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.     

Bolide impacts and the oxidation state of carbon in the Earth's early atmosphere

A one-dimensional photochemical model was used to examine the effect of bolide impacts on the oxidation state of Earth's primitive atmosphere. The impact rate should have been high prior to 3.8 Ga before present, based on evidence derived from the Moon. Impacts of comets or carbonaceous asteroids should have enhanced the atmospheric CO/CO₂ ratio by bringing in CO ice and/or organic carbon that can be oxidized to CO in the impact plume. Ordinary chondritic impactors would contain elemental iron that could have reacted with ambient CO₂ to give CO. Nitric oxide (NO) should also have been produced by reaction between ambient CO₂ and N₂ in the hot impact plumes. High NO concentrations increase the atmospheric CO/CO₂ ratio by increasing the rainout rate of oxidized gases. According to the model, atmospheric CO/CO₂ ratios of unity or greater are possible during the first several hundred million years of Earth's history, provided that dissolved CO was not rapidly oxidized to bicarbonate in the ocean. Specifically, high atmospheric CO/CO₂ ratios are possible if either: (1) the climate was cool (like today's climate), so that hydration of dissolved CO to formate was slow, or (2) the formate formed from CO was efficiently converted into volatile, reduced carbon compounds, such as methane. A high atmospheric CO/CO₂ ratio may have helped to facilitate prebiotic synthesis by enhancing the production rates of hydrogen cyanide and formaldehyde. Formaldehyde may have been produced even more efficiently by photochemical reduction of bicarbonate and formate in Fe⁺⁺-rich surface waters.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

Formation of amino acids from models of Titan and more oxidized atmospheres

Protein and non-protein amino acids were synthesized following hydrolysis of products obtained by high frequency discharge techniques applied to model atmospheres consisting of N₂ as a nitrogen source together with CH₄ and/or CO₂ as a carbon source. Highest yields were obtained in the absence of CO₂ and from mixtures rich in CH₄. Amino acids would indeed be expected on the frozen surface of Titan with its CH₄–N₂ atmosphere.Paper presented at the 6th College Park Colloquium, October 1981.     

CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.     

BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS : I. Protein Amino Acids

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with ¹⁴CO₂ for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of ¹⁴CO₂ into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with ¹⁴CO₂ in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.     

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.     

Light-induced accumulation of lactate and succinate in Anabaena cylindrica

In the cyanobacterium Anabaena cylindrica lactate accumulated in large amounts when the cells were exposed to light. The presence or absence of oxygen, or a change in CO₂ concentration did not affect the lactate accumulation. The cellular succinate level also increased in the light when CO₂ was supplied at the high concentration of 1%. 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), an inhibitor of photosynthetic electron flow, inhibited the increase in the concentration of lactate and succinate. Photosynthesis is a prerequisite for the increase of these organic acids. Thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase, inhibited the increase of succinate, suggesting that the succinate is formed via fumarate by the reverse of reactions of tricarboxylic acid (TCA) cycle. Upon addition of ammonium to the cell suspension in the light under high CO₂ concentration, the increases in the concentrations of lactate and succinate were inhibited while those of glutamine, glutamate and aspartate were stimulated. Ammonium apparently changed the products of metabolism of pyruvate and oxaloacetate from lactate and succinate to amino acids.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - TTFA thenoyltrifluoroacetone - PCA perchloric acid     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-¹⁴C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO₂), hypoxic hypoxia (15 or 10% O₂), and hypoxic hypoxia plus hypercarbia (15 or 10% O₂ with 5% CO₂). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO₂) decreased the incorporation of [U-¹⁴C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO₂) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O₂) or hypoxic hypoxia with 5% CO₂ were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

The Influence of CO2 on the Metabolism of Rhizome Tissue in Iris pseudacorus

In vitro experiments showed that concentrations of CO₂ above 6 per cent inhibited succinate oxidation; 30–10 per cent of succinate oxidation may be blocked in the presence of 12 per cent CO₂. Storage of rhizome cores in CO₂ increased the levels of all the main acids except malic. The amino acids, aspartate, glutamate and alanine, also increased in amounts under these conditions. Cores fix CO₂ probably via the reaction phosphoenolpyruvate to oxaloacetate. Raised CO₂ levels increased the rate of diminution of carbohydrate, but carbon itself was conserved due to a reduction in CO₂ output.     

Utilization of organic nitrogen compounds by Hydrogenomonas eutropha

The chemolithotroph, Hydrogenomonas eutropha, was tested for its ability to utilize a variety of single nitrogen sources during growth in an atmosphere of H₂? O₂? CO₂ The present data show that H. eutropha can utilize the nitrogen from many, but not all, amino acids, several sulfur-containing amino acids, glucosamine, and two aliphatic amides. The nitrogen concentration that supported maximum growth for NH₄Cl, L -glutamate, L -glutamine, urea, and glycine was in the 0.010–0.019M range. H. eutropha failed to remove the nitrogen from primary and secondary amines, eycloleucine, tert-DL -leucine, DL -p-fluorophenylalanine, DL -5-methyltryptophan, creatine, and creatine. This microorganism was able to partially degrade at least six substituted indoles and/or tryptophan catabolites and six substituted imidazoles and/or histidine catabolites. All of a series of 17 dipeptides were able to serve as a nitrogen source for growth in the absence of NH₄Cl. Extracts of H. eutropha were able to catalyze the hydrolysis of 16 α-dipeptides, 2 tripetides, a tetrapeptide, a polypeptide, a β-aspartyl peptide, 2 γ-glutamyl peptides, a N-acetyl amino acid, and 4 amino acid amides. These results emphasize the effectiveness of H. eutropha in utilizing a wide diversity of organic nitrogenous compounds containing amino and amide groups, heterocyclic rings, and peptide bonds.     

Oxalate metabolism by tobacco leaf discs

The turnover rate of oxalate in leaf discs of Nicotiana tabacum, var Havana Seed, during photosynthesis was estimated to be 1 to 2 micromoles per gram fresh weight per hour. Radioactivity from the enzymic oxidation of [¹⁴C]oxalate rapidly appeared in neutral sugars (mainly sucrose), organic acids (mainly malate), and amino acids. Only 5% of the radioactivity was released to the atmosphere as ¹⁴CO₂, and no formate or formaldehyde could be detected. The metabolism of oxalate was not increased by raising the O₂ concentration from 1% to 21% to 60%, nor was the formation of [¹⁴C]oxalate from [2-¹⁴C]glyoxylate changed under the same conditions as was previously observed in vitro (Havir 1983 Plant Physiol 71: 874-878). While oxalate is not an inert end product of the glycolate pathway, it contributes little to the formation of photorespiratory CO₂.     

In vivo utilization ofd(−)-3-hydroxybutyrate by chick brain and spinal cord

The in vivo utilization ofd-3-hydroxy[3-¹⁴C]butyrate for oxidation in the whole animal and for lipid and amino acid synthesis in brain and spinal cord of overnight-fasted 15-day-old chicks has been measured. Appreciable amounts of injected 3-hydroxy[3-¹⁴C]butyrate were expired as¹⁴CO₂ one hour after injection, the total amount of which increased with increasing dosages. Lipid synthesis was high in both brain and spinal cord. Free, cholesterol and phospholipids were the main lipids labeled in both, tissues, increasing with time after injection up to 120 min. The incorporation of radioactivity into triglycerides, esterified cholesterol and free fatty acids was not time-dependent. Increased concentrations of 3-hydroxybutyrate gave rise to higher synthetic rates both in brain and spinal cord The rate of amino acid synthesis was slightly higher in brain than in spinal cord. Glutamate was always the major amino acid formed.     

The synthesis of amino acids and sugars on an inorganic template from constituents of the prebiotic atmosphere

Inelastic Electron Tunnelling Spectroscopy (IETS) has been used to identify the reaction products present on an alumina surface when it is exposed to likely components of the earth's prebiotic atmosphere. The alumina barrier of Al-AlO _x -Pb tunnelling junctions have been exposed to water; aqueous ammonia; wet carbon monoxide gas and to aqueous formaldehyde vapour under normal atmospheric conditions at room temperature. The water spectrum shows strong coincidence with that of a genuine sample of formic acid. It is proposed that atmospheric CO₂ is involved in this surface catalyzed reaction. The aqueous ammonia spectrum is assigned as an amino acid species produced from ammonia, water and atmospheric carbon dioxide. This spectrum compares very closely with the tunnelling spectrum of a genuine sample of glycine. The wet carbon monoxide spectrum and the aqueous formaldehyde spectrum have been produced by an infusion doping process. These spectra of CO and aqueous formaldehyde are assigned as a sugar like polymer or a sugar formed on the alumina surface. A tunnelling spectrum of D(–) fructose has been produced to aid this assignment. The role of an inorganic template such as alumina in the original prebiotic synthesis of amino acids and sugars is considered.     

Carbon Dioxide Fixation in the Carbon Economy of Developing Seeds of Lupinus albus (L.)

The effects of CO₂ concentration and illumination on net gas exchange and the pathway of ¹⁴CO₂ fixation in detached seeds from developing fruits of Lupinus albus (L.) have been studied.

Increasing the CO₂ concentration in the surrounding atmosphere (from 0.03 to 3.0% [v/v] in air) decreased CO₂ efflux by detached seeds either exposed to the light flux equivalent to that transmitted by the pod wall (500 to 600 micro-Einsteins per square meter per second) in full sunlight or held in darkness. Above 1% CO₂ detached seeds made a net gain of CO₂ in the light (up to 0.4 milligrams of CO₂ fixed per gram fresh weight per hour) but ¹⁴CO₂ injected into the gas space of intact fruits (containing around 1.5% CO₂ naturally) was fixed mainly by the pod and little by the seeds.

Throughout development seeds contained ribulose-1,5-bisphosphate carboxylase activity (EC 4.1.1.39), especially in the embryo (up to 99 micromoles of CO₂ fixed per gram fresh weight per hour) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) in both testa (up to 280 micromoles of CO₂ fixed per gram fresh weight per hour) and embryo (up to 355 micromoles of CO₂ fixed per gram fresh weight per hour).

In kinetic experiments the most significant early formed product of ¹⁴CO₂ fixation in both light and dark was malate but in the light phosphoglyceric acid and sugar phosphates were also rapidly labeled. ¹⁴CO₂ fixation in the light was linked to the synthesis of sugars and amino acids but in the dark labeled sugars were not formed.

     

Response of amino acid changes in Aphis gossypii (Glover) to elevated CO2 levels

Effects of elevated CO₂ levels on the amino acid constituents of cotton aphid, Aphis gossypii (Glover), fed on transgenic Bacillus thuringiensis (Berliner) (Bt) cotton [Cryl A(c)], grown in ambient and double‐ambient CO₂ levels in closed‐dynamics CO₂ chambers, were investigated. Lower amounts of amino acids were found in cotton phloem under elevated CO₂ than under ambient CO₂ levels. However, higher amounts of free amino acids were found in A. gossypii fed on elevated CO₂‐grown cotton than those fed ambient CO₂‐grown cotton, and the contents of amino acids in honeydew were not significantly affected by elevated CO₂ levels. A larger amount of honeydew was produced by cotton aphids feeding on leaves under elevated CO₂ treatment than those feeding on leaves under ambient CO₂ treatment, which indicates that A. gossypii ingests more cotton phloem because of the higher C:N ratio of cotton phloem under elevated CO₂ levels. Moreover, the amino acid composition was similar in bodies of aphids ingesting leaves under both CO₂ treatments, except for two alkaline amino acids, lysine and arginine. This suggests that the nutritional constitution of the phloem sap was important for A. gossypii. Our data suggest that more phloem sap will be ingested by A. gossypii to satisfy its nutritional requirement and balance the break‐even point of amino acid in elevated CO₂. Larger amounts of honeydew produced by A. gossypii under elevated CO₂ will reduce the photosynthesis and result in the occurrence of some Entomophthora spp.     

Differential effects of drought and light levels on accumulation of citric and malic acids during CAM in Clusia

Night-time citrate accumulation has been proposed as a response to stress in CAM plants. To address this hypothesis, gas exchange patterns and nocturnal acid accumulation in three species of Clusia were investigated under controlled conditions with regard to water stress and responses to low and high photosynthetic photon flux density (PPFD). Under high PPFD, leaves of Clusia nocturnally accumulated large amounts of both malic and citric acids. Under low PPFD and well-watered conditions, substantial night-time citrate accumulation persisted, whereas malate accumulation was close to zero. Malate accumulation and night-time CO₂ uptake from the atmosphere declined in all three species during prolonged drought periods, whereas citrate accumulation remained similar or increased. Recycling of respiratory CO₂ was substantial for both well-watered and water-stressed plants. The suggestion that citrate accumulation is energetically more favourable than malate accumulation is not supported if the source of CO₂ for the formation of malate is respiratory CO₂. However, the breakdown of citric acid to pyruvate in the light period releases three molecules of CO₂, while the breakdown of malic acid releases only one CO₂ per pyruvate formed. Thus, citric acid should be more effective than malic acid as a mechanism to increase CO₂ concentration in the mesophyll and may help to prevent photoinhibition. Organic acid accumulation also affected the vacuolar pH, which reached values of 2·6–3·0 at dawn. At these pH values, the transport of 2H⁺/ATP is still feasible, suggesting that it is the divalent form of citrate which is being transported in the vacuoles. Since citrate is a well-known buffer, and Clusia spp. show the largest day-night changes in organic acid levels measured in any CAM plant, it is possible that citrate increases the buffer capacity of the vacuoles. Indeed, malate and titratable acidity levels are positively related to citrate levels. Moreover, Clusia species that show the highest nocturnal accumulation of organic acids are also the ones that show the greatest changes in citric acid levels.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.