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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A dynamic protein interactome drives energy conservation and electron flux in Thermococcus kodakarensis

ABSTRACT Life is supported by energy gains fueled by catabolism of a wide range of substrates, each reliant on the selective partitioning of electrons through redox ( red uction and ox idation) reactions. Electron flux through tunable and regulated protein interactions provides dynamic routes for energy conservation, but how electron flux is regulated in vivo , particularly for archaeal metabolisms that support rapid growth at the thermodynamic limits of life, is poorly understood. Identification of bona fide in vivo protein assemblies and how such assemblies dictate the totality of electron flux is critical to our understanding of the regulation imposed on metabolism, energy production, and energy conservation. Here, 25 key proteins in central metabolic redox pathways in the model, genetically accessible, hyperthermophilic archaeon Thermococcus kodakarensis , were purified to reveal an extensive, dynamic, and tightly interconnected network of protein interactions that responds to environmental cues (such as the availability of various reductive sinks) to direct electron flux to maximize energetic gains. Interactions connecting disparate functions suggest many catabolic and anabolic activities occur in spatial proximity in vivo , and while protein complexes have been historically defined under optimal conditions, many of these complexes appear to maintain alternative partnerships in changing conditions. The totality of the results obtained redefines our understanding of in vivo assemblies driving ancient metabolic strategies supporting the growth of modern Archaea. IMPORTANCE Given the potential for rational genetic manipulations of biofuel- and biotech-promising archaea to yield transformative results for major markets, it is a priority to define how the metabolisms of such species are controlled, at least in part, by in vivo protein assemblies, and from such, define routes of energy flux that can be most efficiently altered toward biofuel or biotechnological gains. Proteinaceous electron carriers (PECs, such as ferredoxins) offer the potential for specific protein–protein interactions to coordinate selective reductive flow. Employing the model, genetically accessible, hyperthermophilic archaeon, Thermococcus kodakarensis , we establish the metabolic protein interactome of 25 key redox proteins, revealing that each redox active protein has a dynamic partnership profile, suggesting catabolic and anabolic activities may occur in concert and in temporal and spatial proximity in vivo . These results reveal critical importance in evaluating the newly identified partnerships and their role and utility in providing regulated redox flux in T. kodakarensis .

Williams, Sere A. (ORCID:0000000235509590)↗
NASA NTRS2003

Determining the Biogenicity of Microfossils in the Apex Chert, Western Australia, Using Transmission Electron Microscopy

For over a decade, the oldest evidence for life on this planet has been microfossils in the 3.5 Ga Apex Chert in Western Australia. Recently, the biogenicity of these carbon-rich structures has been called into question through reanalysis of the local geology and reinterpretation of the original thin sections. Although initially described as a stratiform, bedded chert of siliceous clasts, the unit is now thought to be a brecciated hydrothermal vein chert. The high temperatures of a hydrothermal environment would probably have detrimental effects to early non-hyperthermophilic life, compared to that of a shallow sea. Conversely, a hydrothermal origin would suggest that if the microfossils were valid, they might have been hyperthermophilic. Apex Chert controversy. The Apex Chert microfossils were originally described as septate filaments composed of kerogen similar in morphology to Proterozoic and modern cyanobacteria. However new thin section analysis shows that these carbonaceous structures are not simple filaments. Many of the original microfossils are branched and have variable thickness when the plane of focus is changed. Hydrothermal alteration of organic remains has also been suggested for the creation of these strange morphologies. Another point of contention lies with the nature of the carbon material in these proposed microfossils. Kerogen is structurally amorphous, but transforms into well-ordered graphite under high pressures and temperatures. Raman spectrometry of the carbonaceous material in the proposed microfossils has been interpreted both as partially graphitized kerogen and amorphous graphite. However, these results are inconclusive, since Raman spectrometry cannot adequately discriminate between kerogen and disordered graphite. There are also opposing views for the origin of the carbon in the Apex Chert. The carbon would be biogenic if the proposed microfossils are indeed the remains of former living organisms. However, an inorganic Fischer- Tropsch-type synthesis is also a possible explanation for the formation of large-aggregate carbonaceous particles and could also account for the depletion of (13)C observed.

DeGregorio, B. T.↗
NASA NTRS1998

Energetics of amino acid synthesis in hydrothermal ecosystems

Thermodynamic calculations showed that the autotrophic synthesis of all 20 protein-forming amino acids was energetically favored in hot (100 degrees C), moderately reduced, submarine hydrothermal solutions relative to the synthesis in cold (18 degrees C), oxidized, surface seawater. The net synthesis reactions of 11 amino acids were exergonic in the hydrothermal solution, but all were endergonic in surface seawater. The synthesis of the requisite amino acids of nine thermophilic and hyperthermophilic proteins in a 100 degreesC hydrothermal solution yielded between 600 and 8000 kilojoules per mole of protein, which is energy that is available to drive the intracellular synthesis of enzymes and other biopolymers in hyperthermophiles thriving in these ecosystems.

NASA Discipline Exobiology↗
NASA NTRS1996

Hydrothermal systems as environments for the emergence of life

Analysis of the chemical disequilibrium provided by the mixing of hydrothermal fluids and seawater in present-day systems indicates that organic synthesis from CO2 or carbonic acid is thermodynamically favoured in the conditions in which hyperthermophilic microorganisms are known to live. These organisms lower the Gibbs free energy of the chemical mixture by synthesizing many of the components of their cells. Primary productivity is enormous in hydrothermal systems because it depends only on catalysis of thermodynamically favourable, exergonic reactions. It follows that hydrothermal systems may be the most favourable environments for life on Earth. This fact makes hydrothermal systems logical candidates for the location of the emergence of life, a speculation that is supported by genetic evidence that modern hyperthermophilic organisms are closer to a common ancestor than any other forms of life. The presence of hydrothermal systems on the early Earth would correspond to the presence of liquid water. Evidence that hydrothermal systems existed early in the history of Mars raises the possibility that life may have emerged on Mars as well. Redox reactions between water and rock establish the potential for organic synthesis in and around hydrothermal systems. Therefore, the single most important parameter for modelling the geochemical emergence of life on the early Earth or Mars is the composition of the rock which hosts the hydrothermal system.

Non-NASA Center↗
NASA NTRS2013

Does Aspartic Acid Racemization Constrain the Depth Limit of the Subsurface Biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of approximately 89 years for 1 km depth and 27 C and 1-2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

cellular↗
DOE OSTI2023

Candidatus Nanopusillus

The genus Candidatus Nanopusillus is comprised of small coccoid cells (~100–400 nm) that live epibiotically on the surface of archaeal hosts. The first described species, Candidatus Nanopusillus acidilobi, is an anaerobic, hyperthermophilic acidophile whose best growth is observed at 82°C, pH 3.6, cultivated from a hot spring in Yellowstone National Park. Ca. Nanopusillus acidilobi cells associate with the Crenarchaeota host organism Acidilobus sp. 7A. Archaeal flagella (archaella) have been predicted from the genome sequence and shown to be expressed in the proteome. Additionally, a second putative species, Candidatus Nanopusillus massiliensis, was recently reported from human dental plaque and associates with the methanogen Methanobrevibacter oralis. The genome consists of a single scaffold which is highly fragmented by spans of ambiguous nucleotides, with 16S rRNA gene fragments from Bacteria. Both species have small genomes (~0.6 Mb) encoding few biosynthetic genes and no apparent ATP synthase complex genes, suggesting that the nanoarchaeotes rely on their host for the production of major cellular precursors.

acidophile↗
DOE OSTI2026

Rerouting reductant flux via protein tethering enhances biohydrogen production in Thermococcus kodakarensis

Microbes that generate copious amounts of hydrogen (H 2 ) via dark fermentation are a promising means to evolve and improve renewable biofuels. Many anaerobic hyperthermophilic archaea, such as the fast-growing, genetically tractable, heterotroph Thermococcus kodakarensis, produce generous quantities of H2 and provide an idealized platform to further optimize naturally high levels of biohydrogen reduction. Precise genetic manipulations and modifications to growth conditions have already resulted in substantial increases to H2 output but additional improvements are desired. An unexamined and potentially valuable route towards increased H 2 production is to tether select electron donor and acceptor proteins together to reroute and maximize the flow of electrons towards H 2 production. Such strategies have shown promise in Bacteria and Eukarya but have not yet been investigated in thermophilic Archaea. Here, we generate and evaluate twelve novel T. kodakarensis strains wherein a proteinaceous electron carrier (a ferredoxin, Fd) is physically tethered to the membrane-bound-hydrogenase (MBH), the sole H 2 producing enzyme, to direct electron flux towards biohydrogen generation. Growth assessments and H 2 output measurements demonstrate that strains encoding protein-fusions evolve up to ~ 40% more H2 per cell than the host strain. Eliminating H 2 consumption and alternative routes of electron sinks in concert with protein tethering further increased H2 output per cell for a maximum increase of ~ 66% over the host strain. Our results demonstrate that rerouting electron flux via protein tethering coupled with the elimination of reductant sinks is a promising means towards improved biohydrogen production in T. kodakarensis. KEY POINTS: Protein tethering between redox proteins can reroute electron flux in vivo. Enforced protein proximity results in ~ 40% increases in H2 production per cell. Protein-tethering provides a generalizable framework to redirect redox metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Electrochemical Observation and pH Dependence of All Three Expected Redox Couples in an Extremophilic Bifurcating Electron Transfer Flavoprotein with Fused Subunits

Bifurcating enzymes employ energy from a favorable electron transfer to drive unfavorable transfer of a second electron, thereby generating a more reactive product. They are therefore highly desirable in catalytic systems, for example, to drive challenging reactions such as nitrogen fixation. While most bifurcating enzymes contain air-sensitive metal centers, bifurcating electron transfer flavoproteins (bETFs) employ flavins. However, they have not been successfully deployed on electrodes. Herein, we demonstrate immobilization and expected thermodynamic reactivity of a bETF from a hyperthermophilic archaeon, Sulfolobus acidocaldarius (SaETF). SaETF differs from previously biochemically characterized bETFs in being a single protein, representing a concatenation of the two subunits of known ETFs. However, SaETF retains the chemical properties of heterodimeric bETFs, including possession of two FADs: one that undergoes sequential 1-electron (1e) reductions at high E° and forms an anionic semiquinone, and another that is amenable to lower-E° 2e reduction, including by NADH. We found homologous monomeric ETF genes in archaeal and bacterial genomes, accompanied by genes that also commonly flank heterodimeric ETFs, and SaETF’s sequence conservation is 50% higher with bETFs than with canonical ETFs. Thus, SaETF is best described as a bETF. Our direct electrochemical trials capture reversible redox couples for all three thermodynamically expected redox events. We document electrochemical activity over a range of pH values and reveal a conformational change coupled to proton acquisition that affects the electrochemical activity of the higher-E° FAD. Thus, this well-behaved monomeric bETF opens the door to bioinspired bifurcating devices or bifurcation on a chip.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

The Thermococcales as a model system: historical perspectives and emerging tools

Thermococcales are among the most widely studied hyperthermophilic Archaea and have become key models for understanding life at extreme temperatures. Early work in the 1980s culminated in the isolation of novel Thermococcales species from hydrothermal vents that grew rapidly, tolerated extreme heat, and metabolized diverse substrates, making them uniquely amenable for laboratory studies. Their thermostable enzymes and emerging genetic tools facilitated detailed investigations of core processes such as DNA replication, repair, and transcription under conditions that challenge most life forms. These practical advantages, together with the accumulation of tools and protocols, cemented the role of Thermococcales as a model system. Here, we recount how chance discoveries, environmental adaptations, and experimental practicality intersected to establish Thermococcales as a central model for studying archaeal biology and extremophile physiology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

pH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Crystal structure of Methanococcus jannaschii dihydroorotase

In this paper, we report the structural analysis of dihydroorotase (DHOase) from the hyperthermophilic and barophilic archaeon Methanococcus jannaschii . DHOase catalyzes the reversible cyclization of N-carbamoyl-l-aspartate to l-dihydroorotate in the third step of de novo pyrimidine biosynthesis. DHOases form a very diverse family of enzymes and have been classified into types and subtypes with structural similarities and differences among them. This is the first archaeal DHOase studied by x-ray diffraction. Its structure and comparison with known representatives of the other subtypes help define the structural features of the archaeal subtype. The M. jannaschii DHOase is found here to have traits from all subtypes. Contrary to expectations, it has a carboxylated lysine bridging the two Zn ions in the active site, and a long catalytic loop. It is a monomeric protein with a large β sandwich domain adjacent to the TIM barrel. Loop 5 is similar to bacterial type III and the C-terminal extension is long.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Chapter 2: Genetics of Unstudied Thermophiles for Industry

Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.

93 SC - Biological and Environmental Research (BER↗
DOE OSTI2026

Photoinduced Electron Transfer Informs on Pathway Coupling in Flavin-Based Electron Bifurcation

Flavin-based electron bifurcation (FBEB) is an enzymatic mechanism that generates extremely high-energy electrons to drive unfavorable chemical reactions. It is utilized by the NADHdependent ferredoxin:NADP + -oxidoreductase (Nfn) enzyme in hyperthermophile Pyrococcus furiosus to bifurcate electrons from NADPH into the coupled low-potential (endergonic) and highpotential (exergonic) pathways. This process enables P. furiosus to live in harsh and uninviting environments. Despite its biological importance, the mechanisms used by Nfn to facilitate exceptional directional control over short-lived, high-energy electrons and to prevent undesired transfer, particularly along the low-potential pathway, are still not well understood. To elucidate how the protein environment contributes to electronic control in the lowpotential pathway, new techniques must be utilized to probe these unstable intermediates. In this study, we have adapted lowtemperature photoexcitation combined with electron paramagnetic resonance (EPR) to accumulate the short-lived intermediate and place it in the context of the other cofactors involved in the low-potential pathway of Nfn. We observed coincident growth of both the radical intermediate and its nearby [4Fe-4S] cluster over 4.5 h of illumination with NADPH at cryogenic temperatures. The photogenerated paramagnetic species were stable in LN 2 storage indefinitely and recombined when warmed to higher temperatures. The results provide insights into the electron transfer steps and cofactor interactions along the low potential pathway, facilitating a more robust mechanistic understanding of the high-energy events of electron bifurcation. Furthermore, through comparison of cryogenic and room temperature experiments, a potential gating step involving the movement of key residues important for the reversibility of electron flow along this pathway is suggested.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design

In the realm of enzyme engineering, the dual enhancement of thermostability and activity remains a challenge. Herein, we employed a computer-aided approach integrating folding free energy calculations and evolutionary analysis to engineer Paecilomyces thermophila xylanase into a hyperthermophilic enzyme for application in the paper and pulp industry. Through the computational rational design, XynM9 with superior thermostability and enhanced activity was designed. Its optimal reaction temperature increases by 10 °C to 85 °C, its T m increases by 10 °C to 93 °C, and its half-life increases 11-fold to 5.8 h. Additionally, its catalytic efficiency improves by 57% to 3926 s –1 mM –1 . Molecular dynamics simulations revealed that XynM9 is stabilized by more hydrogen bonds and salt bridges than wild-type xylanase. The mutant’s narrower catalytic cleft enhances the substrate-binding affinity, thus improving the catalytic efficiency. In harsh conditions at 80 °C and pH 10, using XynM9 significantly reduced both hemicellulose and lignin, which makes it a good candidate for use in the paper and pulp process. Finally, our study presents an accurate and efficient strategy for the dual enhancement of enzyme properties, guiding further improvement of computational tools for protein stabilization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Conformational Dynamics and Catalytic Backups in a Hyper-thermostable Engineered Archaeal Protein Tyrosine Phosphatase

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid–base catalysis upon loop closure. These enzymes also have a conserved phosphate-binding loop that is typically highly rigid and forms a well-defined anion-binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have often evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP’s high sequence similarity to its natural counterparts, it presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active-site cysteine, mechanistic promiscuity, and, most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8 °C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical, and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme and showcases the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.

archaea↗
DOE OSTI2024

RNA language models predict mutations that improve RNA function

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. RNA structure prediction is not yet possible due to a lack of high-quality reference data associated with organismal phenotypes that could inform RNA function. We present GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences to experimental and predicted optimal growth temperatures of GTDB reference organisms. Using GARNET, we develop sequence- and structure-aware RNA generative models, with overlapping triplet tokenization providing optimal encoding for a GPT-like model. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identify mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

N 4 -acetylcytidine (ac 4 C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. In this work, we report ac 4 C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac 4 C at single-nucleotide resolution. In human and yeast mRNAs, ac 4 C sites are not detected but can be induced—at a conserved sequence motif—via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac 4 C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac 4 C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac 4 C and its potential thermoadaptive role. Our studies quantitatively define the ac 4 C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized -- but only from archaeal methanogens that are anaerobic, O 2 -sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O 2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared with the structures of THI4s from O 2 -sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

59 BASIC BIOLOGICAL SCIENCES↗