No soup for starters? Autotrophy and the origins of metabolism

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T~SS 20 - SEPTEMBER 1995

THE CLA~EAL E:EFaAP~O for the origin of life arose from Opadn's ~ and Urey's z proposals for a reducing early atmosphere in which organic syntheses could take place. ~iller and others s-s dramatically demonstrated that under such conditions, and with suitable energy input, an array of potential building blocks for the origin of life can be produced, it was envisaged that such organic compounds, perhaps supplemented by further supplies from extraterrestrial impactors ~, accumulated in the ocean to form a rich prebiotic 'soup' from which life somehow evolved. ~t was generally supposed that the first organisms were heterotrophs, feed= ing off the soup, and that autotrophy was invented later ?, in time for life to continue after soup depletion. Metabolism was seen as evolving from fermentation (e.g. glycolysis), through the pentose phosphate pathway, photophosphorylation, photosynthesis and, with the advent of oxygen, respiration (for examples see Ref. 8, and Chapter 14 in Ref. 4). Prebiotic development of a genetic system capable of evolution was seen by most workers as prerequisite for the development of metabolism. The discovery of catalytic RNA9 seemed to solve the twin problems of catalysis and genes. RNA, plus a few coenzymes (of unspecified origin), was seen as capable of handling early metabolism as well as its own replication =°.

Was there a soup?D[ffl©ultlesand alternatives Difficulties have been mounting for the soup hypothesis (see Box 1), and alternative pr6posals, differing in detail or in principle from the classical soup scenario, have been put forward. Cairns-Smith n advocated clay crystals as a prebiotic informational ('genetic') surface upon which a primitive metabolism (envisaged in general terms) might have started ~s. Hartman ~- noted that newer geochemical ideas did not support the previously supposed reducing atmosphere (and soup); he therefore conceptually explored an autotrophic origin to metabolism, pointing out the suitability of the (oxidative) citric acid cycle for providing starting compounds for the major biosynthetic pathways. Racker ~3, followed by Hartman ~z and B. E. H. Marion is at the Department of Biochemistry, Universityof Liverpool, PO Box 147, Liverpool, UK L69 3BX. © 1995,ElsevierScienceLtd 0968- 0004/95/$09.50

No sou# start ? Autotrophy and the orSins of {a o i [3. Edward H . ' "Made, "a in recent years, several alternatives to the classical prebiotic-soup model for the origin of fife have boon proposed. Among these, the theory of W~chtersh~user proposes that an archaic version of the autotrophic reductive citric acid cycle, driven by pyrite formation and contained on th~ resulting pyrite surface, was the earliest metabolic cycle, from which the central biosynthetic pathways arose, initially without enzymes or nucleic acids.

de Duve~4'~s, proposed that thioester energy preceded phosphate bond energy for driving various endergonic reactions (such as peptide syntheses). Russell and colleagues R6have explored a novel system in which iron-sulphur membranes axe precipitated under conditions resembling those at deep-ocean hydrothermai outflows. The authors propose that the membranes provide a possibility of very early harnessing of proton motive force in the evocation of metabolism. ~ x &o¢ ~ s t m i ~

But by far the most detailed theory of the origins of metabolism is due to W~chtersh~user. His theory proposes autotrophic origins starting from an archaic, reductive (reverse) citric acid cycle ~7-~-°. The main features of the theory are encapsulated in Box 2 as titles and 'mini-summaries' of the four key papers. The ideas are likely to be of general interest to biochemists who have pondered about the origins of metabolism, and axe briefly reviewed here.

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Early atmosphere. After Earth's formation 4.5billion years ago (4.5 GY)28, the atmosphere was probably C02+C0, 10 bar (lO°Pa) plus N~., 1 bar (lO~Pa) with a liquid H20ocean2g. Prebiotic organic synthesis is poor in CO2+ N2 w=thoutH2 (Ref. 5). Compounds net s ~ i z e d

a~¢ simu~i ~ ¢omiRloM. The following compounds have not been convincingly synthesizerS: the amino acids Arg, Lys and His; straight-chain fatty acids; porphyrins; the coenzymes pyridoxal, thiamine, riboflavin, relic acid, iipoic acid and biotin; rinse and activated nucleotides. Tim=c~le for origins. Life may have originated in a fairly short interval, further decreasing the opportunity for prior accumulation of a rich prebiotic soup. IVlicrofossils occur in 3.5 GY rock3°; there is evidence of biogenic carbon isotope fractionation in 3.8 GY rock3t. Therefore, life with autotrophic carbon fixation is at least 3.8GY old. Heavy meteoritic bombardment occurred between 4.5 and 3.8GY, with probable large-scale ocean-bolting events until somewhere between 4.2 and 3.8GY32'33. Therefore, the interval from the last such 'life.frustrating' event to definite autotrophic organisms is 0.4 GY at most, and probably much less. The deep ocean floor afforded the most protected environment during this period. Universa| ph~dogeny. This suggests that the most ancient organisms were hyperthermophilic

and autotrophic22. T r a M ~ metals and Fe=~ in ~ metab~i=m. Their recruitment is better explained by chemolithoautotrophic origins than by soup-based beterotrophic origins17-2°Prebietl¢ RNA. Ribofuranose contains four chiral centres. These and 3"5' phosphodiester bonding appear to present insuperable difficulties of structural specificity for purely prebiotic synthesis~. The energetics of RNA synthesis are also a major prebiotic problem. RNA catalysis. The catalytic repertoire of RNA is largely confined to ester chemistry and is 141517 20 thought to be too limited to direct an emerging metabolism . ' - •

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TALKINGPOINT HypertlmwJ~, a m t ~ y andsources oe~power There is general agreement that metabolism originated anaerobically, but there is no longer a consensus that the earliest organisms were heterotrophs. All of the deepest roots on the bacterial and especially the archaeal side of the universal tree of life2L22are hyperthermophiles, and many of these are autotrophs. As summarized by Stette~ '...the phylogenetic tree demonstrates that the deepest branches are represented by strictly chemolithoautotrophic organisms... Therefore, autotrophy appears to be a very ancient feature, supporting theories about an autotrophic origin of life.' Autotrophic CO2 fixation needs reducing power. A primordial reducing source must be strong enough to drive overall metabolism reductively28; energy flow must proceed directly from the source into CO2 fixation without the need for intermediate carriers, such as ATP, and should be ldnetically inhibited in the absence of CO2 fixation or other metabolism to avoid depletion. Photoautotrophy based on visible light was not possible before compounds with extended ~r electron systems, such as chlorophyll or carotenoids, evolved as products of metabolism. Mineral (e.g. Fe)-based photo-boosting requires UV light, which would be damaging for emerging life. That leaves the possibility of chemoautotrophlc origin, which must be based on a geochemical source that satisfies the criteria outlined above. Pyrite formation Is proposed 17,2° as uniquely satisfying these criteria,

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TIBS 20 - SEPTEMBER1995

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surfaces (such as clay H) can be ruled out. Anionic bonding to mineral surUnder standard conditions (pH7, 25°C, faces with polyvalent cations, such as 1 M H~S), the free energy for the reac- transition-metal sulphides, and notably tion is -381drool -~, calculated from pyrite, is favourable. For strong surface bonding to pyrite, thermochemical data cited in Ref. 20. This corresponds to a standard reduc- a hydrophilic compound requires at tion potential of -620mV, on the basis least two negative charges on the same of the reduction potential (pH?) for or different groups, for example, -PO32-, 2H÷/H2 of -420 InV. Thus the reaction is -COO- or --S-. The ancient metabolic thermodynamically capable of provid- pathways contain many such polying reducing power for any biochemical anionic metabolites and cofactors, reduction. The reaction remains exer- which are therefore potential surface gonic at geochemically likely concen- bonders. Lipophilicity also contributes trations of H2S (-5raM) and becomes to surface bonding. Surface chemistry is relatively more so at higher temperature, which is of interest given the inference that favourable towards condensations and hyperthermophiles were the earliest polymerizations which, in an aqueous environment, would tend towards forms of life. On its own, the reaction is ldnetically hydrolysis. This is because in solution slow but demonstrable 23. Thus it meets the hydrolysis products gain entropy the criterion of kinetic inhibition in the through extra translational and rotaabsence of coupling, A. simple example tional freedom as compared with surof the thermodynamic feasibility of cou- face reactions. pling to CO2 fixation is the following2°: Lastly and importantly, surface chemistry, and pyrite in particular, may perHCO3- + FeS + H2S~ HCOO- + FeS~+ H20 mit introduction of chirality ~8,2°. where the standard free energy change is -37.1 IOmol-L Surfa©e metabolism Metabolism requires containment. The theory sees primordial containment on a two-dimensional surface. The following considerations favour pyrite as providing that surface. Metabolites must be firmly bound to the surface but capable of lateral migration to permit chemical encounters to occur. Ionic bonding is seen as best satisfying these conditions. The strength of ionic binding increases with the insolubility of the corresponding salt; cationic organic nitrogen bases do not form insoluble salts; therefore cationic bonding to (predominantly) negatively charged

Box 2. W~,htershiuser's theopj: Um four mmin papers 'lb'dte fomatlon, the first eMqff somce for tb: a hYimtl~sb', Pyrite formation from FeS and H2S is prolX),sedas the primordial source of energy and reducing powerlL

'lk~o~ ~ aml totalities: theoff M surface metabolism'. Initial exposition of the theory that metabolism commenced in a two-dimensional system characterized by surface bonding of anionic organic I;gandsto a cationic mineral surface (pyrite)is. proposal that primordial carbon fixation pulled by pyrite formation and whose dicent state to the pyrite surfacezs, for am e v o l ~ bioelmmlMpj:. ~ k~-wlphar world', Development and expansion of the Rroposals in Refs 17-I9 to autotrophic odgins for the whole of central metabolism, includi~ recruitment of the elements, evolution of the sugar pathways, the I.mino acid p a t h S , lipid pathways and membranes, cofactors and purine pathways. :nzymes and nucleic acids are seen as products of, not precursors to, the archaic netabolism2o,

Carbon fieation: the ~edu©tive©itri©acid ©ycie The question of the primordial mode of carbon fixation is central to the theory of autotrophic origins. In contemporary, or extant, metabolism there are three major pathways for autotrophic carbon fixation: the reductive pentose phosphate cycle, the reductive (reverse) citric acid cycle 24 and the reductive acetyl CoA (carbon monoxide dehydrogenase) pathway 25. The reductire pentose phosphate pathway is the major global pathway by which CO2 is fixed in photosynthesis, but it is based on pro-established sugar biochemistry. Therefore it does not satisfy the requirements of primordial (as distinct from extant) autotrophy. The acetyl Cob. pathway, though deeply rooted in various anaerobic Bacteria and Archaea, gives only C2 units (acetate or acetyl CoA), which require further CO~ fixation to be biosynthetically useful, for example for amino acid formation. Therefore this pathway also could not have been the primordial autotrophic pathway. W~chtersh~user proposes that an archaic version of the reductive citric acid cycle, driven by pyrite formation, was the first pathway for autotrophic carbon fixation. The following considerations underly the proposal. The contemporary reductive citric acid cycle occurs in (some) Bacteria and Archaea (Refs 19, 20 and references therein),

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Rgure ~. 'Twisted' or 'figure-of-eight' representation of {a) extant and (b) proposed archaic reductive citric acid cycles. Abbreviations designate citrate cycle intermediates (Su, succinate; 2-KG, 2-ketoglutarate; ICit, isocitrate; Aco, aconitate; Cit, citrate; Fu, fumarate; Ma, malate; and OA, oxaloacetate), pyruvate (Pyr), phosphoenolpyruvate (PEP), reduced ferredoxin (FdH2) and the iron-sulphur enzymes aconitase and fumarate reductase (Fe/S). in (b) compounds with SH groups coexist with those having OH groups (not shown), and carboxyl groups coexist with thio-derivatives (partly shown). Reproduced,with permission, from Ref. 19. carboxylation implying ancient origin. Starting from The extant reductive cycle and the including redudive succinate (four carbon atoms) it hypothetical archaic cycle are shown in (e.g. thiosuccinate to 2-ketoglutarate). doubles itself at every turn, with two Fig. I. This figure shows the cycle in an The carboxyiate products of COz carbon fixations to isocitrate and two unusual, twisted (or 'figure-of-eight') fixation are surface bonded in their more on reductive transformation of notation, to indicate that it comprises nascent state to the growing pyrite acetyl CoA to oxaloacetate 24. Thus it is two similar halves and to show the surface. A small amount of pre-existing acid a highly effective autocatalytic cycle for relationships between the two halves carbon fixation. Citric acid cycle inter- in the extant and archaic cycles. (such as acetate or sucdnate) is mediates are the hub of the central bio- The archaic cycle lacked enzymes required to prime the cycle but, once synthetic pathways (as pointed out in and coenzymes. Where ferredoxin pro- ignited, the cycle is autocatalytic. CAn Ref. 12). The di- and tricarboxylic acids rides reducing power in the extant archaic version of the reductive acetyi are surface bonders that would be re- pathway, it is proposed that pyrite CoA pathway could conceivably have tained on the pyrite surface. Some formation drove the archaic reductions. been a priming pathwaym.) The cycle can expand by branch key enzymes of the contemporary citric In the H~S-rich geochemical setting, reactions into higher homologous several organic compounds were in acid cycle are iron-sulphur proteins (succinate dehydrogenase, fumarate equilibrium with thio-organic hom- cycles and an archaic citrate network, reductase, aconitase) and are seen as ologues, seen as providing key roles in leading in several directions, including having evolved from mineral iron- archaic energetics, such as thioacids compounds with lipophilic properties undergoing thioester-type reactions, and the first membranes. sulphur origins.

339

TALKINGPOINT Methodolol[y of pMhway '~mdk'Uon' Formulation of the archaic citric acid cycle was achieved by a process of conceptual reconstruction, which W~chtersh~user terms 'retrodiction'. Retrodiction consists of making theorybased proposals of an unknown or uncertain past, just as prediction consists of making theory-based statements of an unknown or uncertain future, in Ref. 20, a major section is devoted to theoretical principles and methodology of retrodiction from known contemporary biochemical pathways to unknown archaic pathways. For example, a central proposal is that reductive metabolism in the original FeS/H2S setting preceded the later development of reductive/oxidative metabolism. This proposal requires that the primordial archaic pathways should contain no oxidative steps. Fe--S clusters on pyrite surfaces are seen as evolutionary precursors to those in ironsulphur enzymes. Evolution of pathways proceeds from what is already in place, but nevertheless is saltatory, involving discrete steps, such as pathway extension, lateral branching, recruitment of new starting materials and pathway reversals where equilibria permit. 'Evolved' pathways can be distinguished from (hypothetical) primitive ones by several criteria, such as the involvement of a product of the pathway in an earlier step in the same pathway (for example, tetrahydrofolate in the purine pathway). In many instances, thio-analogues are seen as preceding more familiar contemporary constituents, notably phosphoryl compounds. All contemporary pathways are evolved in the sense that they involve protein catalysts. The coenzymes are seen as originally pre-enzymic. Guided by these principles, W~chtersh~user considers the further evolution of metabolism in detalF. A~luislt~ef Umdm~mts Pyrite formation is seen as providing

the original driving force for thieester activation, and thioesters are seen as predating phosphate-bond energetics. This agrees with, and provides a rationale for, the emergence of the 'thioester world '.4'ts. Nitrogen fixation is also seen as originally driven by pyrite formation, the iron-sulphur-vanadium or iron-sulphur-molybdenum centres of nitrogen fixation evo~ng from there. Phosphate first appears in a surface-bonding role, and is later adapted to energy storage and transductlon.

340

TIBS 20 - SEPTEMBER1995 The recruitment of catalytic metal ions arises from the solubilities (and hence archaic availabilities) of their sulphides: Mn2÷:. Fe~-~> Ni2÷>Co2">Zn2~. Many of the other transition metal sulphides are insoluble and their metal ions later become universal poisons (except copper in aerobic metabolism). Ferredoxins and iron-sulphur-cluster enzymes, which are especially widespread in anaerobic metabolic pathways, are seen as having evolved from archaic pyrite-based reactions.

postulated original pathway and its proposed evolution into the two extant pathways are shown in Fig. 2~

Coe~ymes Many of these are polyanionic potential surface bonders, consistent with roles in surface metabolism. Their evolution is central to metabolism, an(] many workers (for examp|e, Ref. 26) have postulated their emergence preenzymically. Plausible archaic pathways for their synthesis are explored in Ref. 20. Of particular interest is a proposed Sugar pathways archaic tetrapyrrole pathway. This is The sugar pathways are seen as first retrodicted from the extant g]ycine-sucevolving in the synthetic direction from cinyl CoA pathway Olot the Glu tRNA pyruvate, with thio
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¢on©iuding©omments The citric acid cycle is indeed the hub of metabolism in most organisms. The concept of a reductive citric acid cycle at the origins of metabolism carries great appeal, and some of the postulated links betweer: geochemistry and biochemistry are being tested 2a,27. The reader will realize that the theory is not definitive in its many details. To put this present commentary in perspective, it is appropriate to close with an introductory passage from Ref. 20. '...principles are combined to form a coherent methodology of retrodiction. It will be shown how all the pathways of central and universal metabolism can be retrodictively transformed into archaic pathways. These turn out to be mutually compatible, and to be converging with increasing antiquity. The point of convergence turns out to be a simple pyrite-pulled reductive metabolism with thio-organic constituents. In spite of increased concreteness...it would be preposterous to assume that these first attempts at retrodiction are likely to hit upon the true historic sequence of events. The specific merit of such a theory...is its fertility in producing increasingly comprehensive and powerful explanations.

Ultimately, the progressive amendments of the theory...should lead to...a historically ordered table of biochemistry. I expect this may be a precondition for making significant progress in the evolutionary explanation of the genetic machinery, the genetic code and the process of translation.' Acknowledgements I thank GOnter W~chtersh~user for his interest in this article and for several helpful comments, and Beryl Foulkes for the typing of the article.

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