Development of nascent autotrophic carbon fixation systems in various redox conditions of the fluid degassing in early Earth

The origin and development of primary autotrophic metabolism on early Earth 10 were influenced by the two main regimes of degassing of the Earth – reducing (predominance CH4) and oxidative (CO2). Among the existing theories of the autotrophic origin of life in hydrothermal environments, CO2 is usually considered the carbon source for nascent autotrophic metabolism. However, the ancestral carbon used in metabolism may have been derived from CH4 if the outflow of magma fluid to the surface of the Earth consisted mainly 15 of methane. In such an environment, the primary autotrophic metabolic systems had to be methanotrophic. Due to the absence of molecular oxygen in the Archean conditions, this metabolism would have been anaerobic, i.e., oxidation of methane must be realized by inorganic high-potential electron acceptors. In light of the primacy and prevalence of CH4dependent metabolism in hydrothermal systems of the ancient Earth, we propose a model of 20 carbon fixation where the methane is fixed/transformed in a sequence of reactions in an autocatalytic methane-fumarate cycle. Nitrogen oxides are thermodynamically most favorable among possible oxidants of methane; however, even the activity of oxygen created by mineral buffers of iron in hydrothermal conditions is sufficient for methanotrophic acetogenesis. The hydrothermal system model is considered in the form of a phase diagram, which demonstrates 25 the area of redox and P, T conditions favorable for the development of primary methanotrophic metabolism. 1 Deep methane degassing of the early Earth

The global mid-ocean ridge system represents a major site for outgassing of volatiles from the earth's mantle. Methane, which was believed to have a surface origin (low-70 temperature serpentinization ~100° C), apparently is formed at depth, at temperatures of ca.
400° C under redox conditions characterizing intrusive rocks derived from sub-ridge melts (Mével, 2003;Wang et al., 2018). Thus, deep, alkaline-basalt magmatism (elevated alkali content, especially K 2 O), in contrast to basalt-andesitic one, is mainly responsible for methane degassing on the earth's surface. With increasing alkalinity (alkaline slope) in the fluid 75 inclusions of igneous rocks invariably appear with different hydrocarbons (Potter and Konnerup-Madsen, 2003;Nivin et al., 2005). The high content of potassium in the high-silica Hadean crust (Boehnkea et al., 2018) indicates the depth of magmatism and its hydrocarbon specificity.
The model of two-stage development of fluids (I ↔ II) generated by the Earth's core via 80 mantle magma chambers is presented in the phase diagram of the C-H-O compositions, Fig.   1. Fluids ejected from the liquid core were initially saturated with hydrogen, with oxygenic components being of minor importance. However, during the process of the earth's silicate shells (mantle and crust) extension (associated, in our opinion, with a Hadean to Paleoarchean geodynamo (Tarduno et al., 2015)), an increase of fluid permeability stimulates the selective 85 migration of hydrogen (the most mobile component) from it. This process is responsible for hydrogen losing its leading position in ejected fluids and being fundamental to the evolution of low and normal alkalinity magmatism Marakushev, 2008, 2010). In this scenario, the fractionation of chemical components in the fluid would result in rich acidic CO 2 solutions (for example, H 2 +2CO = H 2 O+0.5CO 2 +1.5C and H 2 O+CO 2 = H 2 CO 3 ). These  The transition to compression of silicate shells prevents hydrogen migration from fluids 105 and stimulates the production of hydrocarbons within them; for example, consider the reactions: 3H 2 +CO = H 2 O+CH 4 , 5H 2 +2CO = 2H 2 O+C 2 H 6 ( Fig.1, facies II, reducing conditions). The hydrogen in the reaction like 4H 2 +H 2 CO 3 = 3H 2 O+CH 4 destroys the acid components in fluids, and this determines the alkaline slope in the development of magmatism. This is a two-stage model of the development of the C-H-O system (I ↔ II), 110 which depends on the composition of earth's core fluids, and their transformations in magma chambers.
The existing theories on the origin of autotrophic life mainly identify carbon dioxide as the unique carbon source for metabolism. This autotrophic metabolism should have originated at a high partial pressure of CO 2 in the environment (paragenesis CO 2 + H 2 O, Fig. 1, facies I). 115 We assume that in geodynamic regime II (CH 4 + H 2 O paragenesis), carbon ancestral metabolism could use methane as a carbon source if the flow of free energy from the geochemical environment was coupled with biomass formation reactions. Perhaps, these different regimes of fluid degassing determined the physicochemical conditions of the ambient environment, which, in turn, provided an opportunity for the emergence and 120 development of various systems of ancient autotrophic metabolism. In regime II (Fig. 1), methane and other hydrocarbons could be substrates of the emerging autotrophic metabolism.
The above geochemical and petrological data indicate highly heterogeneous redox conditions between the present-day Earth and conditions that periodically arose in the early Earth. We consider that the anaerobic reductive geochemical conditions of the Archean 125 played a decisive role in the origin and development of carbon and energy metabolism, which were vastly different from those observed in the tops of the branches of the modern phylogenetic tree of prokaryotes. Most metabolically-anaerobic chemoautotrophic organisms are either extinct or strongly limited to narrow anoxic ecological niches. Lateral gene transfer and subsequent phylogenetic divergence erased most evolutionary information recorded in 130 ancestral prokaryotic genomes (Martin et al., 2016).

Anaerobic oxidation of methane
The study of anaerobic oxidation of methane (AOM) in modern oxygen-free 135 environments (marine sedimentary rocks, gas-hydrates, mud volcanoes, black smokers, hydrocarbon seeps) has increased in recent years. This direction was sparked by the discovery of anaerobic methanotrophic archaea (Hinrichs et al., 1999) and, subsequently, their structural consortia with sulfate-reducing bacteria (Knittel and Boetius, 2009). A similar consortia was later discovered in archaea species that function in chemical conjunction with the bacterium 140 Candidatus Methylomirabilis oxyfera, which itself can independently couple AOM to denitrification (Ettwig et al., 2010;Haroon et al 2013). Furthermore, the microbiological AOM was recently shown to be directly associated with the reduction of iron and manganese compounds and minerals (Beal, 2009;Ettwig et al., 2016;Oni and Friedrich, 2017;He et al., 2018), as, for example, in the reaction CH 4 +8Fe 3+ +2H 2 O → CO 2 +8Fe 2+ +8H + (ΔG 0′ = -454 145 kJ/mol CH 4 ).
Recent studies have suggested that both archaea (ANME-2d) (Haroon et al., 2013) and bacteria (Methylobacter) (Martinez-Cruz et al., 2017), without partners, may themselves be versatile methanotrophs capable of using different oxidants as electron acceptors under different environmental conditions. AOM occur by the reversal canonical methanogenesis pathway (Timmers et al., 2017) and, perhaps, the evolution of life periodically includes forward or reverse pathways depending on the substrate (methanogen-methanotroph "switch back" (McGlynn, 2017). For example, nickel enzyme purified from methanogenic archaea can catalyze the oxidation of methane to methyl coenzyme M (CH 4 + CoM-S-S-CoB → CH 3 -S-CoM + HS-CoB; ∆G o' =30±10 kJ), that is the reverse reaction of methyl coenzyme M 155 reductase (Scheller et al., 2010). In general, methano-and methylotrophs use different but often interrelated pathways of carbon fixation (Smejkalová et al., 2010). Newly described methanotrophic anaerobic prokaryotes are frequently discovered in various extreme environmental conditions (Semrau et al., 2008), underscoring the functional and phylogenetic diversity of this group. The search for relict forms of anaerobic methanotrophic metabolism 160 continues.
In 2013, Wolfgang Nitschke and Michael Russell described the possibility of methane assimilation as the sole source of carbon for primordial metabolism (Nitschke and Russell, 2013). They suggested that methanotrophy and not methanogenesis may have been the founding metabolism in the first protocells and presented a model of methanotrophic 165 acetogenesis in which methane, as the carbon source, is assimilated into the biomimetic analogue of the modern reverse acetyl-CoA pathway. The proposed methane oxidant in this pathway of CH 4 fixation was nitric oxide (NO), formed via nitrate/nitrite transformation ('denitrifying methanotrophic acetogenesis') (Russell and Nitschke, 2017). The authors consider the process of low-temperature harzburgite (ophiolites) hydrothermal 170 serpentinization in the presence of carbon oxides served as the main source of methane.
Nevertheless, Wang et al. (2018) argue that there is another unified deep high-temperature process of methane-making for these hydrothermal areas.
In the absence of oxygen, the methane oxidation requires electron acceptors with a high redox potential (such as nitrate, manganese (IV), iron (III), and sulfate). Thermodynamic 175 calculations of anaerobic methanotrophic acetogenesis reactions in aqueous hydrothermal conditions that require oxidized compounds such as sulfur, nitrogen, and iron are considered in Table 1. For example, the free energy of the reaction CH 4 +6Fe 2 O 3 = 0.5CH 3 COOH+H 2 O+4Fe 3 O 4 at 473 К is equal to the sum of the free energy of products formation minus the sum of free energy of the reactants formation at the same temperature 180 (ΔG 473 0 = (0,5ΔG 0 СН3СООН +ΔG 0 Н2О +4ΔG 0 Fe3O4 ) -(ΔG 0 CH4 +6ΔG 0 Fe2O3 ) = -6.49 kJ/mol). fully ionized and non-ionized forms of acetate is presented. Value of ΔG 0 T at 298 and 473 K indicates the advantage of the reactions at low (L) or high (H) temperatures. Free energies of aqueous substances formation at P SAT (Amend and Shock, 2001) were used in calculations. constants from (Amend and Shock, 2001)) than in the process of CO 2 fixation (CO 2 +2H 2 = 0,5CH 3 COOH+H 2 O; ΔG 0 298 = -84.75 kJ). The most favorable reaction CH 4 +2NO = 0,5CH 3 COOH+Н 2 O+N 2 (Table 1) can be represented as a model of methanotrophic acetogenesis, which is part of the reverse acetyl-CoA pathway. The second part of this path is the reaction of CO 2 reduction: СО 2 +2Н 2 = 205 0,5СН 3 СООН+Н 2 О. In sum, this is a very thermodynamically favorable pathway of carbon fixation in the form СН 4 and СО 2 : CH 4 +СО 2 +2NO+2Н 2 = CH 3 COOH+N 2 +2H 2 O. The different stoichiometry of acetogenesis was observed in the archaean Methanosarcina acetivorans, when methane oxidation was associated with the reduction of iron (III) [Soo et al, 2016]. A reaction is proposed in which four methane molecules are oxidized and two CO 2 210 molecules are reduced to form three acetate molecules. Increasing the ratio of СН 4 to СО 2 (4CH 4 +2СО 2 +24Fe 2 O 3 +4Н 2 = 3CH 3 COOH+6H 2 O+16Fe 3 O 4 ) makes the process of anaerobic acetogenesis more thermodynamically favorable (Table. 1, carboxy-methano acetogenesis).

Redox pairs of nitrogen
The LUCA era apparently proceeded in an environment with high CO 2 partial pressure, whereas the pre-LUCA period proceeded in a reducing environment with a significant 215 availability of methane. The question thus arises: was this ancestral reverse acetyl-CoA relic pathway the only metabolic CH 4 fixation system, or were there other proto-biochemical mechanisms for the assimilation of carbon?

The proposed methane-fumarate cycle
Based on the hypothesis of primordial anaerobic methanotrophic metabolism origin, we assume that some components and modules of the metabolic cycles (carboxylic and keto acids, and their associations (parageneses)) may also be relicts of ancient methanotrophic 235 metabolism. One of the few known reactions of CH 4 fixation is the formation of 2methylsuccinate as a result of the reaction: fumarate+CH 4 → 2-methylsuccinate (Thauer and Shima, 2008;Haynes and Gonzalez, 2014), and fumarate addition has been widely proposed as an initial step in the anaerobic oxidation of both aromatic and aliphatic hydrocarbons (Musat, 2015). The reaction of methane with fumarate satisfies the "minimal energy 240 requirements" for autotrophic growth (Beasley and Nanny, 2012), and we consider the possibility of its participation in nascent autotrophic metabolism. We propose a simplified model of the methane-fumarate (MF) cycle, Fig. 2, which could have originated in the reductive Archean hydrothermal systems, at a high partial pressure of endogenous methane (facies II, Figure 1). The cycle is initiated by the reaction of fumarate + methane → 2-245 methylsuccinate. In the hydration and dehydrogenation or anaerobic oxidation reactions, 2methylsuccinate is converted to citramalate, which is disproportionated to acetate and pyruvate with cleavage of a carbon-carbon bond. Pyruvate is an important "hub" metabolite that is a precursor for amino acids, carbohydrates, cofactors, and lipids in an extant metabolic network. The following carbon assimilation reaction in the form of CO 2 with the formation of 250 oxaloacetate is a biomimetic analogue of the reductive tricarboxylic acid (rTCA) cycle reaction. An α-carboxylation of pyruvate is a critical anabolic pathway in modern biochemistry, which resupplies rTCA cycle intermediates. Oxaloacetate is transformed into fumarate in the reactions of the citrate cycle intermediates. The resulting fumarate assimilates methane and begins a new MF cycle, in one turnover of which an acetate molecule is formed 255 from methane and carbon dioxide molecules: CH 4 +CO 2 = CH 3 COOH, Table. 1. The nonenzymatic flow of some reaction sequences of the rTCA cycle, such as oxaloacetate → malate → fumarate → succinate has been recently experimentally confirmed (Muchowska et al., 2017).  Transformation of fumarate into 2-methylsuccinate introduces into the cycle five-carbon intermediates, such as citramalate and mesaconate, functioning, for example, in the reductive 3-hydroxypropionate CO 2 fixation cycle. The autocatalytic nature of the cycle derives from the branching point associated with citramalate cleavage and can be shown by the example of 275 doubling the intermediate as in the reaction: С 4 Н 6 О 5 (malate)+1,5СН 4 +2,5CO 2 = 2С 4 Н 6 О 5 (two malates). This type of autotrophic metabolism, as in the case of the acetyl-CoA pathway, can be defined as carboxy-methanotrophic acetogenesis. As it happened with the archean WL pathway (eg, Soo et al., 2016), methanotrophy probably should be combined with the carboxylation process, therefore nature had to look for all possible sources of carbon dioxide.

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The problem of the most energetically unfavorable reaction of 2-methylsuccinate transformation into citramalate (ΔG 0 298 = 96.57 kJ), Table 2a, can be solved by using oxidants, such as oxides of nitrogen and iron (Fig. 2, inset). Nitric oxide (NO) is the strongest oxidant, but the reaction with Fe 2 O 3 is also favorable at physiological temperatures.    species, promote C-H activation through a metallo-radical pathway. This involves hydrogen radical abstraction from the alkane by the oxo species, followed by a rapid rebound of the radical species onto the metal hydroxo intermediate (Roudesly et al., 2017). The calculation of the potential energy surface showed the thermodynamic possibility of anaerobic oxidation of methane via fumarate addition, in a reaction catalyzed by the glycyl radical (Beasley and 320 Nanny, 2012). The reaction mechanism fumarate+CH 4 → 2-methylsuccinate, fig. 2, seems to be similar to the radical mechanism of breaking the C-H bond with the formation of the C-C bond, catalyzed by benzylsuccinate synthase (Buckel and Golding, 2006;Austin et al., 2011) during microbiological fixation of toluene by fumarate. Radicals of amino acids and dipeptides may be the possible catalysts of methane activation with the formation of methyl 325 radical as an attacking agent. The formation of pyruvate and oxaloacetate in MF cycle, Fig. 2, indicates a very likely formation of amino acids in simple aqueous synthesis, for example: С 3 Н 4 О 3 (pyruvate)+NH 3 = C 3 H 7 O 3 N (serine), ΔG 0 298 = -10,10 kJ or pyruvate+NH 3 +H 2 = C 3 H 7 NO 2 (alanine)+H 2 O, ΔG 0 298 = -124,8 kJ. Barge et al., 2019 show that pyruvate can form the alanine in hydrothermal systems in the presence of mixed-valence iron oxyhydroxides.

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Moreover, the generation of reactive oxygen species H 2 O 2 and OH • from minerals and H 2 O in anaerobic environments (eg. Xian et al., 2019) creates the possibility of various radical mechanisms for the oxidation of substrates in a hydrothermal environment. According to (Weiss et al., 2016), LUCA metabolism had an excess of radical reaction mechanisms, which, in our opinion, could also participate in the reaction of CH 4 fixation in the cycle, overcoming 335 the activation barriers of kinetically unfavorable reaction steps.
Our understanding of the emergence of methanotrophic metabolism is within the framework of the hydrothermal theory for the origin of life (eg, Martin et al., 2008) with all its advantages (continuous flow of energy and matter, the temperature gradient, great possibilities of homogeneous and heterogeneous metal catalysis). Before the occurrence of 340 cellular structures, the primary autotrophic metabolism on the surface of minerals created the chemical space of competing autocatalytic carbon fixation cycles. The accumulation of "biomass" probably led to the emergence of heterotrophic protometabolism and the creation of a certain matrix of the organo-mineral system in which a cascade of proto-biochemical redox reactions could occur, such as in the modern soils (Kéraval et al., 2016). Regardless of 345 the specific mechanism of the functioning of the precellular autotrophic metabolism ("reductive surface pyrite world" (Wächtershäuser, 1988), "hydrothermal reactor" (Russell and Martin, 2004), "organo-mineral matrix" (Kéraval et al., 2016), and others) its origin and development was subject to the laws of aqueous thermodynamics.

Anaerobic methane oxidation in the hydrothermal systems
We represent the hydrothermal system in the form of a phase diagram which displays the chemical potential of oxygen vs. temperature at saturated vapor pressure (P SAT ), where temperatures and pressures are below critical thresholds (647,3 K and 22,1 МПа) (Fig. 3).

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The chemical potentials (µ i ) of components representing its partial energy, the value µ i is expressed through activity α i or fugacity f i as follows: µ i =(µ 0 i ) Т,р + RTlnα i = (µ 0 i ) Т,р + RTln f i . Here numerical values depend on conventional standard states. For activity, the state of pure crystalline substance or unit molal concentration is usually considered as a standard state at a given temperature and pressure. In this state α i = 1 and, hence, µ i = (µ 0 i ) Т,р . The diagram is a 360 two-component system (extensive parameters: C and H), since oxygen become intensive parameters, like the temperature, and pressure. Oxygen is represented by the chemical potential of O 2 in hydrothermal solution (µ Р О2 = RTlna O2 , where a О2 denotes the chemical activity of oxygen). According to the Gibbs' phase rule, at arbitrary pressure, the nonvariant equilibria in the diagram (points) consist of four phases, and the three-phase equilibria (lines) 365 divide the divariant stability fields (facies) of the two-phase equilibria. The equilibrium CH 4 +2O 2 = CO 2 +2H 2 O (bold black line) divides the diagram into the facies of CO 2 (I) and CH 4 (II) (oxic and anoxic areas of the hydrothermal system) and illustrates the two main possibilities for the development of the C-H-O system in the facies 385 carbon dioxide or methane. Intermediates of the MF cycle are acetate, succinate, and fumarate, and we considered their metastable equilibria and parageneses. In all phase space under consideration, there are fumarate facies. The equilibrium of 5Fum+4CH 4 +2O 2 = 6Suc at low-temperature (Fig. 3), is located in the region of very low partial pressures of oxygen, whereas the equilibrium of Fum+2CH 4 +O 2 = 3Acet at high-temperature occurs in facies of 390 high pressures. Acetate and succinate facies (contoured with red and blue equilibria, respectively) completely encompass the equilibrium of CH 4 +O 2 = CO 2 +H 2 O. That is, in hydrothermal solution, the parageneses of some components within the MF cycle are stable in both the CO 2 and the CH 4 facies. The whole system can develop in either of two directions as the chemical potential of oxygen changes: 1. the formation of low-temperature (Suc-H 2 O) and 395 high-temperature (Fum-H 2 O) paragenesis in CO 2 facies (I) and 2. the formation of lowtemperature (Suc-CH 4 ) and high-temperature (Fum-CH 4 ) paragenesis in CH 4 facies (II).
Thus, within these facies, protobiochemical systems supporting carbon fixation in the form of CO 2 or CH 4 can develop, and methane facies (II) represent a broad area of CH 4 assimilation by carboxylic acids in an aqueous environment. The high stability of the succinate-fumarate-400 acetate paragenesis in hydrothermal systems at 200° C (473 K) was experimentally shown (Estrada et al., 2017).
Mineral buffers up to 549 K are located in the facies of succinate, but the equilibrium of HM remains in the area of thermodynamic CO 2 stability (facies I), and PPM and QMF equilibria occur in methane facies II and intersect the fundamental equilibrium of 405 2Suc+2CH 4 +O 2 = 5Acet. Magnetite (Fe 3 O 4 ) facies (between HM and QMF equilibria) encompass CH 4 /CO 2 equilibrium in nearly the entire temperature range of the hydrothermal system under consideration. Thus, the redox areas of magnetite stability correspond to the formation conditions both СО 2 and CH 4 assimilating systems. The presence of magnetite in the early Archean ocean was shown by . Shibuya et al. (2016) also conclude 410 that iron redox reactions probably played an important role in the early evolution of methanotrophic metabolisms in the Hadean alkaline hydrothermal system. The QMF buffer equilibrium is completely located in the methane facies (I), which, according to data (Yang et al., 2014), corresponds on average to the redox conditions of Hadean mantle and crust. Up to the 3.6 billion years ago and maybe even to the great oxidative event of 2.2-2.4 billion years ago on, the earth's surface the oxidation potential of the magnetite redox pairs, apparently, determined the chemical potential of environmental oxygen.

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The cyclic planetary fluid flows (outgassing of volatiles from mantle) drive earth's chemical evolution, leading to the formation of different geobiochemical systems of carbon fixation.
Impulses of CO 2 and CH 4 degassing on our planet must have determined the preference of specific autotrophic carbon fixation metabolism development. It is widely accepted that autotrophic metabolism is the fixation of inorganic carbon solely in the form of CO 2 , but the 425 origin of methane, both on the ancient Earth, and on the planets and satellites (for example, on Titan) is clearly inorganic; therefore, carbon fixation from methane is also a manifestation of autotrophic metabolism (formation of organic compounds from inorganic precursors).
The variety of modern autotrophic carbon fixation seems to be created by the association of the different metabolic associations and modules that, apparently, could function in the 430 ancestral systems of the anaerobic fixation of CH 4 . When approximately ~ 3.6 billion years (Yang et al., 2014), a CO 2 degassing regime became dominant on our planet, the relic methanotrophy systems were forced to die out or be thrown into unusual and extreme ecological niches. If we consider LUCA as a relatively recent player in the evolution of life (Cornish-Bowden and Cárdenas, 2017), the ancestral metabolic systems of carbon fixation in 435 putative pre-LUCA could differ appreciably from modern ones.
In the process of development of CO 2 fixation systems on the Earth, the main problem was the presence of electron donors (therefore, evolution created selective reducing agents: NADH, NADPH, FADH), whereas the fixation of CH 4 essentially depended on the presence of electron acceptors. The oxygen-containing nitrogen compounds are the best oxidants in the 440 hydrothermal systems, but their presence there is unlikely. Nevertheless, the redox pairs of hematite-magnetite and quartz-magnetite-fayalite create a specific area of chemical potential of oxygen that satisfies the thermodynamic requirements of oxidation and assimilation of methane by protometabolic pathways. Hydrothermal systems of ancient Earth may have been very similar to those that currently exist on some extraterrestrial cosmic bodies, such as 445 Europa or Enceladus. The degassing of these cosmic bodies can currently support methane metabolism, but the problem is to know if there are electron acceptors there (Russell et al., 2017).