Prebiotic Synthesis of Methionine and Other Sulfur-Containing Organic Compounds on the Primitive Earth: A Contemporary Reassessment Based on an Unpublished 1958 Stanley Miller Experiment

Original extracts from an unpublished 1958 experiment conducted by the late Stanley L. Miller were recently found and analyzed using modern state-of-the-art analytical methods. The extracts were produced by the action of an electric discharge on a mixture of methane (CH₄), hydrogen sulfide (H₂S), ammonia (NH₃), and carbon dioxide (CO₂). Racemic methionine was formed in significant yields, together with other sulfur-bearing organic compounds. The formation of methionine and other compounds from a model prebiotic atmosphere that contained H₂S suggests that this type of synthesis is robust under reducing conditions, which may have existed either in the global primitive atmosphere or in localized volcanic environments on the early Earth. The presence of a wide array of sulfur-containing organic compounds produced by the decomposition of methionine and cysteine indicates that in addition to abiotic synthetic processes, degradation of organic compounds on the primordial Earth could have been important in diversifying the inventory of molecules of biochemical significance not readily formed from other abiotic reactions, or derived from extraterrestrial delivery.     

Formation of prebiochemical compounds in models of the primitive Earth's atmosphere

In order to understand the formation of organic compounds in the primitive atmosphere, the first steps of evolution in models of the primitive atmosphere were investigated. Mixtures containing C−H−N elements were subjected to a low pressure silent electric discharge for several seconds, and the resulting effluents were analysed mainly by gas chromatography, infrared spectrometry and chemical analysis. The formation of hydrocarbons (i.e. ethylene, acetylene, methylacetylene) and of nitrogen containing compounds (i.e. hydrogen cyanide, cyanogen, saturated nitriles, acrylonitrile, cyanoacetylene) is reported. The influence of the initial mixture composition on the amount of compounds formed was systematically studied. The nature of the nitrogen source (N₂ or NH₃) in the primitive atmosphere has a great influence on the amount and on the very nature of the synthesized products. It is shown that important precursors such as cyanogen and cyanoacetylene are formed only in very rich N₂ mediums. There results show the important role played by the nature of the primitive atmosphere in the determination of the chemical evolution pathways.     

Organic sulfur compounds resulting from the interaction of iron sulfide,hydrogen sulfide and carbon dioxide in an anaerobic aqueous environment

The reaction of iron sulfide (FeS) with H₂S in water, in presence of CO₂ under anaerobic conditions was found to yield H₂ and a variety of organic sulfur compounds, mainly thiols and small amounts of CS₂ and dimethyldisulfide. The same compounds were produced when H₂S was replaced by HCl, in the H₂S-generating system FeS/HCl/CO₂. The identification of the products was confirmed by GC-MS analyses and the incorporation of H₂ in the organic sulfur compounds was demonstrated by experiments in which all hydrogen compounds were replaced by deuterium compounds. Generation of H₂ and the synthesis of thiols were both dependent upon the relative abundance of FeS and HCl or H₂S, i.e. the FeS/HCl- or FeS/H₂S-proportions. Whether thiols or CS₂ were formed as the main products depended also on the FeS/HCl-ratio: All conditions which create a H₂ deficiency were found to initiate a proportional increase in the amount of CS₂. The quantities of H₂ and thiols generated depended on temperature: the production of H₂ was significantly accelerated from 50°C onward and thiol synthesis above 75°C. The yield of thiols increased with the amount of FeS and HCl (H₂S), given a certain FeS/HCl-ratio and a surplus of CO₂. A deficiency of CO₂ results in lower thiol systhesis. The end product, pyrite (FeS₂), was found to appear as a silvery granular layer floating on the aqueous surface. The identity of the thiols was confirmed by mass spectrometry, and the reduction of CO₂ demonstrated by the determination of deuterium incorporation with DCl and D₂O. The described reactions can principally proceed under the conditions comparable to those obtaining around submarine hydrothermal vents, or the global situation about 4 billion years ago, before the dawn of life, and could replace the need for a reducing atmosphere on the primitive earth.     

Addition reactions of malonic nitriles with alkanethiol in aqueous solution

In order to evidence new reactions of prebiotic synthesis in aqueous solution between the S-containing compounds, such as alkanethiols, which can be obtained in gaseous phase during the simulation of the evolution of primitive atmosphere, and the malonic nitriles which are generally considered as important intermediates in the Chemical Evolution, a kinetic study on the eventual reactions of ethanethiol with cyanoacetaldehyde and malononitrile has been carried out.It appears that ethanethiol does not react directly in aqueous solution with cyanoacetaldehyde, but it gives an addition reaction with the double bond of the crotonic dimer of this malonic compound giving a thioether.With malononitrile, ethanethiol reacts directly by addition reaction on the CN group of the monomer, producing an iminothioester. These two reactions are equilibrated. The specific rate constants and the apparent equilibrium constants for these two reactions have been simultaneously studied by UV spectrophotometry at room temperature as a function of pH.The conditions for the formation of iminothioester will be discussed in terms of the respective pKa values of the thiol and of the malonic nitrile.These two addition products, principally the iminothioesters, because of their hydrolysis in thioesters may have played an important role in the prebiochemical evolution.Presented at the second ISSOL meeting and the fifth ICOL in Kyoto 5–10 April, 1977.     

The evolution of the prebiotic atmosphere

Summary High CO₂ levels are required to warm the primitive earth in the face of decreased solar luminosity. The atmosphere should have had an effective stratospheric cold trap, which would have limited the abiotic production rate of oxygen to relatively low values. Photostimulated oxidation of ferrous iron in the oceans should have been the dominant source of atmospheric H₂. Rainout of H₂O₂ would have kept the atmospheric H₂ content high and the O₂ content low, even if other sources of H₂ were small.     

The prospect of life on Jupiter

Recent research into the possibility of there being life in the Solar System other than on the Earth has suggested that Jupiter may be a good candidate. The reactions within its atmosphere, both photolytic and electrical, lead to the formation of many nitrile and amino compounds. We have simulated electrical discharges in the Jovian atmosphere, using anhydrous methane-ammonia mixtures, and shown the formation of simple aliphatic nitriles, amino-nitriles, and their oligomers. Including hydrogen sulfide in the gas mixture, it appears that sulfur-containing amino-nitriles are not formed, since the hydrolysate of the products did not contain the corresponding amino-acids. There is a strong analogy between these reactions and the classical spark reactions simulating the primitive Earth's atmosphere. We are attempting a closer simulation of Jupiter's atmosphere by using appropriate temperature and pressure conditions. It seems that prebiotic synthesis on Jupiter may have reached an advanced state. As an alternative approach we have tested the survival ability of common terrestrial microorganisms in aqueous media at 102 atmospheres pressure and at 20°C. in a simulated Jovian atmosphere.E. coli, S. marcescens, A. aerogenes andB. subtilis will all tolerate 24 h. under these conditions with conditions with little death. The ability of terrestrial organisms to survive Jovian atmospheric conditions, coupled with the likelihood of advanced prebiotic synthesis, suggests that a parallel evolution of life may have occurred on Jupiter and Earth, and that the two forms need not be so different as has been supposed. Lunar Science Institute Contribution.     

Sulfur,ultraviolet radiation,and the early evolution of life

The present biosphere is shielded from harmful solar near ultraviolet (UV) radiation by atmospheric ozone. We suggest here that elemental sulfur vapor could have played a similar role in an anoxic, ozone-free, primitive atmosphere. Sulfur vapor would have been produced photochemically from volcanogenic SO₂ and H₂S. It is composed of ring molecules, primarily S₈, that absorb strongly throughout the near UV, yet are expected to be relatively stable against photolysis and chemical attack. It is also insoluble in water and would thus have been immune to rainout or surface deposition over the oceans. The concentration of S₈ in the primitive atmosphere would have been limited by its saturation vapor pressure, which is a strong function of temperature. Hence, it would have depended on the magnitude of the atmospheric greenhouse effect. Surface temperatures of 45 °C or higher, corresponding to carbon dioxide partial pressures exceeding 2 bars, are required to sustain an effective UV screen. Two additional requirements are that the ocean was saturated with sulfite and bisulfite, and that linear S₈ chains must tend to reform rings faster than they are destroyed by photolysis. A warm, sulfur-rich, primitive atmosphere is consistent with inferences drawn from molecular phylogeny, which suggest that some of the earliest organisms were thermophilic bacteria that metabolized elemental sulfur.     

Catalytic reactions in the solar nebula: Implications for interstellar molecules and organic compounds in meteorites

Organic compounds in meteorites seem to have formed by Fischer-Tropsch-type, catalytic reactions of CO, H₂, and NH₃ in the solar nebula, at 360–400K and (4–10)×10^–6 atm. The onset of these reactions was triggered by the formation of catalytically active grains of magnetite and serpentine at these temperatures.Laboratory experiments show that the Fischer-Tropsch reaction gives a large kineticisotope fractionation of C¹²/C¹³, duplicating the hitherto unexplained fractionation in meteorites. All of the principal compound classes in meteorites are produced by this reaction, or a variant involving a brief excursion to higher temperatures. (1) normal, mono-, and dimethylalkanes (2)arenes andalkylarenes; (3) dimericisoprenoids from C₉ to C₁₄; (4)purines andpyrimidines, such as adenine, guanine, uracil, thymine, xanthine, etc.; (5)amino acids, including tyrosine and histidine; (6)porphyrin-like pigments; (7) aromaticpolymer with –OH and –COOH groups.These reactions may also have played a major role in the evolution of life: first, by converting carbon to a sufficiently non-volatile form to permit its accretion by the inner planets; second, by synthesizing organic compounds on the primitive planets whenever CO, H₂, NH₃, and clay or magnetite particles came together at the right temperature. Similar reactions in other solar nebulae may be the source of interstellar molecules, as first suggested by G. H. Herbig. Ten of the twelve polyatomic interstellar molecules have in fact been seen in these syntheses or in meteorites.This paper is a revised and abridged version of the author's article inScience 182 (1973), 781.     

A Reassessment of Prebiotic Organic Synthesis in Neutral Planetary Atmospheres

The action of an electric discharge on reduced gas mixtures such as H₂O, CH₄ and NH₃ (or N₂) results in the production of several biologically important organic compounds including amino acids. However, it is now generally held that the early Earth’s atmosphere was likely not reducing, but was dominated by N₂ and CO₂. The synthesis of organic compounds by the action of electric discharges on neutral gas mixtures has been shown to be much less efficient. We show here that contrary to previous reports, significant amounts of amino acids are produced from neutral gas mixtures. The low yields previously reported appear to be the outcome of oxidation of the organic compounds during hydrolytic workup by nitrite and nitrate produced in the reactions. The yield of amino acids is greatly increased when oxidation inhibitors, such as ferrous iron, are added prior to hydrolysis. Organic synthesis from neutral atmospheres may have depended on the oceanic availability of oxidation inhibitors as well as on the nature of the primitive atmosphere itself. The results reported here suggest that endogenous synthesis from neutral atmospheres may be more important than previously thought. Stanley L. Miller died May 20, 2007.     

Gas-Phase Reactions in Extraterrestrial Environments: Laboratory Investigations by Crossed Molecular Beams

We have investigated gas-phase reactions of N(²D) with the most abundant hydrocarbons in the atmosphere of Titan by the crossed molecular beam technique. In all cases, molecular products containing a novel CN bond are formed, thus suggesting possible routes of formation of gas-phase nitriles in the atmosphere of Titan and primordial Earth. The same approach has been recently extended to the study of radical–radical reactions, such as the reaction of atomic oxygen with the CH₃ and C₃H₅ radicals. Products other than those already considered in the modeling of planetary atmospheres and interstellar medium have been identified. Presented at: National Workshop On Astrobiology: Search For Life In The Solar System, Capri, Italy, 26 to 28 October, 2005.     

Control of Oxidative Sulfur Metabolism of Chlorobium limicola forma thiosulfatophilum

A metered blend of anaerobic-grade N₂, CO₂, and H₂S gases was introduced into an illuminated, 800-ml liquid volume, continuously stirred tank reactor. The system, described as an anaerobic gas-to-liquid phase fed-batch reactor, was used to investigate the effects of H₂S flow rate and light energy on the accumulation of oxidized sulfur compounds formed by the photoautotroph Chlorobium limicola forma thiosulfatophilum during growth. Elemental sulfur was formed and accumulated in stoichiometric quantities when light energy and H₂S molar flow rate levels were optimally adjusted in the presence of nonlimiting CO₂. Deviation from the optimal H₂S and light energy levels resulted in either oxidation of sulfur or complete inhibition of sulfide oxidation. Based on these observations, a model of sulfide and sulfur oxidases electrochemically coupled to the photosynthetic reaction center of Chlorobium spp. is presented. The dynamic deregulation of oxidative pathways may be a mechanism for supplying the photosynthetic reaction center with a continuous source of electrons during periods of varying light and substrate availability, as in pond ecosystems where Chlorobium spp. are found. Possible applications for a sulfide gas removal process are discussed.     

Reaction of Hydrogen Sulfide with Disulfide and Sulfenic Acid to Form the Strongly Nucleophilic Persulfide

Hydrogen sulfide (H₂S) is increasingly recognized to modulate physiological processes in mammals through mechanisms that are currently under scrutiny. H₂S is not able to react with reduced thiols (RSH). However, H₂S, more precisely HS⁻, is able to react with oxidized thiol derivatives. We performed a systematic study of the reactivity of HS⁻ toward symmetric low molecular weight disulfides (RSSR) and mixed albumin (HSA) disulfides. Correlations with thiol acidity and computational modeling showed that the reaction occurs through a concerted mechanism. Comparison with analogous reactions of thiolates indicated that the intrinsic reactivity of HS⁻ is 1 order of magnitude lower than that of thiolates. In addition, H₂S is able to react with sulfenic acids (RSOH). The rate constant of the reaction of H₂S with the sulfenic acid formed in HSA was determined. Both reactions of H₂S with disulfides and sulfenic acids yield persulfides (RSSH), recently identified post-translational modifications. The formation of this derivative in HSA was determined, and the rate constants of its reactions with a reporter disulfide and with peroxynitrite revealed that persulfides are better nucleophiles than thiols, which is consistent with the α effect. Experiments with cells in culture showed that treatment with hydrogen peroxide enhanced the formation of persulfides. Biological implications are discussed. Our results give light on the mechanisms of persulfide formation and provide quantitative evidence for the high nucleophilicity of these novel derivatives, setting the stage for understanding the contribution of the reactions of H₂S with oxidized thiol derivatives to H₂S effector processes.     

Carbon and energy yields in prebiotic syntheses using atmospheres containing CH4, CO and CO2

Yields based on carbon are usually reported in prebiotic experiments, while energy yields (moles cal^–1) are more useful in estimating the yields of products that would have been obtained from the primitive atmosphere of the earth. Energy yields for the synthesis of HCN and H₂CO from a spark discharge were determined for various mixtures of CH₄, CO, CO₂, H₂, H₂O, N₂ and NH₃. The maximum yields of HCN and H₂CO from CH₄, CO, and CO₂ as carbon sources are about 4×10^–8 moles cal^–1.     

The Use of Ascorbate as an Oxidation Inhibitor in Prebiotic Amino Acid Synthesis: A Cautionary Note

It is generally thought that the terrestrial atmosphere at the time of the origin of life was CO₂-rich and that organic compounds such as amino acids would not have been efficiently formed abiotically under such conditions. It has been pointed out, however, that the previously reported low yields of amino acids may have been partially due to oxidation by nitrite/nitrate during acid hydrolysis. Specifically, the yield of amino acids was found to have increased significantly (by a factor of several hundred) after acid hydrolysis with ascorbic acid as an oxidation inhibitor. However, it has not been shown that CO₂ was the carbon source for the formation of the amino acids detected after acid hydrolysis with ascorbic acid. We therefore reinvestigated the prebiotic synthesis of amino acids in a CO₂-rich atmosphere using an isotope labeling experiment. Herein, we report that ascorbic acid does not behave as an appropriate oxidation inhibitor, because it contributes amino acid contaminants as a consequence of its reactions with the nitrogen containing species and formic acid produced during the spark discharge experiment. Thus, amino acids are not efficiently formed from a CO₂-rich atmosphere under the conditions studied.     

Origin of organic compounds on the primitive earth and in meteorites

The role and relative contributions of different forms of energy to the synthesis of amino acids and other organic compounds on the primitive earth, in the parent bodies or carbonaceous chondrites, and in the solar nebula are examined. A single source of energy or a single process would not account for all the organic compounds synthesized in the solar system. Electric discharges appear to produce amino acids more efficiently than other sources of energy and the composition of the synthesized amino acids is qualitatively similar to those found in the Murchison meteorite. Ultraviolet light is also likely to have played a major role in prebiotic synthesis. Although the energy in the sun's spectrum that can be absorbed by the major constituents of the primitive atmosphere is not large, reactive trace components such as H2S and formaldehyde absorb at longer wavelengths where greater amounts of energy are available and produce amino acids by reactions involving hot hydrogen atoms. The thermal reaction of CO + H2 + NH3 on Fischer-Tropsch catalysts generates intermediates that lead to amino acids and other organic compounds that have been found in meteorites. However, this synthesis appears to be less efficient than electric discharges and to require a special set of reaction conditions. It should be emphasized that after the reactive organic intermediates are generated by the above processes, the subsequent reactions which produce the more complete biochemical compounds are low temperature homogenous reactions occurring in an aqueous environment.     

Prebiotic Synthesis of Protobiopolymers Under Alkaline Ocean Conditions

Clasically, prebiotic chemistry has focused on the production and identification of simple organic molecules, many of them forming part of “intractable polymers” named tholins. In a previous work, we demonstrated that in experiments using an external energy source and inorganic carbon the aqueous aerosols improved the formation of hydrophilic tholins. Herein, we elucidate the role of pH (from 4 to 12) in prebiotic experiments using saline aqueous aerosols, spark discharges and an atmosphere containing CH₄. At all values of pH, the saline aqueous aerosols increased the production of a significant variety of carboxylic acids that could have been present in a primitive Krebs cycle. Moreover, the study for the first time of hydrophilic tholins by 2-D electrophoresis revealed that these are formed by a set of unexpected heavy polymeric species. The initial alkaline conditions significantly increased both the apparent molecular weight of polymeric species up to 80 kDa and their diversity. We propose the term of protobiopolymers to denote those polymeric species fractionated by 2-D electrophoresis since these are formed by biomolecules present in living systems and show diversity in length as well as in functional groups. Thus, aerosols formed in simulated alkaline ocean conditions could provide an optimal medium for the formation of the primeval materials that could be precursors to the emergence of life.     

Geochemical constraints on chemolithoautotrophic reactions in hydrothermal systems

Thermodynamic calculations provide the means to quantify the chemical disequilibrium inherent in the mixing of redeuced hydrothermal fluids with seawater. The chemical energy available for metabolic processes in these environments can be evaluated by taking into account the pressure and temperature dependence of the apparent standard Gibbs free energies of reactions in the S-H₂-H₂O system together with geochemical constraints on pH, activities of aqueous sulfur species and fugacities of H₂ and/or O₂. Using present-day mixing of hydrothermal fluids and seawater as a starting point, it is shown that each mole of H₂S entering seawater from hydrothermal fluids represents about 200,000 calories of chemical energy for metabolic systems able to catalyze H₂S oxidation. Extrapolating to the early Earth, which was likely to have had an atmosphere more reduced than at present, shows that this chemical energy may have been a factor of two or so less. Nevertheless, mixing of hydrothermal fluids with seawater would have been an abundant source of chemical energy, and an inevitable consequence of the presence of an ocean on an initially hot Earth. The amount of energy available was more than enough for organic synthesis from CO₂ or CO, and/or polymer formation, indicating that the vicinity of hydrothermal systems at the sea floor was an ideal location for the emergence of the first chemolithoautotrophic metabolic systems.     

A working hypothesis on the interdependent genesis of nucleotide bases,protein amino acids,and primitive genetic code

In the course of experimental approach to the chemical evolution in the primeval sea, we have found that the main products from formaldehyde and hydroxylamine are glycine, alanine, serine, aspartic acid etc., and the products from glycine and formaldehyde are serine and aspartic acid. Guanine is found in the two-letter genetic codons of all these amino acids.Based upon the finding and taking into consideration the probable synthetic pathways of nucleotide bases and protein amino acids in the course of chemical evolution and a correlation between the two-letter codons and the number of carbon atoms in the carbon skeleton of amino acids, 1 have been led to a working hypothesis on the interdependent genesis of nucleotide bases, protein amino acids, and primitive genetic code as shown in Table I.Protein amino acids can be classified into two groups: Purine Group amino acids and Pyrimidine Group amino acids. Purine bases and Pyrimidine bases are predominant in two-letter codons of amino acids belonging to the former and the latter group respectively.Guanine, adenine, and amino acids of the Purine Group may be regarded as synthesized from C₁ and C₂ compounds and N₁ compounds (including C₁N₁ compunds such as HCN), probably through glycine, in the early stage of chemical evolution.Uracil, cytosine, and amino acids of the Pyrimidine Group may be regarded as synthesized directly or indirectly from three-carbon chain compounds. This synthesis became possible after the accumulation of three-carbon chain compounds and their derivatives in the primeval sea.The Purine Group can be further classified into a Guanine or (Gly+nC₁) Subgroup and an Adenine or (Gly+nC₂) Subgroup or simply nC₂ Subgroup. The Pyrimidine Group can be further classified into a Uracil or C₃C₆C₉ Subgroup and a Cytosine or C₅-chain Subgroup (Table I).It is suggested that the primitive genetic code was established by a specific interaction between amino acids and their respective nucleotide bases. The interaction was dependent upon their concentration in the primeval environments and the binding constants between amino acids and their respective bases.Presented at the International Symposium (Lipmann Symposium) on The Concepts of Chemical Recognition in Biology held in Grignon near Versailles (France) on July 18–20, 1979.     

Organic syntheses from CH4−N2 atmospheres: Implications for Titan

Numerous experiments have already been performed, simulating the evolution of gaseous mixtures containing CH₄ when submitted to energy flux. From their results, it appears that a variety of organic compounds, including unsaturated hydrocarbons and nitriles such as HCN, can be synthesized into noticeable amounts from CH₄–N₂ mixtures. In particular, systematic studies of the influence of the composition of the mixture on the nature and amount of synthesized compounds show that organic volatile nitriles, and particularly cyanoacetylene and cyanogen, are formed only in media rich in nitrogen. Those nitriles have been identified very recently in the atmosphere of Titan, and thus, data from such laboratory experiments may provide important indirect information on the organic chemistry occuring at the periphery of this satellite of Saturn. However, during these experiments, there is a continuous formation and accumulation of molecular hydrogen, which does not occur in the atmosphere of Titan, because of H₂ escape. In order to reassess the data already available from this type of laboratory studies, experiments on CH₄–N₂ atmospheres, with and without H2 escape, have been recently performed. The influence of this parameter on the chemical evolution of the atmosphere and on the nature and relative quantities of organic compounds has been studied.After reviewing these experiments, implications of the obtained results on the organic chemistry at the periphery of Titan are discussed.Paper presented at the 6th College Park Colloquium, October 1981.     

Enhanced Synthesis of Alkyl Amino Acids in Miller’s 1958 H<Subscript>2</Subscript>S Experiment

Stanley Miller’s 1958 H₂S-containing experiment, which included a simulated prebiotic atmosphere of methane (CH₄), ammonia (NH₃), carbon dioxide (CO₂), and hydrogen sulfide (H₂S) produced several alkyl amino acids, including the α-, β-, and γ-isomers of aminobutyric acid (ABA) in greater relative yields than had previously been reported from his spark discharge experiments. In the presence of H₂S, aspartic and glutamic acids could yield alkyl amino acids via the formation of thioimide intermediates. Radical chemistry initiated by passing H₂S through a spark discharge could have also enhanced alkyl amino acid synthesis by generating alkyl radicals that can help form the aldehyde and ketone precursors to these amino acids. We propose mechanisms that may have influenced the synthesis of certain amino acids in localized environments rich in H₂S and lightning discharges, similar to conditions near volcanic systems on the early Earth, thus contributing to the prebiotic chemical inventory of the primordial Earth.