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Results for “flavoenzyme”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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11 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Quantitative measurements of reactive oxygen species partitioning in electron transfer flavoenzyme magnetic field sensing

Biological magnetic field sensing that gives rise to physiological responses is of considerable importance in quantum biology. The radical pair mechanism (RPM) is a fundamental quantum process that can explain some of the observed biological magnetic effects. In magnetically sensitive radical pair (RP) reactions, coherent spin dynamics between singlet and triplet pairs are modulated by weak magnetic fields. The resulting singlet and triplet reaction products lead to distinct biological signaling channels and cellular outcomes. A prevalent RP in biology is between flavin semiquinone and superoxide (O 2 •–) in the biological activation of molecular oxygen. This RP can result in a partitioning of reactive oxygen species (ROS) products to form either O 2 •– or hydrogen peroxide (H 2 O 2 ). Here, we examine magnetic sensing of recombinant human electron transfer flavoenzyme (ETF) reoxidation by selectively measuring O 2 •– and H 2 O 2 product distributions. ROS partitioning was observed between two static magnetic fields at 20 nT and 50 μT, with a 13% decrease in H 2 O 2 singlet products and a 10% increase in O 2 •– triplet products relative to 50 µT. RPM product yields were calculated for a realistic flavin/superoxide RP across the range of static magnetic fields, in agreement with experimental results. For a triplet born RP, the RPM also predicts about three times more O 2 •– than H 2 O 2 , with experimental results exhibiting about four time more O 2 •– produced by ETF. The method presented here illustrates the potential of a novel magnetic flavoprotein biological sensor that is directly linked to mitochondria bioenergetics and can be used as a target to study cell physiology.

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

A catalytic dyad modulates conformational change in the CO 2 -fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO 2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H + and CO 2 to favor the metabolically preferred carboxylation product, acetoacetate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Enzymatic Laser-Induced Graphene Biosensor for Electrochemical Sensing of the Herbicide Glyphosate

Glyphosate is a globally applied herbicide yet it has been relatively undetectable in-field samples outside of gold-standard techniques. Its presumed nontoxicity toward humans has been contested by the International Agency for Research on Cancer, while it has been detected in farmers’ urine, surface waters and crop residues. Rapid, on-site detection of glyphosate is hindered by lack of field-deployable and easy-to-use sensors that circumvent sample transportation to limited laboratories that possess the equipment needed for detection. Herein, the flavoenzyme, glycine oxidase, immobilized on platinum-decorated laser-induced graphene (LIG) is used for selective detection of glyphosate as it is a substrate for GlyOx. The LIG platform provides a scaffold for enzyme attachment while maintaining the electronic and surface properties of graphene. The sensor exhibits a linear range of 10–260 µm, detection limit of 3.03 µm, and sensitivity of 0.991 nA µm -1 . The sensor shows minimal interference from the commonly used herbicides and insecticides: atrazine, 2,4-dichlorophenoxyacetic acid, dicamba, parathion-methyl, paraoxon-methyl, malathion, chlorpyrifos, thiamethoxam, clothianidin, and imidacloprid. Sensor function is further tested in complex river water and crop residue fluids, which validate this platform as a scalable, direct-write, and selective method of glyphosate detection for herbicide mapping and food analysis.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Further, our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nicotinamide Cofactor Biomimetics: Design and Structure Activity Relationships

Biocatalysis is an attractive methodology due to its sustainable metrics, combined with its vaunted regio-, chemo-, and stereoselectivity. Among biocatalysts, oxidoreductases catalyze redox transformations on a wide variety of substrates and are thus particularly attractive for synthetic applications. Many oxidoreductases depend on nicotinamide-based cofactors; however, the high cost and poor atom economy of such cofactors can negate the advantage of biosynthetic processes. The native cofactors (i.e., NADH or NADPH) can be replaced by nicotinamide cofactor biomimetics (NCBs) to address these issues. Additionally, NCBs can enhance enzyme activity due to structures with expanded redox properties. These capabilities open possibilities for reactivity and biorthogonality. Thus, the field has responded with new NCB structures, including variation in their electronic and molecular recognition properties. This perspective focuses on structure-activity relationships of simple NCBs, including their function, stability, and other properties, along with methods for their regeneration in biosynthesis.

agricultural chemistry↗
DOE OSTI2022

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation

Significance Nature has long been an inspiration for materials design, as it exemplifies exquisite control of both matter and energy. Electron bifurcation, a mechanism employed in biological systems to drive thermodynamically unfavorable and energetically challenging chemical reactions, is one such example. A key feature of bifurcating enzymes is the ability of a single redox cofactor to distribute a pair of electrons across two spatially separated electron transfer pathways. Here, we report on the empirical determination of both the one-electron potential and two-electron potential of the bifurcating flavin cofactor in the NADH-dependent ferredoxin-NADP + oxidoreductase I (NfnSL) enzyme. Insights arising from the defined energy landscape of this bifurcation site may underlie the design of synthetic catalysts capable of generating high-energy intermediates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

What Makes a Bifurcase? Insights from a NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase (Nfn) and Homologs

NADH-dependent ferredoxin:NADP+ oxidoreductases (Nfn) is an enzyme family that engage in flavin-based electron bifurcation (FBEB), a mode of energy conservation utilized by life. The protein comprises one large (NfnL) and one small (NfnS) subunits. Thermoanaerobacterium sacchaloryticum (Tsac) is an anaerobic thermophilic bacterium that - with known involvement of Tsac Nfn - can produce ethanol in high, commercially viable concentrations. We sought to investigate the activity and energetic landscape of Tsac Nfn to determine how the enzyme effectuates FBEB. Tsac NfnS, NfnL, and the partner ferredoxin (Tsac Fd) were recombinantly expressed, purified, and reconstituted with iron-sulfur cluster and FAD cofactors. Electron paramagnetic resonance (EPR) was utilized for all proteins. Spectroelectrochemistry was performed with NfnL. Square-wave voltammetry was conducted on NfnL and Fd. Spectrophotometric activity was assayed for NfnL with or without NfnS. Our group continues to investigate the Nfn from Pyrococcus furiosus (Pf Nfn). Unlike Pf NfnS, Tsac NfnS can be stably expressed, purified, and reconstituted in the absence of its partner NfnL, allowing for this subunit to be characterized separately. The energetic profile of FBEB in Tsac Nfn is overall similar to that of Pf NfnL, with some differences: (1) the proximal cluster is at a lower potential (-780 mV vs -711), (2) the bifurcating FAD is at a higher potential (-406 mV vs -436 mV), and (3) Tsac Fd has two [4Fe-4S] clusters at -550 and -410 mV, unlike Pf Fd with a single cluster at approximately 400 mV. Activity assays indicate that the two enzymes perform FBEB in a similar way. Our work continues to build upon the new field of FBEB by demonstrating that the energetic landscape between distantly related archael (Pf) and bacterial (Tsac Nfn) are largely similar. This equips us to understand design principles for FBEB, allowing us to modulate the process in vivo for specific metabolic outcomes.

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

Site-Differentiated Iron–Sulfur Cluster Ligation Affects Flavin-Based Electron Bifurcation Activity

Electron bifurcation is an elegant mechanism of biological energy conversion that effectively couples three different physiologically relevant substrates. As such, enzymes that perform this function often play critical roles in modulating cellular redox metabolism. One such enzyme is NADH-dependent reduced-ferredoxin: NADP+ oxidoreductase (NfnSL), which couples the thermodynamically favorable reduction of NAD+ to drive the unfavorable reduction of ferredoxin from NADPH. The interaction of NfnSL with its substrates is constrained to strict stoichiometric conditions, which ensures minimal energy losses from non-productive intramolecular electron transfer reactions. However, the determinants for this are not well understood. One curious feature of NfnSL is that both initial acceptors of bifurcated electrons are unique iron–sulfur (FeS) clusters containing one non-cysteinyl ligand each. The biochemical impact and mechanistic roles of site-differentiated FeS ligands are enigmatic, despite their incidence in many redox active enzymes. Herein, we describe the biochemical study of wild-type NfnSL and a variant in which one of the site-differentiated ligands has been replaced with a cysteine. Results of dye-based steady-state kinetics experiments, substrate-binding measurements, biochemical activity assays, and assessments of electron distribution across the enzyme indicate that this site-differentiated ligand in NfnSL plays a role in maintaining fidelity of the coordinated reactions performed by the two electron transfer pathways. Given the commonality of these cofactors, our findings have broad implications beyond electron bifurcation and mechanistic biochemistry and may inform on means of modulating the redox balance of the cell for targeted metabolic engineering approaches.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Catalytic Bias of NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase (Nfn) and its Relevance to Ethanol Production in Thermoanaerobacterium Saccharolyticum

NADH-dependent reduced ferredoxin: NADP+ oxidoreductase (Nfn) enzyme catalyzes an energy-conserving flavin-based electron bifurcation (FBEB) reaction. In microbial metabolism, Nfn links redox pools of three electron carriers - ferredoxin (Fd), NAD(H), and NADP(H) - through the following FBEB reaction: 2 NADPH + NAD+ + 2 Fdox 2 NADP+ + NADH + 2 Fdred + H+ The forward reaction is termed electron bifurcation, and the reverse reaction is electron confurcation. Catalytic bias describes an enzyme's tendency to favor one direction of a reversible reaction over the other and is expressed as the ratio of activities in the two directions. Thermoanaerobacterium saccharolyticum (Tsac) is a thermophilic, ethanologenic bacterium that ferments hemicellulose to ethanol at yields above 90%. Its Nfn enzyme is known to support ethanol production, presumably by operating in the confurcating direction to balance cellular cofactors, but this has not previously been demonstrated. To investigate the Tsac Nfn further, we heterologously expressed, purified, and reconstituted the proteins NfnS (NfnA), NfnL (NfnB), and the putative partner Fd with iron-sulfur cluster and/or FAD cofactors. Activity assays monitoring the oxidation or reduction of Fd showed that, across pH 5-10, Tsac Nfn is catalytically biased towards the confurcating direction, favoring NADPH generation over NADPH oxidation by at least fivefold. Using protein electrochemistry, we also determined the reduction potentials of the cofactors in NfnL and Fd. These results indicate that the energetic landscape of FBEB in Tsac Nfn is similar to that in an ortholog. However, Tsac Fd has redox properties distinct from previously assayed Fds, suggesting that the identity and redox properties of Fd may help determine the catalytic bias of Nfn. Additionally, we confirmed the standalone ferredoxin: NADP+ oxidoreductase (FNOR) activity of NfnL but found it to be low and likely insignificant for in vivo redox conversion. We also show how the catalytic bias of Nfn integrates with the hydrogen cycling mechanism proposed in Tsac to better explain cofactor balancing for ethanol production. Our work advances the understanding of electron transfer processes within metabolic networks for the generation of valuable bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗