The Cold Origin of Life: A. Implications Based On The Hydrolytic Stabilities Of Hydrogen Cyanide And Formamide

It has been suggested that hydrogen cyanide(HCN) would not have been present in sufficient concentrationto polymerize in the primitive ocean to produce nucleic acidbases and amino acids. We have measured the hydrolysis ratesof HCN and formamide over the range of 30–150 °C and pH 0–14,and estimated the steady state concentrations in theprimitive ocean. At 100 °C and pH 8, the steady stateconcentration of HCN and formamide were calculated to be7 × 10^-13 M and 1 × 10^-15 M, respectively. Thus, itseems unlikely that HCN could have polymerized in a warmprimitive ocean. It is suggested that eutectic freezing mighthave been required to have concentrated HCN sufficiantly forit to polymerize. If the HCN polymerization was important forthe origin of life, some regions of the primitive earth mighthave been frozen.     

Chemical evolution XXIX. Pyrimidines from hydrogen cyanide.

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 8-9. Hydrolysis of these oligomers at pH 8.5 or with 6 N HCl yields 4,5-dihydroxypyrimidine, as the most abundant pyrimidine product along with orotic acid and 5-hydroxyuracil. These results, together with the earlier data, demonstrate that the three major nitrogen-containing classes of biomolecules could have originated from HCN on the primitive earth. The observation of the formation of orotic acid and 4-aminoimidazole-5-carboxamide by the hydrolysis of the HCN oligomers suggests that once the initially formed pyrimidines and purines were consumed, those life forms persisted which evolved enzymes for conversion of these intermediates to the pyrimidines and purines present in contemporary RNA.     

HCN: A plausible source of purines,pyrimidines and amino acids on the primitive earth

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 9.2. Hydrolysis of these oligomers yields 4,5-dihydroxypyrimidine, orotic acid, 5-hydroxyuracil, adenine, 4-aminoimidazole-5-carboxamide and amino acids. These results, together with the earlier data, demonstrate that the three main classes of nitrogen-containing biomolecules, purines, pyrimidines and amino acids may have originated from HCN on the primitive earth. The observation of orotic acid and 4-aminoimidazole-5-carboxyamide suggests that the contemporary biosynthetic pathways for nucleotides may have evolved from the compounds released on hydrolysis of HCN oligomers.     

Far UV irradiation of model prebiotic atmospheres

UV light has been the most important energy source on the primitive Earth. However, very few experiments have been performed to test directly the possible role of this energy source on the chemical evolution of the primitive atmosphere, mainly on account of experimental difficulties. Experiments are generally performed with other excitations, mainly electric discharge, and it is frequently assumed that UV irradiation would give similar results.As theoretical considerations make this assumption questionable, direct experimental controls have been undertaken: Model primitive atmospheres have been submitted to 147 nm UV light and the gaseous phase has been analysed. Preliminary qualitative results concerning CH₄–NH₃ atmospheres are reported.Irradiation of pure CH₄ gives rise to the synthesis of a large number of hydrocarbons, mainly saturated hydrocarbons but including also unsaturated ones as, C₂H₂, C₂H₄, C₃H₆, C₃H₄. These insaturated hydrocarbons are synthetized at a very low rate when ammonia is present in the medium.Irradiations of CH₄–NH₃ mixtures give rise, in addition to hydrocarbons, to important amounts of HCN (about 0.1%) and to lesser amounts of CH₃CN and C₂H₅CN. No unsaturated nitriles such as acrylonitrile and cyanoacetylene have been detected. Search for amines is in progress.These results evidence that UV irradiation may contribute largely to synthesis of HCN in CH₄–NH₃ atmospheres and, consequently to the synthesis of many biochemical compounds that can be derivated from HCN. However, synthesis of other compounds, such as pyrimidines, which can derivate from other nitriles, such as cyanoacetylene, cannot be initiated only by UV light, contrary to electric discharges. In addition, if electric discharges are very efficient for synthesis of nitriles in CH₄–N₂ atmospheres, there is not yet evidence that UV light is able to do so.Presented at the 2nd ISSOL Meeting and the 5th ICOL in Kyoto, 5–10 April, 1977.     

Origin of terrestrial polypeptides: A theory based on data from discharge-tube experiments

Polymeric materials derived from HCN have been synthesized from reactants containing only carbon, hydrogen and nitrogen, as the solid product formed at high temperature on the walls of a discharge tube, and at room temperature from the gaseous products of that discharge condensed in a cold trap and allowed to warm up in the dark. These compounds were hydrolyzed with acid, and when possible with alkali. Amphoteric molecules were separated from the hydrolysate and examined for amino acids by GLC, after preparation of the TAB derivative. In all cases where nitrogenous solids were hydrolyzed, many natural and a few synthetic amino acids were formed, while blank runs indicated no trace of amino acids under the same treatment. These results have been used to show that many previous experiments in which amino acids have been synthesized from supposed ‘prebiological’ atmospheres have probably all involved the same general reaction steps.

1. Formation of HCN from high-energy reaction of C?H?N(?O) systems.
2. Radical or ionic polymerization or oligomerization of HCN.
3. Hydrolysis of the product.

A new theory for the origin of proteins on the primitive earth has been described in the light of these experimental results. It is suggested that high molecular weight aliphatic and aromatic hydro carbons and polymers derived from HCN are formed in the methane-ammonia atmosphere, and settle on the Earth's surface before the formation of oceans. Rainstorms wash the materials into alkaline pools, where the polymer is hydrolyzed in the presence of other materials necessary for the formation of replicating polypeptides (aromatic hydrocarbons, purines and pyrimidines and possibly phosphate ion). The close similarity between the products of discharge experiments and organic materials found in carbonaceous chondrites has also been discussed.     

Rates of fixation by lightning of carbon and nitrogen in possible primitive atmospheres

A thermochemical-hydrodynamic model of the production of trace species by electrical discharges has been used to estimate the rates of fixation of C and N by lightning in the primitive atmosphere. Calculations for various possible mixtures of CH₄, CO₂, N₂, H₂, and H₂O reveal that the prime species produced were probably HCN and NO and that the key parameter determining the rates of fixation was the ratio of C atoms to O atoms in the atmosphere. Atmospheres with C more abundant than O have large HCN fixation rates, in excess of 10¹⁷ molecules J^–1, but small NO yields. However, when O is more abundant than C, the NO fixation rate approaches 10¹⁷ molecules J^–1 while the HCN yield is small. The implications for the evolution of life are discussed.     

Uracil synthesisvia HCN oligomerization

Uracil is released from HCN oligomers upon acid hydrolysis in concentrations of 0.001% for 1M HCN solutions to 0.005% for 0.1M solutions. This yield is comparable with earlier reported, minor or nonbiological pyrimidines such as 5-hydroxyuracil and orotic acid. This is the first report of uracil itselfvia HCN oligomerization. Data are presented which establish that the observed uracil is not formed by decarboxylation of previously formed orotic acid, butvia acid hydrolysis of at least two other precursors.     

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.     

Uracil synthesis via HCN oligomerization

Uracil is released from HCN oligomers upon acid hydrolysis in concentrations of 0.001% for 1 M HCN solutions to 0.005% for 0.1 M solutions. This yield is comparable with earlier reported, minor or nonbiological pyrimidines such as 5-hydroxyuracil and orotic acid. This is the first report of uracil itself via HCN oligomerization. Data are presented which establish that the observed uracil is not formed by decarboxylation of previously formed orotic acid, but via acid hydrolysis of at least two other precursors.     

Biomolecules from HCN

The mechanism of the condensation of dilute aqueous solutions of HCN and the products formed by these reactions have been investigated. The initial HCN condensation reactions yield3, a compound which is readily oxidized to4. A similar oxidation of5 to6 was also observed. Urea is formed on hydrolysis of4. The oxidation-reduction products formed from HCN may be in part a consequence of the oxidation of3. It has been established by combination GC/MS that the amino acids glycine, diaminosuccinic acid, α-amino-isobutyric acid, aspartic acid, alanine and isoleucine are released on acid hydrolysis of the ‘HCN polymer’. Hydantoin (7), 5,5-dimethylhydantoin (8) and 5-carboxymethyldenehydantoin (10) are also released on acid hydrolysis of the HCN condensation products. The direct conversion of the dicarbonyl derivative, of diaminosuccinic acid to orotic acid via10 at pH 8 has been observed. This conversion suggests a direct route to pyrimidines from HCN.     

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.     

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: Implications for the nature of the first genetic material

Summary Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system.Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template.Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3×10^–6 M), as well as the difficulty of prebiotic activation of phosphates.     

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 X 10(-8) moles cal-1.     

Rates of fixation by lightning of carbon and nitrogen in possible primitive atmospheres

A thermochemical-hydrodynamic model of the production of trace species by electrical discharges has been used to estimate the rates of fixation of C and N by lightning in the primitive atmosphere. Calculations for various possible mixtures of CH4, CO2, CO, N2, H2, and H2O reveal that the prime species produced were probably HCN and NO and that the key parameter determining the rates of fixation was the ratio of C atoms to O atoms in the atmosphere. Atmospheres with C more abundant than O have large HCN fixation rates, in excess of 10(17) molecules J-1, but small NO yields. However, when O is more abundant than C, the NO fixation rate approaches 10(17) molecules J-1 while the HCN yield is small. The implications for the evolution of life are discussed.     

Energy yields for hydrogen cyanide and formaldehyde syntheses: The hcn and amino acid concentrations in the primitive ocean

Prebiotic electric discharge and ultraviolet light experiments are usually reported in terms of carbon yields and involve a large input of energy to maximize yields. Experiments using lower energy inputs are more realistic prebiotic models and give energy yields which can be used to estimate the relative importance of the different energy sources on the primitive earth. Simulated prebiotic atmospheres containing either CH₄, CO or CO₂ with N₂, H₂O and variable amounts of H₂ were subjected to the spark from a high frequency Tesla coil. The energy yields for the synthesis of HCN and H₂CO were estimated. CH₄ mixtures give the highest yields of HCN while H₂CO is most efficiently produced with the CO mixtures. These results are a model for atmospheric corona discharges, which are more abundant than lightning and different in character. Preliminary experiments using artificial lightning are also reported. The energy yields from these experiments combined with the corona discharge available on the earth, allows a yearly production rate to be estimated. These are compared with other experiments and model calculations. From these production rates of HCN (e.g. 100 nmoles cm⁻² yr⁻¹) and the experimental hydrolysis rates, the steady state concentration in the primitive ocean can be calculated (e.g., 4 × 10⁻⁶ M at pH 8 and 0°). A steady state amino acid concentration of 3 × 10⁻⁴ M is estimated from the HCN production rate and the rate of decomposition of the amino acids by passage through the submarine vents.     

Metabolism and motility in prebiotic structures

Easily accessible, primitive chemical structures produced by self-assembly of hydrophobic substances into oil droplets may result in self-moving agents able to sense their environment and move to avoid equilibrium. These structures would constitute very primitive examples of life on the Earth, even more primitive than simple bilayer vesicle structures. A few examples of simple chemical systems are presented that self-organize to produce oil droplets capable of movement, environment remodelling and primitive chemotaxis. These chemical agents are powered by an internal chemical reaction based on the hydrolysis of an oleic anhydride precursor or on the hydrolysis of hydrogen cyanide (HCN) polymer, a plausible prebiotic chemistry. Results are presented on both the behaviour of such droplets and the surface-active properties of HCN polymer products. Such motile agents would be capable of finding resources while escaping equilibrium and sustaining themselves through an internal metabolism, thus providing a working chemical model for a possible origin of life.     

HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were orginally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as I_f, I_Q or I_h. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50–70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.     

Role of Ferrocyanides in the Prebiotic Synthesis of α-Amino Acids

We investigated the synthesis of α-amino acids under possible prebiotic terrestrial conditions in the presence of dissolved iron (II) in a simulated prebiotic ocean. An aerosol-liquid cycle with a prebiotic atmosphere is shown to produce amino acids via Strecker synthesis with relatively high yields. However, in the presence of iron, the HCN was captured in the form of a ferrocyanide, partially inhibiting the formation of amino acids. We showed how HCN captured as Prussian Blue (or another complex compound) may, in turn, have served as the HCN source when exposed to UV radiation, allowing for the sustained production of amino acids in conjunction with the production of oxyhydroxides that precipitate as by-products. We conclude that ferrocyanides and related compounds may have played a significant role as intermediate products in the prebiotic formation of amino acids and oxyhydroxides, such as those that are found in iron-containing soils and that the aerosol cycle of the primitive ocean may have enhanced the yield of the amino acid production.     

Abiotic synthesis of purines and other heterocyclic compounds by the action of electrical discharges

Summary The synthesis of purines and pyrimidines using Oparin-Urey-type primitive Earth atmospheres has been demonstrated by reacting methane, ethane, and ammonia in electrical discharges. Adenine, guanine, 4-aminoimidazole-5-carboxamide (AICA), and isocytosine have been identified by UV spectrometry and paper chromatography as the products of the reaction. The total yields of the identified heterocyclic compounds are 0.0023%. It is concluded that adenine synthesis occurs at a much lower concentration of hydrogen cyanide than has been shown by earlier studies. Pathways for the synthesis of purines from hydrogen cyanide are discussed, and a comparison of the heterocyclic compounds that have been identified in meteorites and in prebiotic reactions is presented.     

Heteropolypeptides on Titan?

Since hydrogen cyanide is a component of Titan's hazy atmosphere, HCN polymers might also be present by way of a low energy pathway leading initially to the synthesis of polyaminomalonitrile. Subsequent reactions of HCN with the activated nitrile groups of this HCN homopolymer would then yield heteropolyamidines, readily converted to heteropolypeptides following contact with frozen water on the surface of Titan.Similar HCN polymers in the reducing atmospheres of Jupiter and Saturn could be major contributors to the yellow-brown-orange appearance of these giant planets.Any detection of such HCN chemistry by the Voyager missions or the pending Galileo probe would constitute evidence for the hypothesis that heteropolypeptides on the primitive Earth were synthesized directly from hydrogen cyanide and water without the intervening formation of -amino acids.Paper presented at the 6th College Park Colloquium, October 1981.