Primordial Bioenergy Sources: the Two Facets of Adenosine Triphosphate

Journal of Inorganic Biochemistry 216 (2021) 111347

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Focused Review Article Primordial bioenergy sources: The two facets of adenosine triphosphate

Juan C. Fontecilla-Camps

Universit´e Grenoble Alpes, CEA, CNRS, IBS, Metalloproteins Unit, F-38044 Grenoble, France

ARTICLE INFO ABSTRACT

Keywords: Life requires energy to exist, to reproduce and to survive. Two major hypotheses have been put forward con Origin of life cerning the source of this energy at the very early stages of life evolution: (i) abiotic organics either brought to Protocells Earth by comets and/or meteorites, or produced at its atmosphere, and (ii) mineral surface-dependent bio Mineral surface bioinorganic chemistry inorganic catalytic reactions. Considering the latter possibility, I propose that, besides being a precursor of Adenosine triphosphate nucleic acids, adenosine triphosphate (ATP), which probably was used very early to improve the fidelity of Helicases ATP synthase nucleic acid polymerization, played an essential role in the transition between mineral-bound protocells and their free counterparts. Indeed, phosphorylation by ATP renders carboxylate groups electrophilic enough to react with nucleophiles such as amines, an effect that, thanks to their Lewis acid character, also have dehydrated metal ions on mineral surfaces. Early ATP synthesis for metabolic processes most likely depended on substrate level + phosphorylation. However, the exaptation of a hexameric helicase-like ATPase and a transmembrane H pump (which evolved to counteract the acidity caused by fermentation reactions within the protocell) generated a much more efficient membrane-bound ATP synthase that uses chemiosmosis to make ATP.

1. Introduction been shown that, upon heating, dehydrated metal ions can store thermal energy through loss of coordinating OH ions and/or water molecules; Life is, by definition, a far-from-equilibrium process that requires and that that energy can be released upon rehydration [10]. 4+ energy to exist, to reproduce and to survive. Hypothesizing about the Dehydrated surface metal ions (like Ti in TiO2) can bind organic nature of the essential energy-related processes of very early life de functions such as carboxylate groups [11]. The Lewis acid character of pends on the choice of their possible settings. the metal renders the carboxylate carbonyl carbon more electrophilic and reactive towards amide synthesis [11,12] (Fig. 1). Similar carbon- 1.1. Primordial energy sources activating reactions, including phosphate and amino acid polymeriza tions, have been reported on an amorphous silica surface [13–15]. Thus, For those who favor the “primordial soup” concept firstdeveloped by these surfaces could have acted as primordial bioinorganic catalysts. A. I. Oparin and J. B. S. Haldane [1–3], which posits that the first Rimola et al. have recently reviewed the possible role of mineral surfaces complex bio-molecules, such as nucleobases and amino acids, had an in prebiotic chemistry using density functional theory [16]. They “ – … abiotic origin, the firstliving organisms should have been heterotrophic concluded that specific organic mineral interactions could organize [4]. This, in turn, implies that energy was extracted from a great variety adsorbed molecules in well-defined orientations and activate them to of organics present in the soup. Their supply had to be constant over ward chemical reactions, leading to an increase in chemical ” time because otherwise the soup would have very rapidly become a non- complexity . viable “low enthalpy, high entropy” system [5]. Consequently, there are rather solid experimental and theoretical — An alternative view considers that the first protocells were autotro bases to postulate that an ensemble of metabolism pathways operating phic and obtained their energy from H oxidation on Fe(Ni)S surfaces on contiguous mineral surfaces with different chemical and redox 2 — [6,7]. Along the same lines, a one-pot carbon fixation of acetylene properties could have generated autocatalytic prebiotic entities [9]. Dry-wet cycles would have favored both polymerizations and the syn (C2H2) and carbon monoxide (CO) by aqueous nickel sulfide(NiS) under ◦ hydrothermal (>100 C) conditions has been recently reported [8]. I thesis and exchange of metabolites between these surfaces [9]. Even have proposed that a second, maybe as important, energy source for tually, with the acquisition of a primitive membrane [17,18], mineral- these autotrophs were dehydrated mineral surfaces [9]. Indeed, it has bound protocells would have emerged who mainly depended on H2

E-mail address: [email protected]. https://doi.org/10.1016/j.jinorgbio.2020.111347 Received 28 September 2020; Received in revised form 14 December 2020; Accepted 21 December 2020 Available online 29 December 2020 0162-0134/© 2020 Elsevier Inc. All rights reserved. J.C. Fontecilla-Camps Journal of Inorganic Biochemistry 216 (2021) 111347

intermediates. Phosphorylation eventually replaced many of the thio lation reactions and a simple acetyl phosphate might have been a very early component of the energetics of protocells (although its usefulness would have been limited because it could not have promoted polymer ization reactions) [27]. As it will be argued below, there are good rea sons to propose, that along with NAD, the essential cofactor adenosine triphosphate (ATP) was a fundamental “energy currency” which, by replacing bioinorganic cofactors, helped the transition between mineral- Fig. 1. Amidation reaction on a mineral surface (adapted with modifications bound and free-living protocells. from [11]). 2. The versatile ATP cofactor oxidation and thermally dehydrated mineral surfaces for their existence. During the peak of the dehydrating phase, protocells would have been in 2.1. ATP mechanisms of action a spore-like state to become active again during the wet phase. It is generally stated that ATP’s energy is released through its hy drolysis to adenosyl diphosphate (ADP) and phosphate. The O~P ester 1.2. Primordial carbon fixation bond broken during this process is weak, because it is a high-energy bond (Fig. 3). In an autotrophic organism a significant fraction of the acquired However, ATP hydrolysis only applies to selected reactions, such as energy is used to fixCO and make energy-rich molecules, such as sugars 2 direct transport of ions and ligands across the cell membrane [28], the and lipids, as well as structural and catalytic proteins. In contemporary modificationof the redox properties of cofactors [29], or to power motor autotrophs the primary energy source is sunlight obtained through proteins [30]. Conversely, in metabolic reactions, ATP is not directly oxygenic photosynthesis, which uses it to split water and energize the hydrolyzed; instead, it is used to either mediate cell regulatory processes released electrons [19]. A simpler and probably older version of this or to activate carbon atoms towards nucleophilic attack, through process was anoxygenic [20] and primordial photosynthesis has been phosphorylation. An example of the latter is the two-step reaction of even proposed in deep-sea hydrothermal vents [21]. glutamate with ammonia to produce glutamine, catalyzed by the However, photosynthesis is inherently complex and, at the early enzyme glutamine synthetase [31] (Fig. 4). stages of life evolution, other energy-demanding CO fixationpathways, 2 In the firststep, glutamate reacts with ATP to form an acyl-phosphate which did not directly depend on sunlight, should have been present. intermediate and ADP. Because it takes less energy to break the weak The most likely candidates are the Wood-Ljungdhal (W-L) pathway of O~P bond (Fig. 3) —to phosphorylate glutamate and release ADP— anaerobic bacterial acetogens and archaeal methanogens [22] and the than the energy released when the stronger O-P bond of the acyl- reverse (reductive) Krebs (RK) cycle, well described in anaerobic phosphate intermediate is formed (Fig. 4), phosphorylation is thermo ε-proteobacteria [23]. Both fix two CO molecules and make acetyl 2 dynamically favorable. The electrophilicity of the carbonyl carbon atom Coenzyme A (CoA) (Fig. 2). bonded to the phosphate group is increased by the electron-withdrawing Remarkably, the W-L pathway uses three enzymes containing the effect of the latter. This, in turn, facilitates the second step of the reac biologically-unusual nickel ion: [NiFe]‑hydrogenase, CO dehydrogenase tion that consists of the nucleophilic attack by the lone pair of the and acetyl CoA synthase [24]. In methanogenic archaea this pathway ammonia N atom to that carbon (Fig. 4, center). The weakened C-O bond differs from its bacterial acetogenic version in the nature of the enzymes is broken, phosphate is released and the C-N bond of the amide moiety of and cofactors that reduce one CO2 to -CH3. This difference suggests that 3 glutamine is formed. Both ADP and the PO4 ion are very good leaving in the last universal common ancestor (LUCA), the W-L pathway used groups because their ions are stabilized by resonance. Without ATP, the abiotically generated -CH [25]. Taken together, these observations 3 synthesis of glutamine from glutamate is thermodynamically unfavor suggest that this pathway, which relies on H oxidation and catalytic 2 able because it takes more energy to break the carboxylate C-O bond protein-bound Fe(Ni)S clusters, played a major role in primordial than the energy released when the C-N bond is formed. Mechanistically, carbon-fixation. Interestingly, this process does not require adenosine the carbon atom of an unreacted carboxylic group is not electrophilic triphosphate (ATP) (Fig. 2A). enough for this reaction to occur. Furthermore, OH is not a good In the ε-proteobacterium Thiomicrospira denitrificans, the RK cycle leaving group (in the reaction shown in Fig. 1, this ion stays bound to the fixesCO using energy from the oxidation of thiosulfate (S O2 ), which 2 2 3 mineral surface until the next dehydration step). Similarly, in the reac furnishes reduced (iron‑sulfur)-ferredoxin, reduced NAD(P)H, reduced tion of ATP citrate lyase the phosphorylated carbonyl carbon of the in flavin-adeninedinucleotide (FADH ) and ATP (Fig. 2B) [23]. Like in the 2 termediate citryl-phosphate undergoes the nucleophilic attack of the W-L pathway, the use of an inorganic energy source by the RK cycle is thiolate S atom of CoA and the resulting citryl CoA is cleaved to acetyl consistent with its expected very old origin. CoA and oxaloacetate [32] (Fig. 2B). The activation effect of phos phorylation has been recently described in a theoretical study of peptide 1.3. From minerals to organic cofactors bond formation between two glycine molecules mediated by ATP under abiotic conditions [33]. Autotrophic protocells that relied on the W-L pathway and the RK Regulatory phosphorylations follow a very similar mechanism cycle for carbon fixation would have been still partially bound to a except that they are reversible reactions; serine, threonine and tyrosine mineral surface and dependent of it for catalysis and energy generation. residues are phosphorylated by enzymes called kinases and dephos The next crucial step was their liberation from that surface and its bio phorylated by phosphatases thus mediating signal transduction in cell inorganic contribution to metabolism. At that point, organic hydride processes such as differentiation, division and proliferation [34]. carriers and thiolating or/and phosphorylating organic cofactors Another series of synthetic reactions, which involve nucleotide replaced inorganic equivalent species [9]. A remarkable observation in triphosphate hydrolysis to pyrophosphate, are nucleic acid polymeri this regard is that nicotinamide adenine dinucleotide (NAD) can capture zations by polymerases and amino acid activation by aminoacyl-tRNA a metal-bound hydride from a soluble piano-stool iridium organome synthetases. In the first case the nucleophile is a ribose hydroxyl tallic complex and be reduced to NADH [26]; this suggests that nico group and the substrate the α-P atom of an NTP (any of the four nu tinamide, or one of its organic precursors, was already capable of cleotides of either DNA or RNA). Amino acid activation involves the transferring hydrides from a mineral surface to relevant metabolic phosphorylation of one of its carboxylic oxygen atoms by the α-P atom of

2 J.C. Fontecilla-Camps Journal of Inorganic Biochemistry 216 (2021) 111347

Fig. 2. A. The Wood-Ljungdhal pathway in methanogens (adapted from https://en.wikipedia.org/wiki/Wood-Ljungdahl_pathway). B. The reverse or reductive Krebs cycle.

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DNA). Consequently, it must have already been present at the onset of genetics. It is remarkable that, with the exception of the S-adenosyl-L-methi onine (SAM) cofactor of radical metalloenzymes, where triphosphate is replaced by methionine [42], the (phospho-) riboadenosine moiety is not directly involved in the reactivity of any of the cofactors where it is present. This, in turn, suggests that initially this moiety served mostly as a soluble, maybe polypeptide binding, module connected to a functional group. From a purely geochemical standpoint the evolutionary choice of phosphate as the crucial component of ATP has been considered puz Fig. 3. The structure of ATP. The blue arrow indicates the weak high-energy zling [43]. In fact, phosphorus bioavailability during Earth’s history has O~P bond that is broken to generate ADP and PO3 (see text). The adenine 4 been probably very limited because of its tendency to form insoluble ring is biosynthesized from simple intermediates: red = N from glutamine, green = N from aspartate, gray = C from formate, purple = C from bicarbonate compounds with, for instance, iron hydroxides and calcium ions and blue = fragment from glycine (see text). [43,44]. For this reason, it has been postulated that initial energy pro cesses in proto-metabolisms could have been based on thioesters instead ′ of phosphoesters [45,46]. Using systems biology approaches, Goldford ATP forming a 5 -aminoacyl adenylate. These reactions do not require et al. [47] have characterized what they called a “cryptic phosphate- substrate activation and they probably did not involve mineral surfaces independent core metabolism”, enriched for enzymes carrying in primordial settings. Furthermore, here ATP (or NTP) is a substrate not ′ iron‑sulfur clusters. However, it is obvious that at one point during life a cofactor. Conversely, the reaction of 5 -aminoacyl adenylate with one evolution phosphorus was bioavailable enough to become essential. of the ribosyl -OH groups of tRNA is reminiscent of the activation of acyl Several possibilities have been advanced to solve this conundrum. groups by ATP in metabolic reactions. + Herschy et al. [44] has argued that in anoxic Archean oceans, Fe2 could 3 2 have reduced phosphate (PO4 ) to phosphite (HPO3 ), a phosphorus 2.2. The origin of ATP source that would have been stable and more soluble. Conversely, Toner and co-workers [43] have shown that carbonate-rich lakes could have Starting from phosphoribosyl pyrophosphate (PRPP), the synthesis been a reasonable source of soluble phosphate because of local depletion of the adenine nucleobase requires 12 steps [9,35–37]. Nine of these of calcium ions due to their precipitation as carbonates (calcite, arago steps involve nucleophilic -N: attacks on carbons sequentially phos nite). Another hypothesis proposes that the prebiotic source of phos phorylated by 8 ATPs and 1 guanosine triphosphate (GTP); accordingly, phorus could have been diamidophosphate (DAP), which is not readily the biosynthesis of adenine is autocatalytic. All the C-N bonds of the precipitated by divalent ions and is known to phosphorylate sugars, adenine ring, excepting the one already contained in the glycine-derived nucleotides, peptides and lipids in water [48]. fragment (Fig. 3), are formed by these reactions, which are equivalent to Given the intrinsically complex nature of research on the origin(s) of the already described glutamine synthesis from ammonia, glutamate life, we will probably never exactly know how phosphate became and ATP (Fig. 4). I have suggested [9] that similar biomimetic reactions essential to proto-organisms. Maybe a bit less problematic is to under could have synthesized adenine, and the other nucleobases, from simple stand why phosphate was naturally selected to be the essential compo “ ” precursors on dehydrated mineral surfaces, like TiO2 and silica [11–13]. nent of the main energy currency of the cell. As mentioned above, Indeed, there are clear similarities between the activation of carbonyl phosphates and pyrophosphates are very good leaving groups. Another carbon atoms by phosphorylation and by binding to dehydrated mineral important point, raised by Westheimer [49], is that phosphates and surfaces (Figs. 1 and 4). Thus, these surfaces could have behaved as ATP- phosphorylated compounds are normally ionized at physiological pH, like cofactors being regenerated during the dry phase of dry-wet cycles which will prevent them from diffusing across a hydrophobic cell in primordial settings [9]. membrane. This property could have been essential to protocells with The next issue to consider is whether ATP was initially formed by the relatively leaky membranes. Westheimer [49] also gives the example of association of adenine, ribose and triphosphate or there was an alter histidine synthesis where every intermediate is either positively or native way to synthesize it. The simplest and biomimetic way would be negatively charged. So, ionized molecules would have stayed within direct synthesis from its three components. Concerning this possibility, cells. Having phosphate groups as linkers for nucleic acids may also be Akouche et al. have reported the remarkable synthesis of adenosine related to their ionization properties and their lower propensity to hy monophosphate (AMP) from adenine, phosphate and ribose on a fumed drolysis. In a later review, Kamerlin et al. [50] have used theoretical silica surface [38]. Furthermore, the same authors have shown the sta approaches to further justify the natural choice of phosphate. They bilization of β-D-ribofuranose, the form of ribose that is found in ribo conclude that “phosphates are negatively charged and the resulting nucleotides, on the same mineral surface [39]. Taken together, these charge-charge repulsion with the attacking nucleophile contributes to observations suggest that adenine was synthetized very early and that its the very high barrier for hydrolysis, making phosphate esters among the association with ribose —also postulated to be an early metabolite ob most inert compounds known. However, …the same charge-charge tained from the polymerization of formaldehyde [40,41]— and phos repulsion that makes phosphate ester hydrolysis so -kinetically- unfa phate, could have occurred before the formation of the first protocells. vorable also makes it possible to regulate, by exploiting the electro Indeed, as discussed below, ATP, along with the other NTPs, is a sub statics” [50]. Regulation is, of course, an essential property of life. strate for RNA polymerization (and its deoxyribose version in the case of

Fig. 4. Reaction of glutamate with ammonia to produce glutamine. The electrophilic phosphate-bound carbon of the intermediate is readily attacked by the nucleophilic N atom of ammonia (blue).

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2.3. ATP synthesis now and then OP reactions are the ones of aerobic glucose respiration, where the final electron acceptor is O2: In contemporary organisms, ATP can be synthesized from ADP and 6 O2 + C6H12O6 + 38 ADP + 38 Pi→38 ATP + 6 CO2 + 6 H2O (3) phosphate through two widely different processes: substrate-level phosphorylation (SLP) and ATP synthase coupled to a transmembrane Several anaerobic microorganisms can respire using other final electrochemical gradient [51,52]. SLP is generally an outcome of the electron acceptors, such as nitrate or sulfate ions, or even CO2. Because fermentation of glucose (C6H12O6). In acetogenic bacteria this process the reduction potentials of these species are more negative —or less generates a net energy gain of 8 [H] reducing equivalents and 4 ATP positive— than that of the O2/H2O couple, they are less effective in molecules [53]: making ATP. But anaerobic respiration is still a much better ATP pro ducer than fermentation and, because of its clear selective advantage, it C6H12O6 + 4 ADP + 4 Pi→2 CH3COOH + 2 CO2 + 4 ATP + 8 [H] (1) probably appeared before the Great Oxidation Event caused by photo In these strict anaerobes the energy needed to synthetize glucose synthetic cyanobacteria about 2.5 billion years ago [56]. comes from the oxidation of either formate or hydrogen [53]. As dis In bacteria, ATP synthase is made of two essential components called cussed above, the resulting electrons are used by the W-L pathway to Fo and F1 [57]. The c-ring of Fo is an integral membrane protein reduce two CO2 molecules and generate the acetyl CoA intermediate composed, depending on the species, of either 10 or 15 identical sub (Fig. 2A). In some anaerobes this intermediate species can transfer its units, capable of sequentially translocate protons from the extracellular acetyl moiety to oxaloacetate to generate citrate as part of the oxidative to the intracellular space. This sequence generates a rotation that is Krebs cycle (Fig. 2B, clockwise) [54]. This version of the cycle can be transmitted to the F1 component by the γ subunit. The three αβ subunits described as the stepwise oxidization of citrate back to oxaloacetate and of F1 form a ring and are kept fixedby the stator made of subunits Comic two CO2 molecules with the generation of reducing power (NADH and Sans MS (Fig. 5). Each β subunit can bind ADP + Pi and make one ATP FADH2) and one additional SLP reaction: molecule. However, ATP will not be released unless the β subunit un dergoes a conformational change caused by the rotation of the γ subunit. Acetyl CoA + 3 NAD+ + FAD + ADP + P + 2 H O→2 CO + 3 NADH i 2 2 In this way, the mechanical rotational energy caused by proton trans + FADH + ATP + 2H+ + CoA 2 location is indirectly transformed to chemical energy. Similar ATP (2) synthases are also found in archaea, mitochondria and chloroplasts, and + + The coupling of the W-L pathway to the oxidative Krebs cycle —via some archaeal enzymes can use Na instead of H , or both [58]. Which of these two ATP synthetic metabolic processes was the firstto acetyl CoA— effectively transfers the inorganic reducing power of H2 operate during life evolution? The fermentative SLP is the better (via the F420H2 cofactor, Fig. 2A) to the biological NADH and FADH2 cofactors. candidate because it is much simpler than electrochemical gradient- Photosynthesis and oxidative phosphorylation are much more effi based ATP synthesis. When ATP synthase evolved, ATP must already cient than fermentation at making ATP. In oxygenic photosynthesis, the have been the energy currency of protocells, playing a very early and sunlight-energized electrons obtained from water splitting are used to central role in most anabolic and catabolic reactions. Indeed, fermen reduce NAD(P) to NAD(P)H, which, in turn, can be used to synthesize tation is the reverse of gluconeogenesis [59] and ATP modulates the essential biomolecules through the Calvin cycle [19]. The concomitantly reactivity of the intermediates in both processes. As already mentioned, generated protons will establish an electrochemical gradient (ΔΨ) ATP is also extensively involved in purine synthesis (Fig. 3) and is across the cell or organelle membrane. The ATP synthase enzyme instrumental in pyrimidine synthesis [60]. In addition, as we will see + (Fig. 5) uses this H gradient to make ATP. Anoxygenic photosynthetic below, it plays a fundamental role in assuring nucleic acid replication organisms use essentially the same strategy albeit with a simpler reac fidelity.If ATP had not been already essential for the protocell it would tion chain [55]. be not have been selectively advantageous to generate a complex motor Oxidative phosphorylation (OP) also uses an electrochemical protein such as ATP synthase, which does not perform any directly gradient to synthesize ATP by a homologous ATP synthase. However, in useful function for the cell. this case the gradient is generated by the oxidation of NADH and FADH2, produced by the oxidative Krebs cycle. Some of the best characterized 2.4. The origin of ATP synthase + An often and widely discussed hypothesis postulates that H gradi ents operating in hydrothermal vents were a primordial energy source for protocells [5] [21,61–63]. This idea is, in principle, appealing because these gradients are similar to those found across membranes in relevant contemporary cells and organelles. Less discussed, although a central issue in this context, is the necessary transition from geochemical to biological electrochemical gradients as energy source for a protocell. Lane et al. have proposed that the ATP synthase enzyme had no simpler intermediates and, consequently, it could have been “invented” de novo [61]. They have compared this possibility with the evolution of the ribosome. This comparison is reminiscent of W. Paley’s argument in “Natural Theology” (1803) [64] about what would be the use of half a watch, as discussed by R. Dawkins in “The Blind Watchmaker” with respect to the evolution of the eye [65]. Like a primitive eye that could already detect light, a primordial ribosome could already have per formed peptide synthesis [66]. Conversely, like a half watch that could not measure time, half ATP synthase could not have made ATP by chemiosmosis. A more parsimonious view is to consider that ATP synthase emerged through exaptation. The question is from which proteins? The F1 sub

Fig. 5. Bacterial FoF1 ATP synthase. The proton concentration is higher in the complex of ATP synthase (Fig. 5) is functionally and structurally ho extracellular space. The black arrows indicate the rotation sense. mologous to protein ATPases involved in assuring high-fidelity DNA

5 J.C. Fontecilla-Camps Journal of Inorganic Biochemistry 216 (2021) 111347 replication [30]. In contemporary metabolism one such protein is the 3. ATP and the “RNA world” bacterial RecA [67-69], which, like the F1 subcomplex of ATP synthase, can form a hexameric ring structure with a central hole [70]; RecA 3.1. Origin of the RNA world concept mediates homologous recombination of DNA through a mechanism that involves (i) the formation of a filament composed of this protein, ATP The discovery that natural RNAs not only carry information but can and single-stranded (ss)DNA, (ii) the binding of double-stranded (ds) also catalyze several reactions, prompted the speculation that, during DNA to this filament,(iii) the search for homology between strands and the Archean eon, there existed an “RNA world” [81–83] where organ (iv) exchange of the complementary strand generating a new hetero isms devoid of proteins called ribocytes lived [84]. It has also been duplex [71,72]. ATP binds at the interface of two RecA molecules and its proposed that the numerous ribonucleotide-containing biological co hydrolysis is used to detach the heteroduplex from the filament [71]. factors, such as ATP and NAD, are “leftovers” of that world [85]. But, During this process RecA functions as a motor protein [67]. In addition how likely an RNA world could have been? Because in contemporary to its recombination activity, RecA is involved in repairing stalled organisms, with the notable exception of the ribosome, RNAs carry out replication forks, double-strand breaks and stress response to ssDNA intramolecular, single-turnover, reactions [86], a larger catalytic gaps, which do not require ATP hydrolysis [67]. When DNA damage is repertoire has been sought, and in many cases found, using in vitro se extensive, bacteria use low-fidelity Y-family polymerases to replicate lection approaches [87]. However, it is still not clear what was the DNA across the lesion. A 30 residue-long C-terminal extension found in source of essential biological building blocks, such as the nucleobases these polymerases is homologous to the N-terminal oligomerization and amino acids, in an RNA world [88]. This problem has prompted domain of RecA, suggesting that the latter plays a fundamental role in some authors to suggest the existence of a pre-RNA world where the regulating Y-family polymerase activity [73]. Because, as discussed synthesis of nucleobases and other metabolites firstevolved [89]. Other above, at the very early stages of life evolution, improving nucleic acid proposed sources of primordial biologically-relevant organics are their polymerization fidelitymust have been under strong selective pressure, continuous arrival in meteorites and/or comets [90] and their genera the contemporary association of a corrective ATPase and a polymerase tion by lightening in Earth’s atmosphere [91]. Consequently, the RNA might have very deep evolutionary roots. It also suggests that ATPase world hypothesis is often associated with the “primordial soup” concept. motor activity initially evolved to improve nucleic acid replication Indeed, the existence of such a world implies that ribonucleotides were fidelity. abundant in some locations; even though the in vitro triphosphorylation Other RecA-like ATPases involved in nucleic acid replication are the of free nucleosides by a ribozyme has not been achieved [92]. Pearce hexameric helicases [70,74]. Mulkidjanian et al. have put forward the et al. [90] have put forward an extreme view of this possibility by idea, based on amino acid sequence and structural homologies, that ATP arguing that nucleobases of meteoritic origin rapidly gave rise to RNA synthase originated from the evolution of an RNA helicase and a polymers in warm ponds that underwent wet-dry cycles. membrane channel through intermediate RNA and protein translocases [75]. However, translocases derive from the bacterial flagellum [76], 3.2. How plausible is the RNA world? which is a highly energy-demanding motor complex. Consequently, it seems unlikely that it could have been ancestral to ATP synthase at a In spite of its popularity, the RNA world hypothesis is confronted time when metabolic ATP, obtained by SLP, could have been relatively with several potential problems. Firstly, like the primordial soup, it does scarce. A more plausible scheme places the hexameric helicases as direct not readily explain the origin of reliable and reproducible energy sour ancestors of the F1 subcomplex of ATP synthase [77]. These motor ces. Muller [93] has proposed a cyclic “thermosynthesis” process for proteins couple the hydrolysis of nucleoside triphosphates to nucleic energy generation in such a world, which coupled ATP synthesis by an acid unwinding. This function is essential for nucleic acid replication ancestor of the β subunit of the F1 component of ATP synthase (Fig. 5), and requires energy to disrupt the extensive hydrogen-bonding network with ATP dissociation by thermal unfolding. However, as discussed formed between the two strands. Hexameric helicases dissociate them above, this idea tacitly implies that ATP was already the “energy cur by forcing one of the strands to go through the central hole of the ring- rency”; otherwise, there would be no reason for a protocell to evolve shaped hexamer, possible through rotation [78], in a process called such a complicated “heat engine” that does not perform any directly “steric exclusion” [79]. Importantly, this arrangement is reminiscent of relevant metabolic function. the one formed by the three αβ subunits and the central γ subunit of ATP Secondly, from what we know about organismal evolution, the less synthase (Fig. 5). specialized a living organism is, the higher the chances it will be able to Conversely, the Fo subcomplex ancestor was likely to be a multi- diversify. This notion has been formulated as “Cope’s law of the Un subunit membrane protein that first evolved to pump out the excess of specialized” [94]. Also, in agreement with this idea, it has been found + H ions resulting from fermentation [51]. Indeed, when in acidic media, that generalist bacterial species have 19-fold higher speciation rates + contemporary bacteria keep their pH homeostasis using a H ATPase than specialist ones [95]. Based on these observations, it is not easy to pump, homologous to the ATP synthase, to promote proton efflux[ 80]. picture an already sophisticated RNA world, which would have included Similarly, the association of the multi-subunit membrane rotor with the ribocytes [84], evolving to become the DNA/RNA-protein world we hexameric helicase-like ATPase motor —and other essential subunits— know. would have been efficient enough to keep pH homeostasis within the Thirdly, the RNA world hypothesis is confronted with the so-called protocell. Eventually, the evolution of membrane-bound, proton- “Eigen paradox”, which states that a primordial RNA must be long exporting electron transfer chains could have reduced intracellular enough to code for a protein that will improve its replication fidelity + acidification while the primordial H ATPase pump generated an elec [96]. But, before proteins, what would have been the probability of trochemical gradient with higher proton concentrations in the extra having an error-free RNA polymerase ribozyme? A very recent result cellular space [51]. This new gradient would have reversed the rotation concerning this question has been reported by Tjhung et al. [97], who of the Fo subcomplex ancestor. The direct coupling of this motion to used a class I ligase as template to generate an RNA polymerase ribo conformational changes in the hexameric helicase-like ATPase motor zyme that could synthesize its own ancestor. However, the necessary would have synthesized ATP from ADP and phosphate. It is remarkable complexity of the replicative process introduced enough disabling mu that for ATPase/synthase to be reversible, both Fo and F1 had to be tations to the full-length products as to render the majority of them molecular motors. The emergence of electron transport chains, which inactive [97]. The observed intrinsic low replication fidelityof ribozyme allowed the coupling of the oxidative Krebs cycle with ATP production, polymerases makes the notion of a full-fledgedRNA world problematic. was a major event in the evolution of energy metabolism. Furthermore, there is no guaranty that the ribozyme catalytic repertoire artificially obtained through in vitro directed evolution experiments,

6 J.C. Fontecilla-Camps Journal of Inorganic Biochemistry 216 (2021) 111347 could have ever been expressed by abiotically-generated oligonucleo dehydrated mineral surface (orange rectangle) that acts as a bio tides in primordial settings [98]. inorganic cofactor and its carbonyl carbon is attacked by the amino In a recent paper, Wills & Carter have used a mathematical approach group of another, closely bound amino acid (top left (2)) [9]. The to argue that the genetic code could not have emerged in an RNA world nascent polypeptide has limited polymerase and helicase (i.e. strand [99]. separation) activities and stimulates polymerization of the oligonucle otide which complementary strand (blue figure,top right (3)) folds into 4. A possible “RNA/protein world” a structure that also binds four amino acids that could be different or, if there are the same, would bind to codon-like pockets instead (sense- 4.1. RNA-protein copolymerization: A possible “frozen” accident antisense ancestral protein coding has been discussed in [100]). This complementary oligonucleotide also binds to the dehydrated An alternative to a full-fledged RNA world that would make more mineral surface and the bound amino acids polymerize (bottom right evolutionary sense, is a more generalized system composed of inter (4)). It then generates the “gray” oligonucleotide (1) closing the cycle. acting polypeptides and oligonucleotides [100,101]. In a world with Many copies of identical or similar polypeptides and oligonucleotides low-fidelityreplicating RNAs and either randomly made polypeptides or are made by mutually enhanced coded polymerization. Although it is just free amino acids, most of these interactions would not be subjected difficult to predict how and when ATP got involved in these processes, to Darwinian evolution. For evolution to get started, an interaction be the transition proposed above between mineral-bound and free proto tween polypeptides and oligonucleotides should, (i) increase the RNA cells could have played a central role in it. In this scheme, ATP replaced polymerization fidelity, (ii) improve the RNA polymerization rates and the mineral surface as a cofactor of peptide formation through phos (iii) either code for the bound polypeptide sequence or specifically phorylation and, at the same time, became involved in nucleic acid polymerize sequentially-bound amino acids. By its very nature, this replication fidelity.In a previous review article I discussed the evolution initial event might have been fortuitous, maybe even a “frozen accident” of the genetic code starting from this “stereochemical origin” [104] (and [101]. Specific oligonucleotide recognition of amino acids might have references therein). Intriguingly, Martínez-Rodríguez et al. have found existed very early. For instance, Johnson and Wang [102] have shown that ATP-binding 46mers from Class I and II amino acid activating en that Phe, Trp, Asp, Ile, Gln, Tyr, Leu, His, Leu, Arg and Met residues from zymes can be coded by opposite strands of the same gene [105]. The ribosomal proteins are found close to their respective anticodon and scheme described in Fig. 6 is consistent with that finding; ATP binding codon nucleotide sequences in ribosomal RNAs. A similar result has would have been the next step. been obtained by M. Yarus for amino acid recognition by oligonucleo Remarkably, in contemporary metabolism both polypeptide and tide aptamers using directed evolution [103]. These specificamino acid nucleic acid polymerization reactions rely, for the most part, on the side chain-trinucleotide interactions could have increased the poly correct positioning of the reactants. Consequently, they are mainly en merization fidelity and improved RNA polymerization rates by helping tropy and acid-base dependent and do not require protein functional the correct positioning of reactants. The same could be argued in the groups to lower the activation energy, or to form covalent intermediates. opposite direction, where the oligonucleotide would have positioned For this reason, these polymerization reactions appear to be simpler than neighboring amino acids in the right orientation and distance to form those of many contemporary enzymes, as might be expected from their peptide bonds. ancient origin. Competition among nascent co-polymers for selected amino acids and nucleotides, along with the occurrence of relevant mutations, would have been subjected to natural selection. These ideas 4.2. A possible scenario for RNA/protein evolution are similar to those previously proposed by Carter et al. and Kunin [100,101,106], but introduce a new concept, based on the reported Fig. 6 depicts the above ideas in a very simple form. In a primordial specific anticodon/codon-amino acid interactions [102,103]. setting, several different amino acids (colored geometrical figures) are If, as proposed, nucleic acids and proteins have an extensively shared recognized by their corresponding anticodons in a folded oligonucleo past, then ATP, like the other adenine-based cofactors, besides being a tide (gray figure, bottom left (1)). Peptides bonds are formed when the precursor of nucleic acids, was associated very early to polypeptides as carboxylate group of one of the bound amino acids interacts with a an essential element in many reactions (consequently, it would not be a leftover from an RNA world, as proposed [85]).

5. Conclusions

Several observations make it plausible for protocells undergoing dry- wet cycles to have obtained their energy from inorganic sources (H2 oxidation, thermal storage by dehydration) while being attached to mineral surfaces. In order to be freed from these surfaces, they had to replace these inorganic catalysts and cofactors with their bioorganic equivalents [9]. Among others, two of the latter may have combined a readily available AMP fragment with a functional one: NAD, with nicotinamide as a hydride carrier, and ATP, with labile O~P bonds to either hydrolyze or phosphorylate and activate carbon atoms. ATP functions can be divided into metabolic and chemomechanical. Of these, the former are very likely to be older because they are simpler and more essential [52]. However, ATP chemomechanical-related activity should also have evolved early, due to strong selective pressure to improve nucleic acid polymerization fidelity. This is strongly suggested by the Fig. 6. Simplified scheme of possible early amino acid-oligonucleotide in teractions and co-polymerization (see text). Different amino acids (colored rather poor performance of a contemporary in vitro-selected nucleotide figures) are shown bound to the original (gray) and complementary (blue) ol polymerizing ribozyme [97]. igonucleotides. However, this did not have to be always the case, as several When exaptation is considered, there is no reason to conclude that amino acids recognize both their corresponding anticodons and co the evolution of a biological transmembrane electrochemical gradient dons [102,103]. had to take place in a hydrothermal vent as often proposed

7 J.C. Fontecilla-Camps Journal of Inorganic Biochemistry 216 (2021) 111347

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