolyses, 30 decarboxylations, 35 ring closure reactions, 31 aromatic ring formations, and 44 carbon reductions by reduced nicotinamide, flavins, ferredoxin, or formate. The 402 reactions of the biosynthetic core trace to the last universal common ancestor (LUCA), and reveal that synthesis of LUCA's chemical constituents required no external energy inputs such as electric discharge, UV-light or phosphide minerals. The biosynthetic reactions of LUCA uncover a natural thermodynamic tendency of metabolism to unfold from energy released by reactions of H 2 , CO 2 , NH 3 , H 2 S, and phosphate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wimmer, Xavier, Vieira, Pereira, Leidner, Sousa, Kleinermanns, Preiner and Martin.)

The possible evolutionary significance of pyrophosphate (PP i ) has been discussed since the early 1960s. Lipmann suggested that PP i could have been an ancient currency or a possible environmental source of metabolic energy at origins, while Kornberg proposed that PP i vectorializes metabolism because ubiquitous pyrophosphatases render PP i forming reactions kinetically irreversible. To test those ideas, we investigated the reactions that consume phosphoanhydride bonds among the 402 reactions of the universal biosynthetic core that generates amino acids, nucleotides, and cofactors from H 2 , CO 2 , and NH 3 . We find that 36% of the core's phosphoanhydride hydrolyzing reactions generate PP i , while no reactions use PP i as an energy currency. The polymerization reactions that generate ~80% of cell mass - protein, RNA, and DNA synthesis - all generate PP i , while none use PP i as an energy source. In typical prokaryotic cells, aminoacyl tRNA synthetases (AARS) underlie ~80% of PP i production. We show that the irreversibility of the AARS reaction is a kinetic, not a thermodynamic effect. The data indicate that PP i is not an ancient energy currency and probably never was. Instead, PP i hydrolysis is an ancient mechanism that imparts irreversibility, as Kornberg suggested, functioning like a ratchet's pawl to vectorialize the life process toward growth. The two anhydride bonds in nucleoside triphosphates offer ATP-cleaving enzymes an option to impart either thermodynamic control (P i formation) or kinetic control (PP i formation) upon reactions. This dual capacity explains why nature chose the triphosphate moiety of ATP as biochemistry's universal energy currency., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wimmer, Kleinermanns and Martin.)

The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here, we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial-derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal-derived and eukaryote-specific genes. The surplus of bacterial-derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage-specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggests that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Bacteria are the most abundant cells on Earth. They are generally regarded as ancient, but due to striking diversity in their metabolic capacities and widespread lateral gene transfer, the physiology of the first bacteria is unknown. From 1089 reference genomes of bacterial anaerobes, we identified 146 protein families that trace to the last bacterial common ancestor, LBCA, and form the conserved predicted core of its metabolic network, which requires only nine genes to encompass all universal metabolites. Our results indicate that LBCA performed gluconeogenesis towards cell wall synthesis, and had numerous RNA modifications and multifunctional enzymes that permitted life with low gene content. In accordance with recent findings for LUCA and LACA, analyses of thousands of individual gene trees indicate that LBCA was rod-shaped and the first lineage to diverge from the ancestral bacterial stem was most similar to modern Clostridia, followed by other autotrophs that harbor the acetyl-CoA pathway.

The metabolism of cells contains evidence reflecting the process by which they arose. Here, we have identified the ancient core of autotrophic metabolism encompassing 404 reactions that comprise the reaction network from H 2 , CO 2 , and ammonia (NH 3 ) to amino acids, nucleic acid monomers, and the 19 cofactors required for their synthesis. Water is the most common reactant in the autotrophic core, indicating that the core arose in an aqueous environment. Seventy-seven core reactions involve the hydrolysis of high-energy phosphate bonds, furthermore suggesting the presence of a non-enzymatic and highly exergonic chemical reaction capable of continuously synthesizing activated phosphate bonds. CO 2 is the most common carbon-containing compound in the core. An abundance of NADH and NADPH-dependent redox reactions in the autotrophic core, the central role of CO 2 , and the circumstance that the core's main products are far more reduced than CO 2 indicate that the core arose in a highly reducing environment. The chemical reactions of the autotrophic core suggest that it arose from H 2 , inorganic carbon, and NH 3 in an aqueous environment marked by highly reducing and continuously far from equilibrium conditions. Such conditions are very similar to those found in serpentinizing hydrothermal systems.