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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Mechanism of Radical Initiation in the Radical SAM Enzyme Superfamily

Radical S-adenosylmethionine (SAM) enzymes use a site-differentiated [4Fe-4S] cluster and SAM to initiate radical reactions through liberation of the 5'-deoxyadenosyl (5'-dAdo•) radical. They form the largest enzyme superfamily, with more than 700,000 unique sequences currently, and their numbers continue to grow as a result of ongoing bioinformatics efforts. The range of extremely diverse, highly regio- and stereo-specific reactions known to be catalyzed by radical SAM superfamily members is remarkable. The common mechanism of radical initiation in the radical SAM superfamily is the focus of this review. Most surprising is the presence of an organometallic intermediate, Ω, exhibiting an Fe–C5'-adenosyl bond. Regioselective reductive cleavage of the SAM S–C5' bond produces 5'-dAdo• to form Ω, with the regioselectivity originating in the Jahn–Teller effect. Ω liberates the free 5'-dAdo• as the catalytically active intermediate through homolysis of the Fe–C5' bond, in analogy to Co–C5' bond homolysis in B 12 , which was once viewed as biology's choice of radical generator.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Radical SAM enzymes: Nature's choice for radical reactions

Enzymes that use a [4Fe-4S] 1+ cluster plus S-adenosyl-L-methionine (SAM) to initiate radical reactions (radical SAM) form the largest enzyme superfamily, with over half a million members across the tree of life. This review summarizes recent work revealing the radical SAM reaction pathway, which ultimately liberates the 5´-deoxyadenosyl (5´-dAdo•) radical to perform extremely diverse, highly regio- and stereo-specific, transformations. Most surprising was the discovery of an organometallic intermediate Ω exhibiting an Fe-C5´-adenosyl bond. Ω liberates 5´-dAdo• through homolysis of the Fe-C5´ bond, in analogy to Co-C5´ bond homolysis in B12, previously viewed as biology’s paradigmatic radical generator. The 5´-dAdo• has been trapped and characterized in radical SAM enzymes via a recently-discovered photoreactivity of the [4Fe- 4S] + /SAM complex, and has been confirmed as a catalytically active intermediate in enzyme catalysis. The regioselective SAM S-C bond cleavage to produce 5´-dAdo• originates in the JahnTeller effect. The simplicity of SAM as a radical precursor, and the exquisite control of 5´-dAdo• reactivity in radical SAM enzymes, may be why radical SAM enzymes pervade the tree of life, while B12 enzymes are only a few.

deoxyadenosyl radical↗
DOE OSTI2024

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein–protein and protein–peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe 4 S 4 . Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.

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

Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase

ABSTRACT Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, we show that two different MgmA homologs are able to methylate MCR in vivo when expressed in Methanosarcina acetivorans , an organism that does not normally possess this PTM. M. acetivorans strains expressing MgmA showed small, but significant, reductions in growth rates and yields on methylotrophic substrates. Structural characterization of the Ni(II) form of Gln-methylated M. acetivorans MCR revealed no significant differences in the protein fold between the modified and unmodified enzyme; however, the purified enzyme contained the heterodisulfide reaction product, as opposed to the free cofactors found in eight prior M. acetivorans MCR structures, suggesting that substrate/product binding is altered in the modified enzyme. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation. IMPORTANCE Methane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies.

Rodriguez Carrero, Roy J. (ORCID:0000000184475641)↗
DOE OSTI2022

The Function of Two Radical‐SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]‐Hydrogenase Cofactor

Abstract In the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol ( 1 ) is 3‐methylated to form 2 , then 4‐guanylylated to form 3 , and converted into the full cofactor. HcgA‐G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S ‐adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis Δ hcgA strain was complemented with HcgA or precursors 1 , 2 or 3 . The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1 . We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the Δ hcgG system was recovered by HcgG but not by 3 , which indicated that HcgG catalyzes the reactions after the biosynthesis of 3 . The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

Arriaza‐Gallardo, Francisco J.↗
DOE OSTI2022

The Function of Two Radical‐SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]‐Hydrogenase Cofactor

Abstract In the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol ( 1 ) is 3‐methylated to form 2 , then 4‐guanylylated to form 3 , and converted into the full cofactor. HcgA‐G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S ‐adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis Δ hcgA strain was complemented with HcgA or precursors 1 , 2 or 3 . The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1 . We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the Δ hcgG system was recovered by HcgG but not by 3 , which indicated that HcgG catalyzes the reactions after the biosynthesis of 3 . The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

[FeFe]-Hydrogenase In Vitro Maturation

The [FeFe]-hydrogenase H-cluster is a complex organometallic cofactor whose assembly and installation requires three dedicated accessory proteins referred to as HydE, HydF, and HydG. The roles of these maturases and the precise mechanisms by which they synthesize and insert the H-cluster are not fully understood. This Minireview will focus on new insights into the [FeFe]-hydrogenase maturation process that have been provided by in vitro approaches in which the biosynthetic pathway has been partially or fully reconstructed using semisynthetic and enzyme-based approaches. Specifically, the application of these in vitro, semisynthetic, and fully defined approaches has shed light on the roles of individual maturation enzymes, the nature of H-cluster assembly intermediates, the molecular precursors of H-cluster ligands, and the sequence of steps involved in [FeFe]-hydrogenase maturation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Peptide Selenocysteine Substitutions Reveal Direct Substrate–Enzyme Interactions at Auxiliary Clusters in Radical S -Adenosyl- L -methionine Maturases

Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe–4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C–H to initiate catalysis, followed by formation of a C–S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se–C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea

Methylthiotransferases (MTTases) are radical S-adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH 3 ) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (ms 2 A) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein. Although the functions of MTTases in bacteria and eukaryotes have been established via detailed genetic and biochemical studies, MTTases from the archaeal domain of life are understudied and the substrate specificity determinants of MTTases remain unclear. Here, we report the in vitro enzymatic activities of an MTTase (C4B56_06395) from a thermophilic Ca. Methanophagales anaerobic methanotroph (ANME) as well as the MTTase from a hyperthermophilic methanogen – MJ0867 from Methanocaldococcus jannaschii. Both enzymes catalyze the methylthiolation of N 6 -threonylcarbamoyladenosine (t 6 A) and N 6 -hydroxynorvalylcarbamoyladenosine (hn 6 A) residues to produce 2-methylthio-N 6 -threonylcarbamoyladenosine (ms 2 t 6 A) and 2-methylthio-N 6 -hydroxynorvalylcarbamoyladenosine (ms 2 hn 6 A), respectively. To further assess the function of archaeal MTTases, we analyzed select tRNA modifications in a model methanogen – Methanosarcina acetivorans – and generated a deletion of the MTTase-encoding gene (MA1153). We found that M. acetivorans produces ms 2 hn 6 A in exponential phase of growth, but does not produce ms 2 t 6 A in detectable amounts. Upon deletion of MA1153, the ms 2 A modification was absent, thus confirming the function of MtaB-family MTTases in generating ms 2 hn 6 A modified nucleosides in select tRNAs.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1989

Electron Spin Resonance (ESR) detection of active oxygen species and organic phases in Martian soils

The presence of active oxygen species (O(-), O2(-), O3(-)) and other strong oxidants (Fe2O3 and Fe3O4) was invoked in interpretations of the Viking biological experiments and a model was also suggested for Martian surface chemistry. The non-biological interpretations of the biological results gain futher support as no organic compounds were detected in the Viking pyrolysis-gas chromatography mass spectrometer (GCSM) experiments at concentrations as low as 10 ppb. Electron spin resonance (ESR) measures the absorption of microwaves by a paramagnetic and/or ferromagnetic center in the presence of an external field. In many instances, ESR has the advantage of detailed submicroscopic identification of the transient species and/or unstable reaction intermediates in their environments. Since the higly active oxygen species (O(-), O2(-), O3(-), and R-O-O(-)) are all paramagnetic in nature, they can be readily detected in native form by the ESR method. Active oxygen species likely to occur in the Martian surface samples were detected by ESR in UV-irradiated samples containing MgO. A miniaturized ESR spectrometer system can be developed for the Mars Rover Sample Return Mission. The instrument can perform the following in situ Martian samples analyses: detection of active oxygen species; characterization of Martian surface chemistry and photooxidation processes; and searching for organic compounds in the form of free radicals preserved in subsoils, and detection of microfossils with Martian carbonate sediments.

Tsay, Fun-Dow↗
NASA NTRS2019

Fusion-Enabled Pluto Orbiter and Lander

Direct Fusion Drive (DFD) is a unique fusion engine concept based on the Princeton Field-Reversed Configuration (PFRC) fusion reactor conceived by Dr. Sam Cohen of the Princeton Plasma Physics Laboratory. DFD would enable the Pluto orbiter and lander context mission and more broadly enable true "rapid transit" to outer-planet and near interstellar space. The truly game-changing levels of thrust and power in a modestly sized package could integrate with our current launch infrastructure while radically expanding the science capability of these missions. Our Phase I was our first funded work on the DFD, with previous work at PSS occurring only under internal R&D. We established the feasibility of our Pluto mission trajectories using straight-line and planar models, including a departure spiral from Earth and insertion at Pluto. We developed our first thrust and specific impulse model using the results of the UEDGE multi-fluid code. Our specific power model was improved. During this Phase II effort, we continued our efforts to increase the fidelity of the designs for the RF, magnet, and shielding subsystems. Dedicated thrust augmentation experiments were run on the PFRC experiment, using a supersonic gas puffing valve. For the first time, we analyzed the design of a closed-loop operation mode and estimated the hardware that would be required for a dual-mode engine. In an exciting new development, we have invented a new thermophotovoltaic thermal conversion method that has the potential to have efficiencies of a Brayton or Stirling system. Our report presents details of these analyses. Our roadmap to bringing DFD to flight predicts that with sufficient support, a first flight unit could be built by 2040. We anticipate that three machine generations are required before this point: a ~1 T PFRC-3 machine hitting new plasma temperature and density levels, a ~5 T PFRC-4 machine with first demonstration of D-3He fusion, and a flight. (The current experiment, PFRC-2, is limited to about 0.1 T). In order to achieve a flight in 2035-2040, the TRL of the supporting systems must be increased in parallel, including low mass radiators, cryogenic propellant storage, and large (>100 kW) thermal conversion systems. Fortunately, many of these systems are dual-use and are required for other technologies including fission systems, so DFD would contribute to and benefit from those programs. This NIAC support and the results of our work have led to multiple follow-on contracts. We won two NASA STTRs focused on DFD subsystems, one on the RF system and one on the superconducting magnets, and our magnet STTR is now midway through a Phase II. We will be receiving a superconducting test magnet for experiments at PPPL this summer. In addition, we won an ARPA-E OPEN grant; this is the first time that OPEN has included fusion technologies, and we are part of a cohort of three alternative fusion companies now supported directly by DOE. We are very optimistic that if we are able to meet our experimental milestones in the next 12-18 months, we will be competitive for additional DOE grants to build PFRC-3.

Propulsion↗
NASA NTRS1989

ESR Measurement Of Crystallinity In Semicrystalline Polymers

Photogenerated free radicals decay at different rates in crystalline and amorphous phases. Degree of crystallinity in polymer having both crystalline and amorphous phases measured indirectly by technique based in part on electron-spin-resonance (ESR) spectroscopy. Accuracy of crystallinity determined by new technique equals or exceeds similar determinations by differential scanning calorimetry, wide-angle x-ray scattering, or measurement of density.

Kim, Soon Sam↗
DOE OSTI2023

Spin–Orbit versus Hyperfine Coupling-Mediated Intersystem Crossing in a Radical Pair

While spin–orbit coupling (SOC) is typically the dominant interaction that couples singlet and triplet states within individual chromophores, hyperfine coupling (HFC) becomes important in multichromophoric systems, particularly in relation to the radical pair mechanism. Here, we use TD-DFT to calculate the spin–orbit coupling and hyperfine coupling between the first singlet and triplet charge transfer states of the radical pair 2 Pyrene– and 2 N,N-dimethylaniline + . We show that, as the intermolecular donor–acceptor distance grows, SOC decays to zero (as one would expect) because singlet and triplet states are characterized by identical orbitals in space, while the HFC remains comparatively constant. Here, the switching region occurs around 4 Å, beyond which HFC dominates over SOC as far as defining the rate of intersystem crossing (ISC).

74 ATOMIC AND MOLECULAR PHYSICS↗
NASA NTRS1989

Electron Spin Resonance (ESR) studies of returned comet nucleus samples

The most important objective of the Comet Nucleus Sample Returm Mission is to return samples which could reflect formation conditions and evolutionary processes in the early solar nebula. It is expected that the returned samples will consist of fine-grained silicate materials mixed with ices composed of simple molecules such as H2O, NH3, CH4 as well as organics and/or more complex compounds. Because of the exposure to ionizing radiation from cosmic-ray, gamma-ray, and solar wind protons at low temperature, free radicals are expected to be formed and trapped in the solid ice matrices. The kind of trapped radical species together with their concentration and thermal stability can be used as a dosimeter as well as a geothermometer to determine thermal and radiation histories as well as outgassing and other possible alternation effects since the nucleus material was formed. Since free radicals that are known to contain unpaired electrons are all paramagnetic in nature, they can be readily detected and characterized in their native form by the Electron Spin Resonance (ESR) method. In fact, ESR has been shown to be a non-destructive, highly sensitive tool for the detection and characterization of paramagnetic, ferromagnetic, and radiation damage centers in terrestrial and extraterrestrial geological samples. The potential use of ESR as an effective method in the study of returned comet nucleus samples, in particular, in the analysis of fine-grained solid state icy samples is discussed.

Tsay, Fun-Dow↗
NASA NTRS1997

Electron Spin Resonance (ESR) Studies of Returned Comet Nucleus Samples

Electron Spin Resonance (ESR) studies have been carried out on organic and inorganic free radicals generated by gamma-ray and/or UV-irradiation and trapped in ice matrices. It is suggested that the concentration of these free radicals together with their thermal stability can be used as an accurate built-in geothermometer and radiation probe for returned comet nucleus sample studies. ESR studies have also been carried out on paramagnetic (Mn(2+), Ti(3+), and Fe(3+)) and ferromagnetic (ferric oxide and metallic iron) centers known to be present in terrestrial and extraterrestrial samples. The presence or absence of these magnetic centers coupled with their characteristic ESR lineshape can be used to investigate the shock effects, quenching/cooling rate and oxidation-reduction conditions in the formation and subsequent evolution of returned comet nucleus samples.

Tsay, Fun-Dow↗
DOE OSTI2023

Solution Structure and Scaling Laws of Cylindrical and Tapered Bottlebrush Polymers

Bottlebrush polymers are a unique class of macromolecular architectures with a plethora of potential industrial and pharmaceutical applications that critically depend on the bottlebrush shape and dimensions. Here, a systematic series of 12 cylindrical and 12 cone-shaped (tapered) bottlebrush polymers with poly(tert-butyl acrylate) (PtBA) or polystyrene (PS) side chains were synthesized using the sequential addition of macromonomers ring-opening metathesis polymerization (SAM-ROMP) grafting-through method. Small-angle neutron scattering (SANS) studies on dilute solutions of the two types of bottlebrush polymers provided noninvasive characterization of their structural dimensions and chain conformations. Simulated SANS traces, generated using coarse-grained molecular dynamics simulations, reproduced the distinctive features observed in the experimental SANS signals and provided necessary validation for data modeling. The combined analysis of experimental and simulated SANS signals yielded key structural and conformational parameters, including the bottlebrush radius, length, and Kuhn length as well as the excluded volume parameter and the correlation length of the polymer side chains. Importantly, the obtained structural parameters followed well-defined scaling laws as a function of the backbone and side chain degrees of polymerization, as predicted by mean field theories. In conclusion, these findings provide clear experimental and computational evidence of the interdependence of structural and conformational properties in an important class of polymer architectures.

36 MATERIALS SCIENCE↗