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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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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Mechanistic assessment of tolerance to iron deficiency mediated by Trichoderma harzianum in soybean roots

Abstract Aims Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. Methods and Results Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2•–) and hydrogen peroxide (H2O2) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. Conclusions TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. Significance and Impact of the Study These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A nitrogenase-like enzyme system catalyzes methionine, ethylene, and methane biogenesis

Bacterial production of hydrocarbon gases like ethylene and methane affects soil ecosystems and the climate. This study reveals that methane and ethylene from land and freshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway. This pathway employs a nitrogenase-like reductase that is distinct from known nitrogenases and nitrogenase-like reductases. The nitrogenase-like methylthio-alkane reductase specifically functions in carbon-sulfur bond breakage to reduce ubiquitous and significant volatile organic sulfur compounds such as dimethyl sulfide and (2-methylthio)ethanol. The liberated methanethiol serves as the immediate precursor to methionine. Whereas ethylene or methane is released into the environment. This pathway provides a biological route to the synthesis of ethylene in the absence of oxygen. Finally, anaerobic ethylene production by this pathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depleted soils Methane production reveals an additional bacterial pathway that is distinct from archaeal methanogenesis.

10 SYNTHETIC FUELS↗
DOE OSTI2022

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media

Abstract Background The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Results Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets ( ACL1 and ACC1 , improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1 ) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1 Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. Conclusions The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1 , or the deletion of DGA1 combined with overexpression of ACC1 and ACL1 . Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for Metabolic Engineering of Rhodotorula toruloides IFO0880 Improves C16 and C18 Fatty Alcohol Production from Synthetic Media

The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Conversion↗
DOE OSTI2023

The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family ( e . g ., CcoA) are required for Cu import and heme-Cu B center biogenesis of the cbb 3 -type cytochrome c oxidases ( cbb 3 -Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo 3 -type quinol oxidases ( bo 3 -Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo 3 -Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E . coli bo 3 -Qox. When respiratory growth is dependent on bo 3 -Qox, because of deletion of the bd -type Qox enzymes, three candidate genes, yhjE , ydiM , and yfcJ , were found to be critical for E . coli growth. Radioactive metal uptake assays showed that Δ ydiM has a slower 64 Cu uptake, whereas Δ yhjE accumulates reduced 55 Fe in the cell, while no similar uptake defect is associated with Δ ycfJ . Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biochemical responses of hairgrass (Deschampsia caespitosa) to hydrological change

Plant growth and development are closely related to water availability. Water deficit and water excess are detrimental to plants, causing a series of damage to plant morphology, physiological and biochemical processes. In the long evolutionary process, plants have evolved an array of complex mechanisms to combat against stressful conditions. In the present study, the duration-dependent changes in ascorbate (AsA) and glutathione (GSH) contents and activities of enzymes involved in the AsA-GSH cycle in hairgrass ( Deschampsia caespitosa ) in response to water stress was investigated in a pot trial using a complete random block design. The treatments were as follows: (1) heavily waterlogging, (2) moderate waterlogging, (3) light waterlogging, (4) light drought, (5) moderate drought, (6) heavily drought, and (7) a control (CK) with plant be maintained at optimum water availability. The hairgrass plants were subjected to waterlogging or drought for 7, 14, 21 and 28 days and data were measured following treatment. Results revealed that hairgrass subjected to water stress can stimulate enzymatic activities of ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and L-galactono-1, 4-lactone dehydrogenase (GalLDH), switched on the ascorbate-glutathione (AsA-GSH) cycle and the L-galactose synthesis, up-regulated the contents of AsA and GSH, and maintained higher ratios of ascorbate to dehydroascorbate (AsA/DHA) and reduced glutathione to oxidized glutathione (GSH/GSSG) to alleviate potential oxidative damage. However, the light waterlogging did not induce hairgrass under stress to switch on the AsA-GSH pathway. In general, the critic substances and enzyme activities in AsA-GSH metabolic pathway increased as the increase of water stress intensity. As the increase of exposure duration, the critic antioxidant substances content and enzyme activities increased first and then maintained a relatively stable higher level. Our findings provide comprehensive information on biochemical responses of hairgrass to hydrological change, which would be a major step for accelerating ecological restoration of degradation alpine marshes in the Qinghai-Tibetan Plateau.

Luo, Qiaoyu↗
DOE OSTI2022

Exogenous Hemin alleviated cadmium stress in maize (Zea mays L.) by enhancing leaf photosynthesis, AsA-GSH cycle and polyamine metabolism

Cadmium (Cd) stress is one of the principal abiotic stresses that inhibit maize growth. The research was to explore (hemin chloride) Hemin (100 μmol L −1 ) on photosynthesis, ascorbic acid (AsA)-glutathione (GSH) cycle system, and polyamine metabolism of maize under Cd stress (85 mg L −1 ) using nutrient solution hydroponics, with Tiannong 9 (Cd tolerant) and Fenghe 6 (Cd sensitive) as experimental materials. The results showed that Hemin can increase leaf photosynthetic pigment content and ameliorate the ratio of Chlorophyll a/chlorophyll b ( Chla / Chlb ) under Cd stress. The values of ribose 1, 5-diphosphate carboxylase/oxygenase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPCase), and total xanthophyll cycle pool [(violoxanthin (V), antiflavin (A) and zeaxanthin (Z)] increased, which enhancing xanthophyll cycle (DEPS) de-epoxidation, and alleviating stomatal and non-stomatal limitation of leaf photosynthesis. Hemin significantly increased net photosynthetic rate ( P n ), stomatal conductance ( g s ), transpiration rate ( T r ), photochemical quenching coefficient ( qP ), PSII maximum photochemical efficiency ( F v /F m ), and electron transfer rate ( ETR ), which contributed to the improvement of the PSII photosynthetic system. Compared with Cd stress, Hemin can reduce thiobartolic acid reactant (TBARS) content, superoxide anion radical (O 2 − ) production rate, hydrogen peroxide (H 2 O 2 ) accumulation, and the extent of electrolyte leakage (EL); decreased the level of malondialdehyde (MDA) content and increased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); slowed the decrease in dehydroascorbic acid reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activity and the increase in glutathione reductase (GR) and ascorbate peroxidase (APX) activity in leaves; promoted the increase in AsA and GSH content, decreased dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and increased AsA/DHA and GSH/GSSG ratios under Cd stress. Hemin promoted the increase of conjugated and bound polyamine content, and the conversion process speed of free putrescine (Put) to free spermine (Spm) and spermidine (Spd) in maize; decreased polyamine oxidase (PAO) activity and increased diamine oxidase (DAO), arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) enzyme activities in leaves under Cd stress.

Piao, Lin↗
NASA NTRS1986

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

NASA Center ARC↗
DOE OSTI2020

Synthetic pathway for biological carbon dioxide sequestration

This invention relates to methods for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell heterologous polynucleotides encoding (1) a succinyl CoA synthetase, (2) a 2-oxoglutarate:ferredoxin oxidoreductase, (3) a 2-oxoglutarate carboxylase, (4) an oxalosuccinate reductase, or (5) an isocitrate lyase, or (6) a succinyl CoA synthetase and a 2-oxoglutarate:ferredoxin oxidoreductase, (7) a 2-oxoglutarate carboxylase and an oxalosuccinate reductase polypeptide, and/or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotides are from a bacterial and/or an archaeal species. Additionally, transformed plants, plant parts, and/or plant cells are provided as well as products produced from the transformed plants, plant parts, and/or plant cells.

09 BIOMASS FUELS↗
DOE OSTI2026

Catabolism of lignin-related methoxylated compounds in white-rot fungi utilizes non-canonical oxidoreductases

White-rot fungi (WRF) are the most effective lignin-degrading organisms in nature, making them essential to Earth’s carbon cycle. Lignin is a highly methoxylated, heterogeneous biopolymer derived from plants. However, the pathways WRF use to metabolize methoxylated aromatic monomericcompounds as carbon sources remain unidentified. Here, we employ a systems biology approach to elucidate the intracellular catabolism of vanillate – a monomethoxylated aromatic compound – in two white-rot fungi (WRF), Gelatoporia subvermispora and Trametes versicolor. We identified and biochemically validated a four-enzyme pathway that converts vanillate into ß-ketoadipate – a metabolite that enters central carbon metabolism. This pathway deviates from typical bacterial pathways, where vanillate is initially demethylated and ring-cleaved by intradiol dioxygenases; instead, oxidative decarboxylation occurs prior to ring cleavage by extradiol dioxygenases. Thus, we conducted an in-depth investigation of ring cleavage and further downstream catabolism by the identified fungal enzymes using biochemical and structural approaches. This revealed non-canonical enzymes, including a highly substrate-specific extradiol dioxygenase and a metal-free, promiscuous reductase, the latter capable of acting on catabolic intermediates derived from both methoxylated and non-methoxylated aromatic compounds. This work emphasizes the potential of WRF and their enzymes to advance lignin valorization and enhance our understanding of their role during wood decay.

dioxygenase↗
DOE OSTI2023

Tissue-preferential recruitment of electron transfer chains for cytochrome P450-catalyzed phenolic biosynthesis

Cytochrome P450 system consists of P450 monooxygenase and redox pattern(s). While the importance of monooxygenases in plant metabolism is well documented, the metabolic roles of the related redox components have been largely overlooked. Here, we show that distinct electron transfer chains are recruited in phenylpropanoid-monolignol P450 systems to support the synthesis of different classes of phenolics in different plant tissues. While Arabidopsis cinnamate 4-hydroxylase adopts conventional NADPH-cytochrome P450 oxidoreductase (CPR) electron transfer chain for its para-hydroxylation, ferulate 5-hydroxylase employs both NADPH-CPR cytochrome b 5 (CB5) and NADH-cytochrome b 5 reductase (CBR)-CB5 chains to support benzene ring 5-hydroxylation. Moreover, the former route is primarily recruited in the stem for syringyl lignin synthesis, while the latter dominates in the syntheses of 5-hydroxylated phenolics in seeds and seed coat suberin. The availability of reductants NADPH and NADH and the catalytic properties of both reductases to CB5 contribute to the tissue-preferential recruitment of electron transfer systems. Our study unveils an additional layer of complexity and versatility of P450 system that the higher plants evolved for diversifying phenolic repertoires.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Reaction pathways and Sb(III) minerals formation during the reduction of Sb(V) by Rhodoferax ferrireducens strain YZ-1

Antimony (Sb), a non-essential metalloid, can be released into the environment through various industrial ac-tivities. Sb(III) is considered more toxic than Sb(V), but Sb(III) can be immobilized through the precipitation of insoluble Sb 2 S 3 or Sb 2 O 3 . In the subsurface, Sb redox chemistry is largely controlled by microorganisms; how-ever, the exact mechanisms of Sb(V) reduction to Sb(III) are still unclear. Here, in this study, a new strain of Sb(V)-reducing bacterium, designated as strain YZ-1, that can respire Sb(V) as a terminal electron acceptor was iso-lated from Sb-contaminated soils. 16S-rRNA gene sequencing of YZ-1 revealed high similarity to a known Fe(III)-reducer, Rhodoferax ferrireducens. XRD and XAFS analyses revealed that bioreduction of Sb(V) to Sb(III) proceed through a transition from amorphous valentinite to crystalline senarmontite (allotropes of Sb 2 O 3 ). Genomic DNA sequencing found that YZ-1 possesses arsenic (As) metabolism genes, including As(V) reductase arsC. The qPCR analysis showed that arsC was highly expressed during Sb(V)-reduction by YZ-1, and thus is proposed as the potential Sb(V) reductase in YZ-1. This study provides new insight into the pathways and products of microbial Sb(V) reduction and demonstrates the potential of a newly isolated bacterium for Sb bioremediation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

The oleaginous yeast Cutaneotrichosporon oleaginosum modifies corn stover alkali lignin

The current paradigm in synthetic biology for lignin bioconversion platforms includes primarily bacteria and filamentous fungi. Yeast are notoriously understudied for their role in lignin degradation and utilization, despite their ubiquity in saprophytic microbial communities. A few publications report lignin-modifying yeasts, but investigations to date have relied on model aromatic compounds or lignin-containing substrates replete with other carbon sources. In this work, we use a suite of analytical tools to evaluate interactions between corn stover-extracted lignin and the oleaginous yeast Cutaneotrichosporon oleaginosum. Notably, 2D-NMR analysis showed a significant decrease in the H-lignin component as well as resinol (β-β) and phenylcoumaran (β-5) linkages. Using super-resolution fluorescence microscopy, we demonstrated that this yeast may uptake polymeric lignin and/or undertakes interactions at the cellular envelope. To explore mechanisms of lignin modification, transport, and aromatics catabolism, extensive secretomics and proteomics analyses were conducted. Compared to carbon-limited glucose and “No Carbon” controls, several putative laccases, quinone reductases, superoxide dismutases, and glyoxal/oxalate oxidases were upregulated in the lignin condition. Excitingly, two ferric reductases and an oxalate exchanger were only observed in the lignin condition. These results indicate that C. oleaginosum may perform extracellular quinone redox cycling to generate lignin-modifying reactive oxygen species. These findings enhance our understanding of yeast-lignin interactions and provide valuable insights for validation studies and metabolic engineering.

09 BIOMASS FUELS↗
DOE OSTI2024

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

Abstract Background Cost-effective production of biofuels from lignocellulose requires the fermentation of d -xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD + to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae . Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans , which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis . Conclusion Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

09 BIOMASS FUELS↗
DOE OSTI2020

Utilization of protein film electrochemistry to characterize the mechanisms imparting aerotolerance and bidirectionality in soluble, multimeric [NiFe]-hydrogenases (Final Report for Grant No. DE-SC0008074)

The overall goal of this project was to better understand how structural features control functional aspects of enzymes involved in catalysis in which a substrate is oxidized or reduced. Two types of model enzymes were explored: the soluble [NiFe]-hydrogenase from Pyrococcus furiousus and the thiosulfate reductase from Pyrobaculum aerophilum. Both are models for complex, multielectron transformations such as nitrogen and carbon dioxide reduction, and parts of important biogeochemical cycles. In this project, electrochemical methods were used to define the activities of these enzymes and targeted variants designed to modulate activity. We showed that the catalytic activity of the soluble hydrogenase from Pyrococcus furiosus can be modulated by removing large parts of the enzyme far from the active site. This surprising result suggests that interactions between functional centers far apart within the enzyme may be essential for controlling and tuning activity. We also showed that the catalytic activity of thiosulfate reductase from Pyrobaculum aerophilum can be modulated by changing the metal at the active site to be either molybdenum or tungsten. These results contribute to our understanding of how the protein environment can tune the reactivity of an embedded metal, catalytic center. The results were and are being published in peer-reviewed, scientific publications.

08 HYDROGEN↗
DOE OSTI2023

The N‐terminal intrinsically disordered region of Ncb5or docks with the cytochrome b 5 core to form a helical motif that is of ancient origin

Abstract NADH cytochromeb 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochromeb 5 (b 5 ) and cytochromeb 5 reductase (b 5 R) domains separated by a CHORD‐Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter‐domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full‐length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high‐resolution atomic structures of theb 5 domain and a construct containing the CS andb 5 R domains (CS/b 5 R). Ncb5or also contains an N‐terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to ab 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N‐terminal region naturally fused to theb 5 domain, we were able to obtain a high‐resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11‐residue α‐helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in theb 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N‐terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi‐domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochromeb 5 proteins from three kingdoms among eukaryotes.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Tuning Local Atomic Structures in MoS 2 Based Catalysts for Electrochemical Nitrate Reduction

In recent years, there has been a substantial surge in the investigation of transition-metal dichalcogenides such as MoS 2 as a promising electrochemical catalyst. Inspired by denitrification enzymes such as nitrate reductase and nitrite reductase, the electrochemical nitrate reduction catalyzed by MoS 2 with varying local atomic structures is reported. Further, it is demonstrated that the hydrothermally synthesized MoS 2 containing sulfur vacancies behaves as promising catalysts for electrochemical denitrification. With copper doping at less than 9% atomic ratio, the selectivity of denitrification to dinitrogen in the products can be effectively improved. X-ray absorption characterizations suggest that two sulfur vacancies are associated with one copper dopant in the MoS 2 skeleton. DFT calculation confirms that copper dopants replace three adjacent Mo atoms to form a trigonal defect-enriched region, introducing an exposed Mo reaction center that coordinates with Cu atom to increase N 2 selectivity. Apart from the higher activity and selectivity, the Cu-doped MoS 2 also demonstrates remarkably improved tolerance toward oxygen poisoning at high oxygen concentration. Finally, Cu-doped MoS 2 based catalysts exhibit very low specific energy consumption during the electrochemical denitrification process, paving the way for potential scale-up operations.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Active-Site Environmental Factors Customize the Photophysics of Photoenzymatic Old Yellow Enzymes

The development of non-natural photoenzymatic systems has reinvigorated the study of photoinduced electron transfer (ET) within protein active sites, providing new and unique platforms for understanding how biological environments affect photochemical processes. Here, we use ultrafast spectroscopy to compare the photoinduced electron transfer in known photoenzymes. 12-Oxophytodienoate reductase 1 (OPR1) is compared to Old Yellow Enzyme 1 (OYE1) and morphinone reductase (MR). The latter enzymes are structurally homologous to OPR1. We find that slight differences in the amino acid composition of the active sites of these proteins determine their distinct electron-transfer dynamics. Our work suggests that the inside of a protein active site is a complex/heterogeneous dielectric network where genetically programmed heterogeneity near the site of biological ET can significantly affect the presence and lifetime of various intermediate states. Our work motivates additional tunability of Old Yellow Enzyme active-site reorganization energy and electron-transfer energetics that could be leveraged for photoenzymatic redox approaches.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗