Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Serpentinization as the source of energy, electrons, organics, catalysts, nutrients and pH gradients for the origin of LUCA and life.

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 H2. Molecular hydrogen can, in turn, serve as a freely diffusible source of electrons for the reduction of CO2 to organic compounds, provided that suitable catalysts are present. Using catalysts that are naturally synthesized in hydrothermal vents during serpentinization H2 reduces CO2 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 CO2 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 H2-dependent CO2 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.

Ambient temperature CO2 fixation to pyruvate and subsequently to citramalate over iron and nickel nanoparticles.

The chemical reactions that formed the building blocks of life at origins required catalysts, whereby the nature of those catalysts influenced the type of products that accumulated. Recent investigations have shown that at 100 °C awaruite, a Ni3Fe alloy that naturally occurs in serpentinizing systems, is an efficient catalyst for CO2 conversion to formate, acetate, and pyruvate. These products are identical with the intermediates and products of the acetyl-CoA pathway, the most ancient CO2 fixation pathway and the backbone of carbon metabolism in H2-dependent autotrophic microbes. Here, we show that Ni3Fe nanoparticles prepared via the hard-templating method catalyze the conversion of H2 and CO2 to formate, acetate and pyruvate at 25 °C under 25 bar. Furthermore, the 13C-labeled pyruvate can be further converted to acetate, parapyruvate, and citramalate over Ni, Fe, and Ni3Fe nanoparticles at room temperature within one hour. These findings strongly suggest that awaruite can catalyze both the formation of citramalate, the C5 product of pyruvate condensation with acetyl-CoA in microbial carbon metabolism, from pyruvate and the formation of pyruvate from CO2 at very moderate reaction conditions without organic catalysts. These results align well with theories for an autotrophic origin of microbial metabolism under hydrothermal vent conditions.

Role of geochemical protoenzymes (geozymes) in primordial metabolism: specific abiotic hydride transfer by metals to the biological redox cofactor NAD.

Hydrogen gas, H2 , is generated in serpentinizing hydrothermal systems, where it has supplied electrons and energy for microbial communities since there was liquid water on Earth. In modern metabolism, H2 is converted by hydrogenases into organically bound hydrides (H- ), for example, the cofactor NADH. It transfers hydrides among molecules, serving as an activated and biologically harnessed form of H2 . In serpentinizing systems, minerals can also bind hydrides and could, in principle, have acted as inorganic hydride donors-possibly as a geochemical protoenzyme, a 'geozyme'- at the origin of metabolism. To test this idea, we investigated the ability of H2 to reduce NAD+ in the presence of iron (Fe), cobalt (Co) and nickel (Ni), metals that occur in serpentinizing systems. In the presence of H2 , all three metals specifically reduce NAD+ to the biologically relevant form, 1,4-NADH, with up to 100% conversion rates within a few hours under alkaline aqueous conditions at 40 °C. Using Henry's law, the partial pressure of H2 in our reactions corresponds to 3.6 mm, a concentration observed in many modern serpentinizing systems. While the reduction of NAD+ by Ni is strictly H2 -dependent, experiments in heavy water (2 H2 O) indicate that native Fe can reduce NAD+ both with and without H2 . The results establish a mechanistic connection between abiotic and biotic hydride donors, indicating that geochemically catalysed, H2 -dependent NAD+ reduction could have preceded the hydrogenase-dependent reaction in evolution.

Energy at Origins: Favorable Thermodynamics of Biosynthetic Reactions in the Last Universal Common Ancestor (LUCA).

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, ΔG, which determine whether a reaction can go forward or not, should help specify the source. By calculating values of ΔG across the conserved and universal core of 402 individual reactions that synthesize amino acids, nucleotides and cofactors from H2, CO2, NH3, H2S and phosphate in modern cells, we find that 95-97% of these reactions are exergonic (ΔG ≤ 0 kJ⋅mol-1) at pH 7-10 and 80-100°C under nonequilibrium conditions with H2 replacing biochemical reductants. While 23% of the core's reactions involve ATP hydrolysis, 77% are ATP-independent, thermodynamically driven by ΔG 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 H2, CO2, NH3, H2S, and phosphate.

The Autotrophic Core: An Ancient Network of 404 Reactions Converts H2, CO2, and NH3 into Amino Acids, Bases, and Cofactors.

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 H2, CO2, and ammonia (NH3) 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. CO2 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 CO2, and the circumstance that the core's main products are far more reduced than CO2 indicate that the core arose in a highly reducing environment. The chemical reactions of the autotrophic core suggest that it arose from H2, inorganic carbon, and NH3 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.

The ambivalent role of water at the origins of life.

Life as we know it would not exist without water. However, water molecules not only serve as a solvent and reactant but can also promote hydrolysis, which counteracts the formation of essential organic molecules. This conundrum constitutes one of the central issues in origin of life. Hydrolysis is an important part of energy metabolism for all living organisms but only because, inside cells, it is a controlled reaction. How could hydrolysis have been regulated under prebiotic settings? Lower water activities possibly provide an answer: geochemical sites with less free and more bound water can supply the necessary conditions for protometabolic reactions. Such conditions occur in serpentinising systems, hydrothermal sites that synthesise hydrogen gas via rock-water interactions. Here, we summarise the parallels between biotic and abiotic means of controlling hydrolysis in order to narrow the gap between biochemical and geochemical reactions and briefly outline how hydrolysis could even have played a constructive role at the origin of molecular self-organisation.

A hydrogen-dependent geochemical analogue of primordial carbon and energy metabolism.

Hydrogen gas, H2, is generated by alkaline hydrothermal vents through an ancient geochemical process called serpentinization, in which water reacts with iron-containing minerals deep within the Earth's crust. H2 is the electron donor for the most ancient and the only energy-releasing route of biological CO2 fixation, the acetyl-CoA pathway. At the origin of metabolism, CO2 fixation by hydrothermal H2 within serpentinizing systems could have preceded and patterned biotic pathways. Here we show that three hydrothermal minerals-greigite (Fe3S4), magnetite (Fe3O4) and awaruite (Ni3Fe)-catalyse the fixation of CO2 with H2 at 100 °C under alkaline aqueous conditions. The product spectrum includes formate (up to 200 mM), acetate (up to 100 µM), pyruvate (up to 10 µM), methanol (up to 100 µM) and methane. The results shed light on both the geochemical origin of microbial metabolism and the nature of abiotic formate and methane synthesis in modern hydrothermal vents.

The Future of Origin of Life Research: Bridging Decades-Old Divisions.

Research on the origin of life is highly heterogeneous. After a peculiar historical development, it still includes strongly opposed views which potentially hinder progress. In the 1st Interdisciplinary Origin of Life Meeting, early-career researchers gathered to explore the commonalities between theories and approaches, critical divergence points, and expectations for the future. We find that even though classical approaches and theories-e.g. bottom-up and top-down, RNA world vs. metabolism-first-have been prevalent in origin of life research, they are ceasing to be mutually exclusive and they can and should feed integrating approaches. Here we focus on pressing questions and recent developments that bridge the classical disciplines and approaches, and highlight expectations for future endeavours in origin of life research.

Catalysts, autocatalysis and the origin of metabolism.

If life on Earth started out in geochemical environments like hydrothermal vents, then it started out from gasses like CO2, N2 and H2. Anaerobic autotrophs still live from these gasses today, and they still inhabit the Earth's crust. In the search for connections between abiotic processes in ancient geological systems and biotic processes in biological systems, it becomes evident that chemical activation (catalysis) of these gasses and a constant source of energy are key. The H2-CO2 redox reaction provides a constant source of energy and anabolic inputs, because the equilibrium lies on the side of reduced carbon compounds. Identifying geochemical catalysts that activate these gasses en route to nitrogenous organic compounds and small autocatalytic networks will be an important step towards understanding prebiotic chemistry that operates only on the basis of chemical energy, without input from solar radiation. So, if life arose in the dark depths of hydrothermal vents, then understanding reactions and catalysts that operate under such conditions is crucial for understanding origins.

Serpentinization: Connecting Geochemistry, Ancient Metabolism and Industrial Hydrogenation.

Rock⁻water⁻carbon interactions germane to serpentinization in hydrothermal vents have occurred for over 4 billion years, ever since there was liquid water on Earth. Serpentinization converts iron(II) containing minerals and water to magnetite (Fe3O4) plus H2. The hydrogen can generate native metals such as awaruite (Ni3Fe), a common serpentinization product. Awaruite catalyzes the synthesis of methane from H2 and CO2 under hydrothermal conditions. Native iron and nickel catalyze the synthesis of formate, methanol, acetate, and pyruvate-intermediates of the acetyl-CoA pathway, the most ancient pathway of CO2 fixation. Carbon monoxide dehydrogenase (CODH) is central to the pathway and employs Ni° in its catalytic mechanism. CODH has been conserved during 4 billion years of evolution as a relic of the natural CO2-reducing catalyst at the onset of biochemistry. The carbide-containing active site of nitrogenase-the only enzyme on Earth that reduces N2-is probably also a relic, a biological reconstruction of the naturally occurring inorganic catalyst that generated primordial organic nitrogen. Serpentinization generates Fe3O4 and H2, the catalyst and reductant for industrial CO2 hydrogenation and for N2 reduction via the Haber⁻Bosch process. In both industrial processes, an Fe3O4 catalyst is matured via H2-dependent reduction to generate Fe5C2 and Fe2N respectively. Whether serpentinization entails similar catalyst maturation is not known. We suggest that at the onset of life, essential reactions leading to reduced carbon and reduced nitrogen occurred with catalysts that were synthesized during the serpentinization process, connecting the chemistry of life and Earth to industrial chemistry in unexpected ways.

Something special about CO-dependent CO2 fixation.

Carbon dioxide enters metabolism via six known CO2 fixation pathways, of which only one is linear, exergonic in the direction of CO2 -assimilation, and present in both bacterial and archaeal anaerobes - the Wood-Ljungdahl (WL) or reductive acetyl-CoA pathway. Carbon monoxide (CO) plays a central role in the WL pathway as an energy rich intermediate. Here, we scan the major biochemical reaction databases for reactions involving CO and CO2 . We identified 415 reactions corresponding to enzyme commission (EC) numbers involving CO2 , which are non-randomly distributed across different biochemical pathways. Their taxonomic distribution, reversibility under physiological conditions, cofactors and prosthetic groups are summarized. In contrast to CO2 , only 15 reaction classes involving CO were detected. Closer inspection reveals that CO interfaces with metabolism and the carbon cycle at only two enzymes: anaerobic carbon monoxide dehydrogenase (CODH), a Ni- and Fe-containing enzyme that generates CO for CO2 fixation in the WL pathway, and aerobic CODH, a Mo- and Cu-containing enzyme that oxidizes environmental CO as an electron source. The CO-dependent reaction of the WL pathway involves carbonyl insertion into a methyl carbon-nickel at the Ni-Fe-S A-cluster of acetyl-CoA synthase (ACS). It appears that no alternative mechanisms to the CO-dependent reaction of ACS have evolved in nearly 4 billion years, indicating an ancient and mechanistically essential role for CO at the onset of metabolism.

The last universal common ancestor between ancient Earth chemistry and the onset of genetics.

All known life forms trace back to a last universal common ancestor (LUCA) that witnessed the onset of Darwinian evolution. One can ask questions about LUCA in various ways, the most common way being to look for traits that are common to all cells, like ribosomes or the genetic code. With the availability of genomes, we can, however, also ask what genes are ancient by virtue of their phylogeny rather than by virtue of being universal. That approach, undertaken recently, leads to a different view of LUCA than we have had in the past, one that fits well with the harsh geochemical setting of early Earth and resembles the biology of prokaryotes that today inhabit the Earth's crust.

Native metals, electron bifurcation, and CO2 reduction in early biochemical evolution.

Molecular hydrogen is an ancient source of energy and electrons. Anaerobic autotrophs that harness the H2/CO2 redox couple harbour ancient biochemical traits that trace back to the universal common ancestor. Aspects of their physiology, including the abundance of transition metals, radical reaction mechanisms, and their main exergonic bioenergetic reactions, forge links between ancient microbes and geochemical reactions at hydrothermal vents. The midpoint potential of H2 however requires anaerobes that reduce CO2 with H2 to use flavin based electron bifurcation-a mechanism to conserve energy as low potential reduced ferredoxins via soluble proteins-for CO2 fixation. This presents a paradox. At the onset of biochemical evolution, before there were proteins, how was CO2 reduced using H2? FeS minerals alone are probably not the solution, because biological CO2 reduction is a two electron reaction. Physiology can provide clues. Some acetogens and some methanogens can grow using native iron (Fe0) instead of H2 as the electron donor. In the laboratory, Fe0 efficiently reduces CO2 to acetate and methanol. Hydrothermal vents harbour awaruite, Ni3Fe, a natural compound of native metals. Native metals might have been the precursors of electron bifurcation in biochemical evolution.