Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Role of geochemical protoenzymes (geozymes) in primordial metabolism: specific abiotic hydride transfer by metals to the biological redox cofactor NAD.

Hydrogen gas, H2 , is generated in serpentinizing hydrothermal systems, where it has supplied electrons and energy for microbial communities since there was liquid water on Earth. In modern metabolism, H2 is converted by hydrogenases into organically bound hydrides (H- ), for example, the cofactor NADH. It transfers hydrides among molecules, serving as an activated and biologically harnessed form of H2 . In serpentinizing systems, minerals can also bind hydrides and could, in principle, have acted as inorganic hydride donors-possibly as a geochemical protoenzyme, a 'geozyme'- at the origin of metabolism. To test this idea, we investigated the ability of H2 to reduce NAD+ in the presence of iron (Fe), cobalt (Co) and nickel (Ni), metals that occur in serpentinizing systems. In the presence of H2 , all three metals specifically reduce NAD+ to the biologically relevant form, 1,4-NADH, with up to 100% conversion rates within a few hours under alkaline aqueous conditions at 40 °C. Using Henry's law, the partial pressure of H2 in our reactions corresponds to 3.6 mm, a concentration observed in many modern serpentinizing systems. While the reduction of NAD+ by Ni is strictly H2 -dependent, experiments in heavy water (2 H2 O) indicate that native Fe can reduce NAD+ both with and without H2 . The results establish a mechanistic connection between abiotic and biotic hydride donors, indicating that geochemically catalysed, H2 -dependent NAD+ reduction could have preceded the hydrogenase-dependent reaction in evolution.

Energy at Origins: Favorable Thermodynamics of Biosynthetic Reactions in the Last Universal Common Ancestor (LUCA).

Though all theories for the origin of life require a source of energy to promote primordial chemical reactions, the nature of energy that drove the emergence of metabolism at origins is still debated. We reasoned that evidence for the nature of energy at origins should be preserved in the biochemical reactions of life itself, whereby changes in free energy, ΔG, which determine whether a reaction can go forward or not, should help specify the source. By calculating values of ΔG across the conserved and universal core of 402 individual reactions that synthesize amino acids, nucleotides and cofactors from H2, CO2, NH3, H2S and phosphate in modern cells, we find that 95-97% of these reactions are exergonic (ΔG ≤ 0 kJ⋅mol-1) at pH 7-10 and 80-100°C under nonequilibrium conditions with H2 replacing biochemical reductants. While 23% of the core's reactions involve ATP hydrolysis, 77% are ATP-independent, thermodynamically driven by ΔG of reactions involving carbon bonds. We identified 174 reactions that are exergonic by -20 to -300 kJ⋅mol-1 at pH 9 and 80°C and that fall into ten reaction types: six pterin dependent alkyl or acyl transfers, ten S-adenosylmethionine dependent alkyl transfers, four acyl phosphate hydrolyses, 14 thioester hydrolyses, 30 decarboxylations, 35 ring closure reactions, 31 aromatic ring formations, and 44 carbon reductions by reduced nicotinamide, flavins, ferredoxin, or formate. The 402 reactions of the biosynthetic core trace to the last universal common ancestor (LUCA), and reveal that synthesis of LUCA's chemical constituents required no external energy inputs such as electric discharge, UV-light or phosphide minerals. The biosynthetic reactions of LUCA uncover a natural thermodynamic tendency of metabolism to unfold from energy released by reactions of H2, CO2, NH3, H2S, and phosphate.

Pyrophosphate and Irreversibility in Evolution, or why PPi Is Not an Energy Currency and why Nature Chose Triphosphates.

The possible evolutionary significance of pyrophosphate (PPi) has been discussed since the early 1960s. Lipmann suggested that PPi could have been an ancient currency or a possible environmental source of metabolic energy at origins, while Kornberg proposed that PPi vectorializes metabolism because ubiquitous pyrophosphatases render PPi forming reactions kinetically irreversible. To test those ideas, we investigated the reactions that consume phosphoanhydride bonds among the 402 reactions of the universal biosynthetic core that generates amino acids, nucleotides, and cofactors from H2, CO2, and NH3. We find that 36% of the core's phosphoanhydride hydrolyzing reactions generate PPi, while no reactions use PPi as an energy currency. The polymerization reactions that generate ~80% of cell mass - protein, RNA, and DNA synthesis - all generate PPi, while none use PPi as an energy source. In typical prokaryotic cells, aminoacyl tRNA synthetases (AARS) underlie ~80% of PPi production. We show that the irreversibility of the AARS reaction is a kinetic, not a thermodynamic effect. The data indicate that PPi is not an ancient energy currency and probably never was. Instead, PPi hydrolysis is an ancient mechanism that imparts irreversibility, as Kornberg suggested, functioning like a ratchet's pawl to vectorialize the life process toward growth. The two anhydride bonds in nucleoside triphosphates offer ATP-cleaving enzymes an option to impart either thermodynamic control (Pi formation) or kinetic control (PPi formation) upon reactions. This dual capacity explains why nature chose the triphosphate moiety of ATP as biochemistry's universal energy currency.

The Autotrophic Core: An Ancient Network of 404 Reactions Converts H2, CO2, and NH3 into Amino Acids, Bases, and Cofactors.

The metabolism of cells contains evidence reflecting the process by which they arose. Here, we have identified the ancient core of autotrophic metabolism encompassing 404 reactions that comprise the reaction network from H2, CO2, and ammonia (NH3) to amino acids, nucleic acid monomers, and the 19 cofactors required for their synthesis. Water is the most common reactant in the autotrophic core, indicating that the core arose in an aqueous environment. Seventy-seven core reactions involve the hydrolysis of high-energy phosphate bonds, furthermore suggesting the presence of a non-enzymatic and highly exergonic chemical reaction capable of continuously synthesizing activated phosphate bonds. CO2 is the most common carbon-containing compound in the core. An abundance of NADH and NADPH-dependent redox reactions in the autotrophic core, the central role of CO2, and the circumstance that the core's main products are far more reduced than CO2 indicate that the core arose in a highly reducing environment. The chemical reactions of the autotrophic core suggest that it arose from H2, inorganic carbon, and NH3 in an aqueous environment marked by highly reducing and continuously far from equilibrium conditions. Such conditions are very similar to those found in serpentinizing hydrothermal systems.

The ambivalent role of water at the origins of life.

Life as we know it would not exist without water. However, water molecules not only serve as a solvent and reactant but can also promote hydrolysis, which counteracts the formation of essential organic molecules. This conundrum constitutes one of the central issues in origin of life. Hydrolysis is an important part of energy metabolism for all living organisms but only because, inside cells, it is a controlled reaction. How could hydrolysis have been regulated under prebiotic settings? Lower water activities possibly provide an answer: geochemical sites with less free and more bound water can supply the necessary conditions for protometabolic reactions. Such conditions occur in serpentinising systems, hydrothermal sites that synthesise hydrogen gas via rock-water interactions. Here, we summarise the parallels between biotic and abiotic means of controlling hydrolysis in order to narrow the gap between biochemical and geochemical reactions and briefly outline how hydrolysis could even have played a constructive role at the origin of molecular self-organisation.

A hydrogen-dependent geochemical analogue of primordial carbon and energy metabolism.

Hydrogen gas, H2, is generated by alkaline hydrothermal vents through an ancient geochemical process called serpentinization, in which water reacts with iron-containing minerals deep within the Earth's crust. H2 is the electron donor for the most ancient and the only energy-releasing route of biological CO2 fixation, the acetyl-CoA pathway. At the origin of metabolism, CO2 fixation by hydrothermal H2 within serpentinizing systems could have preceded and patterned biotic pathways. Here we show that three hydrothermal minerals-greigite (Fe3S4), magnetite (Fe3O4) and awaruite (Ni3Fe)-catalyse the fixation of CO2 with H2 at 100 °C under alkaline aqueous conditions. The product spectrum includes formate (up to 200 mM), acetate (up to 100 µM), pyruvate (up to 10 µM), methanol (up to 100 µM) and methane. The results shed light on both the geochemical origin of microbial metabolism and the nature of abiotic formate and methane synthesis in modern hydrothermal vents.

Catalysts, autocatalysis and the origin of metabolism.

If life on Earth started out in geochemical environments like hydrothermal vents, then it started out from gasses like CO2, N2 and H2. Anaerobic autotrophs still live from these gasses today, and they still inhabit the Earth's crust. In the search for connections between abiotic processes in ancient geological systems and biotic processes in biological systems, it becomes evident that chemical activation (catalysis) of these gasses and a constant source of energy are key. The H2-CO2 redox reaction provides a constant source of energy and anabolic inputs, because the equilibrium lies on the side of reduced carbon compounds. Identifying geochemical catalysts that activate these gasses en route to nitrogenous organic compounds and small autocatalytic networks will be an important step towards understanding prebiotic chemistry that operates only on the basis of chemical energy, without input from solar radiation. So, if life arose in the dark depths of hydrothermal vents, then understanding reactions and catalysts that operate under such conditions is crucial for understanding origins.

Serpentinization: Connecting Geochemistry, Ancient Metabolism and Industrial Hydrogenation.

Rock⁻water⁻carbon interactions germane to serpentinization in hydrothermal vents have occurred for over 4 billion years, ever since there was liquid water on Earth. Serpentinization converts iron(II) containing minerals and water to magnetite (Fe3O4) plus H2. The hydrogen can generate native metals such as awaruite (Ni3Fe), a common serpentinization product. Awaruite catalyzes the synthesis of methane from H2 and CO2 under hydrothermal conditions. Native iron and nickel catalyze the synthesis of formate, methanol, acetate, and pyruvate-intermediates of the acetyl-CoA pathway, the most ancient pathway of CO2 fixation. Carbon monoxide dehydrogenase (CODH) is central to the pathway and employs Ni° in its catalytic mechanism. CODH has been conserved during 4 billion years of evolution as a relic of the natural CO2-reducing catalyst at the onset of biochemistry. The carbide-containing active site of nitrogenase-the only enzyme on Earth that reduces N2-is probably also a relic, a biological reconstruction of the naturally occurring inorganic catalyst that generated primordial organic nitrogen. Serpentinization generates Fe3O4 and H2, the catalyst and reductant for industrial CO2 hydrogenation and for N2 reduction via the Haber⁻Bosch process. In both industrial processes, an Fe3O4 catalyst is matured via H2-dependent reduction to generate Fe5C2 and Fe2N respectively. Whether serpentinization entails similar catalyst maturation is not known. We suggest that at the onset of life, essential reactions leading to reduced carbon and reduced nitrogen occurred with catalysts that were synthesized during the serpentinization process, connecting the chemistry of life and Earth to industrial chemistry in unexpected ways.

DNA photoreacts by nucleobase ring cleavage to form labile isocyanates.

Differential infrared absorption spectroscopy was used to study the formation of isocyanates and further photo-products in the oligonucleotides dG10, dC10 and dT10 and in their mononucleosides by ultraviolet light at 266 nm. We find that α-cleavage takes place in oligonucleotides and mononucleosides both in films and in solution. The very intense and spectrally isolated isocyanate (N=C=O) asymmetric stretch vibration at 2277 cm(-1) is used as a spectroscopic marker for detection of the photo-product. The band disappears upon reaction with small amounts of water vapour as expected for isocyanates. Quantum yields for isocyanate formation by nucleobase ring cleavage in the α-position to the carbonyl group are ∼5 × 10(-5) in the mononucleosides and up to 5 × 10(-4) in the oligonucleotides. In the mixed oligonucleotides dG10/dC10 and dA10/dT10 the quantum yield of α-cleavage drops by a factor of 10 compared to the single oligonucleotides. Implications for DNA repair and photo-induced DNA-protein cross-linking via isocyanate reaction with NH2 groups of amino acids are discussed.

Isomer-selective vibrational spectroscopy of jet-cooled phenol-acetylene aggregates.

The structures of the phenol (Ph)-acetylene (A) clusters PhA1,2,3 and Ph2A1 are assigned on the basis of isomer and mass specific IR-UV double resonance spectroscopy and compared to the structure of the PhA cocrystal. The structures of the PhA1,2,3 clusters are dominated by phenol-acetylene π-hydrogen bonds whereas Ph2A1 binds via OH···OH···C≡C interaction with dominating Ph-Ph hydrogen bond like in the phenol dimer and acetylene attached to the free OH group of the proton acceptor phenol. The macroscopic crystal is a clathrate of phenol with acetylene with hydrogen bridges only between the phenol molecules and not between phenol and acetylene. (1) A possible aggregation pathway is proposed in which larger phenol clusters like Ph6 are cyclic with no free OH available anymore to which acetylene could attach as proton acceptor.

UV-light-induced hydrogen transfer in guanosine-guanosine aggregates.

Aggregates of a lipophilic guanine (G) derivative have been studied in n-hexane by femtosecond-to-microsecond UV-visible broadband transient absorption, stationary infrared and UV-visible spectroscopy and by quantum chemical calculations. We report the first time-resolved spectroscopic detection of hydrogen transfer in GG aggregates, which leads to (G-H)(·) radicals by means of G(+)G(-) charge transfer followed by proton transfer. These radicals show a characteristic electronic spectrum in the range 300-550 nm. The calculated superimposed spectrum of the species that result from NH⋅⋅⋅N proton transfer agrees best with the experimental spectrum.

Acetylation makes the difference: a joint experimental and theoretical study on low-lying electronically excited states of 9H-adenine and 9-acetyladenine.

Vibronic spectra of 9H-adenine, 9-acetyladenine and several alkyladenines have been recorded by resonant two-photon ionization spectroscopy of the laser-desorbed molecules, entrained in a molecular beam. While adenine and the alkyladenines exhibit similar electronic spectra, 9-acetyladenine behaves considerably different. Theoretical absorption spectra of 9H-adenine and 9-acetyladenine were calculated using the combined density functional theory/multi-reference configuration interaction approach and using second order coupled cluster theory, in order to explain striking differences in the experimental spectra. The major differences between the 9H-adenine and the 9-acetyladenine absorption spectra can be traced back to the different configurations, which contribute to the excitations, both of the lowest ππ* and the nπ* states. While the excitations in 9H-adenine are localized in the chromophore, they show considerable charge transfer character from the chromophore to the acetyl group in the case of 9-acetyladenine.

Towards a spectroscopic and theoretical identification of the isolated building blocks of the benzene-acetylene cocrystal.

Isomer- and mass-selective UV and IR-UV double resonance spectra of the BA3, B2A, and B2A2 clusters of benzene (B) and acetylene (A) are presented. Cluster structures are assigned by comparison with the UV and IR spectra of benzene, the benzene dimer, as well as the BA, BA2, and B2A clusters. The intermolecular vibrations of BA are identified by dispersed fluorescence spectroscopy. Assignment of the cluster structures is supported by quantum chemical calculations of IR spectra with spin-component scaled second-order Møller-Plesset (SCS-MP2) theory. Initial propositions for various structures of the BA3 and B2A2 aggregates are generated with model potentials based on density functional theory combined with the symmetry-adapted perturbation theory (DFT-SAPT) approach. Shape and relative cluster stabilities are then confirmed with SCS-MP2. T-shaped geometries are the dominant structural motifs. Higher-energy isomers are also observed. The detected cluster structures are correlated with possible cluster formation pathways and their role as crystallization seeds is discussed.

Characterization of maleimide dimers in photo-cross-linked copolyimide films.

Copolyimide membranes are established materials for the separation of gaseous and liquid mixtures. Cross-linking of the polymer strands improves the physical and chemical stability. The photo-cross-linking of a 6FDA-ODA/6FDA-DABA 4 : 1 copolyimide membrane containing maleimide side groups as linker was investigated by FTIR spectroscopy. IR absorption spectra of the copolyimide backbone, 3-hydroxypropyldimethyl maleimide and the copolyimide functionalized with 3-hydroxypropyldimethyl maleimide were measured before and after different irradiation times and compared to each other. For band assignment a normal mode analysis was performed. The backbone of the polymer and the maleimide linker can be well distinguished due to their different spectral band positions. Only the films containing a maleimide moiety perform a photoreaction, the polymer backbone does not interfere. Based on the difference spectra and the results of the DFT calculations it was shown that the trans- and the cis-cycloadduct as well as the previously suggested 2-2'-adduct without a cyclobutane ring are formed upon UV irradiation. Evidence for an oxetane-like photoproduct was not found. Different time constants for the increase of the product bands were observed. The cycloadduct accumulates with a shorter time constant (τ = 2 to 5 min) than the 2-2'-adduct (τ = 75 min). The yield of the photo-cross-linking reaction was determined by spectral deconvolution and kinetic fitting of several marker bands. For the copolyimide synthesized in this work, a maximum value of 6% was reached. The stiffness of the copolyimide backbone inhibits further photo-cross-linking.

Phenothiazinyl rhodanylidene merocyanines for dye-sensitized solar cells.

Phenothiazinyl rhodanylidene acetic acid merocyanine dyes with variable substitution pattern on the peripheral benzene ring were synthesized in good to excellent yields by Knoevenagel condensation of the corresponding phenothiazinyl aldehydes and rhodanine-N-acetic acid. The electronic properties were investigated by cyclic voltammetry, absorption, and fluorescence spectroscopy. Electron releasing substitution leads to an appreciable lowering of the oxidation potential, bathochromic shift of the absorption band, and minimization of the emission quantum yield. Not least as a consequence of these properties, the compounds are interesting for use as chromophores in dye-sensitized solar cells (DSSC). DSSCs were constructed and successfully tested by determining the characteristic parameters such as incident-photon-to-electron conversion efficiency (IPCE), fill factor (FF), and overall efficiency.

Investigation of cross-linked and additive containing polymer materials for membranes with improved performance in pervaporation and gas separation.

Pervaporation and gas separation performances of polymer membranes can be improved by crosslinking or addition of metal-organic frameworks (MOFs). Crosslinked copolyimide membranes show higher plasticization resistance and no significant loss in selectivity compared to non-crosslinked membranes when exposed to mixtures of CO2/CH4 or toluene/cyclohexane. Covalently crosslinked membranes reveal better separation performances than ionically crosslinked systems. Covalent interlacing with 3-hydroxypropyldimethylmaleimide as photocrosslinker can be investigated in situ in solution as well as in films, using transient UV/Vis and FTIR spectroscopy. The photocrosslinking yield can be determined from the FTIR-spectra. It is restricted by the stiffness of the copolyimide backbone, which inhibits the photoreaction due to spatial separation of the crosslinker side chains. Mixed-matrix membranes (MMMs) with MOFs as additives (fillers) have increased permeabilities and often also selectivities compared to the pure polymer. Incorporation of MOFs into polysulfone and Matrimid® polymers for MMMs gives defect-free membranes with performances similar to the best polymer membranes for gas mixtures, such as O2/N2 H2/CH4, CO2/CH4, H2/CO2, CH4/N2 and CO2/N2 (preferentially permeating gas is named first). The MOF porosity, its particle size and content in the MMM are factors to influence the permeability and the separation performance of the membranes.

Excited state proton transfer is not involved in the ultrafast deactivation of Guanine-Cytosine pair in solution.

Different derivatives of Guanine (G) and Cytosine (C), which sterically enforce the Watson-Crick (WC) conformer, have been studied in CHCl(3) by means of broad-band transient absorption spectroscopy. Our experiments rule out the involvement of an Excited State Proton Transfer (ESPT), which dominates the excited state decay of GC in the gas phase. Instead, the ultrafast dynamics via internal conversion occurs in a polar environment mainly by relaxation in the monomer moieties. Time-dependent density functional theory (TD-DFT) calculations in solution indeed indicate that population transfer from the bright excited states toward the charge transfer state is not effective in CHCl(3) and a noticeable energy barrier is associated with the ESPT reaction. ESPT is therefore not expected to be a main deactivation route for GC pairs within DNA.

IR spectroscopy of isolated neutral and protonated adenine and 9-methyladenine.

IR spectroscopy is employed to study isolated adenine and its derivative 9-methyladenine in both their neutral and protonated forms. The IR spectra of neutral adenine and 9-methyladenine are measured in a molecular beam expansion via IR-UV ion-dip spectroscopy in the 525 to 1750 cm(-1) region. For adenine, UV excitation selects the 9H tautomer to give a conformer-selective IR spectrum. For 9-methyladenine, only one tautomer exists because of the methyl substitution at the N(9) position. The experimental spectra agree closely with spectra computed for these tautomers at the B3LYP/6-311++G(df,pd) level of theory. These spectra complement previous tautomer-specific IR spectra in the hydrogen stretching range. The 9H-adenine spectrum obtained is compared to a previously recorded FTIR spectrum of adenine at 280 °C, which shows close agreement, although the 7H tautomer cannot be excluded from contributing. Protonated adenine and 9-methyladenine are generated by electrospray ionization and studied via IR multiple-photon dissociation (IRMPD) spectroscopy. Comparison of the experimental spectra with computed spectra allows identification of the protonation site, which suggests that the 1-9 tautomer is the dominant contributor to the spectra.

Fourier transform infrared spectroscopy of 2'-deoxycytidine aggregates in CDCl3 solutions.

We investigated the self-aggregation of 2'-deoxy-3',5'-bis(tert-butyldimethylsilyl)-cytidine dC(TBDMS)(2) in CDCl(3) solutions by Fourier transform infrared (FT-IR) spectroscopy and report the formation of larger aggregates than dimers in this solvent for the first time. The hydrogen bonding patterns in these complexes, which occur with increasing concentration may serve as a model for DNA super-structures such as triplexes. From the IR spectra, wavelength dependent absolute extinction coefficients of the monomer, dimer as well as a contribution from larger clusters which are supposedly trimers are deduced on the basis of a simple deconvolution method. Our results are supported by RI-B3LYP/TZVP calculations within the conductorlike screening model framework, to account for solvent effects in the ab initio calculations.