Redox Proteins Research Articles

Redox Protein Thioredoxin Mediates Neurite Outgrowth in Primary Cultured Mouse Cerebral Cortical Neurons

Thioredoxin system plays an important role in maintaining the cellular redox balance. Recent evidence suggests that thioredoxin (Trx) system may promote cell survival and neuroprotection. In this study, we explored the role of thioredoxin system in neuronal differentiation using a primary mouse cortical neuronal cell culture. First, Trx and Trx reductase (TrxR) protein levels were analyzed in cultured neurons from 1 to 32 days in vitro (DIV). The result showed that Trx and TrxR protein levels time-dependently increased in the neuron cell culture from 1 to 18 DIV. To establish the role of Trx in neuronal differentiation, Trx gene expression was knockdown in cultured neurons using Trx sgRNA CRISPR/Cas9 technology. Treatment with CRISPR/Cas9/Trx sgRNA decreased Trx protein levels and caused a reduction in dendritic outgrowth and branching of cultured neurons. Then, primary cortical neurons were treated with the Trx inhibitor PX12 to block Trx reducing activity. Treatment with PX12 also reduced dendritic outgrowth and branching. Furthermore, PX12 treatment reduced the ratio of phosphorylated cyclic AMP response element-binding protein (CREB)/total CREB protein levels. To investigate whether CREB phosphorylation is redox regulated, SH-SY5Y cells were treated with H2O2, which reduced phosphorylated CREB protein levels and increased CREB thiol oxidation. However, treatment with CB3, a Trx-mimetic tripeptide, rescued H2O2-decreased CREB phosphorylation. Our results suggest that Trx regulates neuronal differentiation and maturation of primary mouse cortical neurons by targeting CREB neurotrophic pathway. Trx may regulate CREB activation by maintaining the cellular redox balance.

Low-Molecular Thiols as a Factor Improving the Sensitivity of Escherichia coli Mutants with Impaired ADP–Heptose Synthesis to Antibiotics

—Low molecular-weight thiols as glutathione and cysteine are an important part of the cell’s redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichiacoli with a gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (∆cysB and ∆cysE) and inhibition of glutathione synthesis (∆gshAB) lead to a decrease in the sensitivity of the ∆gmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (∆eamA) or increasing import (Ptet-tcyP) into cells of the oxidized form of cysteine–cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols, or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low-molecular-weight thiols saves NADPH equivalents and limits the processes of protein redox modification. This leads to increasing of resistance of the ∆gmhA strain to antibiotics. An increase in low-molecular-weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ∆gmhA strain to antibiotics.

Harnessing redox proteomics to study metabolic regulation and stress response in lignin-fed Rhodococci

BackgroundRhodococci are studied for their bacterial ligninolytic capabilities and proclivity to accumulate lipids. Lignin utilization is a resource intensive process requiring a variety of redox active enzymes and cofactors for degradation as well as defense against the resulting toxic byproducts and oxidative conditions. Studying enzyme expression and regulation between carbon sources will help decode the metabolic rewiring that stymies lignin to lipid conversion in these bacteria. Herein, a redox proteomics approach was applied to investigate a fundamental driver of carbon catabolism and lipid anabolism: redox balance.ResultsA consortium of Rhodococcus strains was employed in this study given its higher capacity for lignin degradation compared to monocultures. This consortium was grown on glucose vs. lignin under nitrogen limitation to study the importance of redox balance as it relates to nutrient availability. A modified bottom–up proteomics workflow was harnessed to acquire a general relationship between protein abundance and protein redox states. Global proteomics results affirm differential expression of enzymes involved in sugar metabolism vs. those involved in lignin degradation and aromatics metabolism. As reported previously, several enzymes in the lipid biosynthetic pathways were downregulated, whereas many involved in β-oxidation were upregulated. Interestingly, proteins involved in oxidative stress response were also upregulated perhaps in response to lignin degradation and aromatics catabolism, which require oxygen and reactive oxygen species and generate toxic byproducts. Enzymes displaying little-to-no change in abundance but differences in redox state were observed in various pathways for carbon utilization (e.g., β‑ketoadipate pathway), lipid metabolism, as well as nitrogen metabolism (e.g., purine scavenging/synthesis), suggesting potential mechanisms of redox-dependent regulation of metabolism.ConclusionsEfficient lipid production requires a steady carbon and energy flux while balancing fundamental requirements for enzyme production and cell maintenance. For lignin, we theorize that this balance is difficult to establish due to resource expenditure for enzyme production and stress response. This is supported by significant changes to protein abundances and protein cysteine oxidation in various metabolic pathways and redox processes.

Promotion of Direct Electron Transfer to Cytochrome c by Functionalized Thiophene‐based Conducting Polymers

AbstractControlling direct electron transfer (DET) to redox proteins is of great interest for fundamental studies on biochemical processes and the development of biotechnological devices, such as biosensors or enzymatic fuel cells. Cytochrome c is a classical model protein for studying DET reactions that plays a key role in the onset of cellular apoptosis and the mitochondrial respiratory chain. In this contribution, we explored DET between cyt c and conducting polymers bearing the chemical structure of thiophene, specifically PEDOT, and its OH‐containing derivative, PHMeEDOT. The combination of electrochemistry and in situ FTIR spectroscopy allowed us to gain more insight into the inner mechanism of DET at physiological pH. Hydrophilic interactions favour the correct orientation of the heme crevice of cytochrome c towards the polymer surface. When a positive charge is injected into the conducting polymer, the increasing electrostatic repulsion between protein and surface induces the desorption of lysine residues near the heme group and stimulates protein flipping. This effect was more pronounced at PEDOT‐ than PHMeEDOT‐modified electrodes since the latter shows stronger interactions with lysine residues, partially hindering protein rotation at moderate potential. The potential‐induced reorientation process was similar on both polymer surfaces, only at high positive potentials.

Structural conservation in the glutathione binding in Sphingomonas sp. glutaredoxin Grx3 and variations for cold adaptation

Glutaredoxin 3 (Grx3), a redox protein with a thioredoxin-fold structure, maintains structural integrity and glutathione (GSH) binding capabilities across varying habitat temperatures. The cis-Pro loop, essential for GSH binding, relies on the Arg-Asp salt bridge (α2-α3) and Gln-His hydrogen bond (β3-β4) for its conformation. In some psychrophilic Grx3 variants, Arg in α2 is replaced with Tyr, and His in β4 is replaced with Phe. This study examines the roles of these bonds in Grx3's structure, function, and cold adaptation, using SpGrx3 from the Arctic bacterium Sphingomonas sp. Despite its cold habitat, SpGrx3 maintains the Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond. The R51Y substitution disrupts the α2-α3 salt bridge, while the H63F and H63Y substitutions hinder the salt bridge through cation-π interactions with Arg51, involving Phe63/Tyr63, thereby enhancing flexibility. Conversely, mutations that disrupt the hydrogen bond (Q56A, H63A, and H63F) reduce thermal stability. In the psychrophilic Grx3 configuration A48T/R51Y/H63F, a Thr48-Gln56 hydrogen bond stabilizes the cis-Pro loop, enhancing flexibility by disrupting both bonds. Furthermore, all mutants exhibit reduced α-helical content and catalytic efficiency. In summary, the highly conserved Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond are crucial for stabilizing the cis-Pro loop and catalytic activity in SpGrx3. His63 is favored as it avoids cation-π interactions with Arg51, unlike Phe63/Tyr63. Psychrophilic Grx3 variants have adapted to cold environments by reducing GSH binding and increasing structural flexibility. These findings deepen our understanding of the structural conservation in Grx3 for GSH binding and the critical alterations required for cold adaptation.

Low-Molecular Thiols as a Factor Improving the Sensitivity of <i>Escherichia coli</i> Mutants with Impaired Synthesis ADP-Heptose to Antibiotics

Low molecular weight thiols as glutathione and cysteine are an important part of the cell’s redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichia coli during gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (∆cysB and ∆cysE) and inhibition of glutathione synthesis (∆gshAB) lead to a decrease in the sensitivity of the ∆gmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (∆eamA) or increasing the of the import (Ptet-tcyP) into cells of oxidized form of cysteine -cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low molecular weight thiols saves NADPH equivalents and limits the processes of protein redox modification. It leads to increasing of resistance of the ∆gmhA strain to antibiotics. On the contrary, an increase in low molecular weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ∆gmhA strain to antibiotics.

Cloning, expression and functional characterization of recombinant tumor necrosis factor α1 (TNFα1) from white crucian carp in gut immune regulation

TNFα is one of important cytokines belonging to TNF superfamily, which can exhibit a pleiotropic effect in immune modulation, homeostasis as well as pathogenesis. However, its immunoregulatory function on mucosal immunity in fish gut are still unclear. In this study, we aimed to investigated the immunoregulatory role of TNFα1 in midgut of white crucian carp (WCC). WCC-TNFα1 sequence and its deduced structure were firstly identified in WCC. Then, tissue-specific analysis revealed that high-level WCC-TNFα1 expression was detected in gill. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulated, increased trends of WCC-TNFα1 expressions were detected in immune-related tissues and cultured fish cells, respectively. WCC anal-intubated with WCC-TNFα1 fusion protein showed the increased levels of edema and fuzzy appearance in impaired villi, along with atrophy and reduction of goblet cells (GC). Moreover, the expression levels of tight junction (TJ) genes and mucin genes were consistently lower than those of the control (P < 0.05). WCC-TNFα1 treatment could sharply decrease antioxidant status in midgut, while the expression levels of caspase (CASP) genes, unfolded protein response (UPR) genes and redox response genes increased dramatically. Our results suggested that WCC-TNFα1 could exhibit a detrimental effect on antioxidant and mucosal immune regulation in midgut of WCC.

Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation.

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from - 150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool. This article is protected by copyright. All rights reserved.

Exploring the impact of incubation times and concentrations of self-assembled monolayers on electron transfer in biosensing

This study investigates the impact of different incubation times and concentrations of a self-assembled monolayer (SAMs) of 3-mercaptopropionic acid (MPA) on the rate of electron transfer in redox processes. The aim is to understand how these parameters can affect the sensitivity and efficiency of biosensors based on direct electron transfer in redox proteins. Through a series of experiments, different incubation times and concentrations of MPA were examined to determine their impact on the electron-transfer rate. Using methylene blue MB molecules as a model system and employing the EC-SPR technique, the reflectance differences (ΔR) between the reduced and oxidized states of MB were analyzed, serving as an indicator of the electron transfer rate. The results revealed significant variations in the rate depending on the incubation times and concentrations of the MPA. It was determined that a combination of 1 mM MPA concentration and 6-hour incubation time provided optimal conditions for maintaining a significant (ΔR). These findings have important implications for optimizing sensor surfaces in biosensors based on direct electron transfer in redox proteins.

PATHOGENIC SPECIES OF CLOSTRIDIA AND THEIR ANTIBIOTIC RESISTANCE, VIRULENCE FACTORS, AND GENOMIC FEATURES

Scientific data on the diversity of dangerous clostridia species causing infectious diseases in farm animals are presented. Current knowledge about the pathogenicity and virulence factors of clostridia and their harmful effects on the organisms of highly productive animals. Information is provided on anaerobic bacteria that can form capsules and biofilm structures, which are important determinants of virulence that block the action of the immune systems of macroorganisms, antibacterial agents, and various disinfectants. Data on the phenotypic and molecular-genetic stability of such significant clostridia as C. perfringens and C. difficile, found in published research results, are presented in tables. Current information on the determinants of virulence identified in C. septicum, C. sordellii, C. sporogenes, and C. tetani from various biological materials from different animals is presented in the article. Mechanisms of resistance to antibiotics, altered expression of redox proteins, DNA repair, mutation genes responsible for antibiotic resistance, formation of biofilms, and the presence of a matrix that hinders the penetration of antimicrobial agents into bacteria are described, as well as their prevalence among pathogenic clostridia worldwide. Modern available therapy methods and antimicrobial agents are outlined as an alternative to therapeutic agents used to treat diseases in humans, animals, and poultry caused by clostridia.

The Protein Matrix of Plastocyanin Supports Long-Distance Charge Transport with Photosystem I and the Copper Ion Regulates Its Spatial Span and Conductance.

Charge exchange is the fundamental process that sustains cellular respiration and photosynthesis by shuttling electrons in a cascade of electron transfer (ET) steps between redox cofactors. While intraprotein charge exchange is well characterized in protein complexes bearing multiple redox sites, interprotein processes are less understood due to the lack of suitable experimental approaches and the dynamic nature of the interactions. Proteins constrained between electrodes are known to support electron transport (ETp) through the protein matrix even without redox cofactors, as the charges housed by the redox sites in ET are furnished by the electrodes. However, it is unknown whether protein ETp mechanisms apply to the interprotein medium present under physiological conditions. We study interprotein charge exchange between plant photosystem I (PSI) and its soluble redox partner plastocyanin (Pc) and address the role of the Pc copper center. Using electrochemical scanning tunneling spectroscopy (ECSTS) current-distance and blinking measurements, we quantify the spatial span of charge exchange between individual Pc/PSI pairs and ETp through transient Pc/PSI complexes. Pc devoid of the redox center (Pcapo) can exchange charge with PSI at longer distances than with the copper ion (Pcholo). Conductance bursts associated with Pcapo/PSI complex formation are higher than in Pcholo/PSI. Thus, copper ions are not required for long-distance Pc/PSI ETp but regulate its spatial span and conductance. Our results suggest that the redox center that carries the charge in Pc is not necessary to exchange it in interprotein ET through the aqueous solution and question the canonical view of tight complex binding between redox protein partners.

Impairment in photosynthesis induced by CAT inhibition depends on the intensity of photorespiration and peroxisomal APX expression in rice

We have previously shown that rice plants silenced for peroxisomal ascorbate peroxidase (OsAPX4-RNAi) display higher resilience to photosynthesis under oxidative stress and photorespiratory conditions. However, the redox mechanisms underlying that intriguing response remain unknown. Here, we tested the hypothesis that favorable effects triggered by peroxisomal APX deficiency on photosynthesis resilience under CAT inhibition are dependent on the intensity of photorespiration associated with the abundance of photosynthetic and redox proteins. Non-transformed (NT) and OsAPX4-RNAi silenced rice plants were grown under ambient (AC) or high CO2 (HC) conditions and subjected to 3-amino-1,2,4-triazole (3-AT)-mediated CAT activity inhibition. Photosynthetic measurements evidenced that OsAPX4-RNAi plants simultaneously exposed to CAT inhibition and HC lost the previously acquired advantage in photosynthesis resilience displayed under AC. Silenced plants exposed to environment photorespiration and CAT inhibition presented lower photorespiration as indicated by smaller Gly/Ser and Jo/Jc ratios and glycolate oxidase activity. Interestingly, when these silenced plants were exposed to HC and CAT-inhibition, they exhibited an inverse response compared to AC in terms of photorespiration indicators, associated with higher accumulation of proteins. Multivariate and correlation network analyses suggest that the proteomics changes induced by HC combined with CAT inhibition are substantially different between NT and OsAPX4-RNAi plants. Our results suggest that the intensity of photorespiration and peroxisomal APX-mediated redox signaling are tightly regulated under CAT inhibition induced oxidative stress, which can modulate the photosynthetic efficiency, possibly via a coordinated regulation of protein abundance and rearrangement, ultimately triggered by crosstalk involving H2O2 levels related to CAT and APX activities in peroxisomes.

Functional Association between Storage Protein Mobilization and Redox Signaling in Narrow-Leafed Lupin (Lupinus angustifolius L.) Seed Germination and Seedling Development.

(1) Background: Seed storage mobilization, together with oxidative metabolism, with the ascorbate-glutathione (AsA-GSH) cycle as a crucial signaling and metabolic functional crossroad, is one of the main regulators of the control of cell morphogenesis and division, a fundamental physiological process driving seed germination and seedling growth. This study aims to characterize the cellular changes, composition, and patterns of the protein mobilization and ROS-dependent gene expression of redox metabolism in Lupinus angustifolius L. (narrow-leafed lupin, NLL) cotyledons during seed germination. (2) Methods: We performed gene expression analyses via RT-qPCR for conglutins α (1, 2, and 3), β (1, 2, and 5), γ (1, 2), and δ (2 and 4), including a ubiquitin gene as a control, and for redox metabolism-related genes; GADPH was used as a control gene. A microscopic study was developed on cotyledon samples from different germination stages, including as IMB (imbibition), and 2-5, 7, 9, and 11 DAI (days after imbibition), which were processed for light microscopy. SDS-PAGE and immunocytochemistry assays were performed using an anti-β-conglutin antibody (Agrisera), and an anti-rabbit IgG Daylight 488-conjugated secondary antibody. The controls were made while omitting primary Ab. (3) Results and Discussion: Our results showed that a large amount of seed storage protein (SSP) accumulates in protein bodies (PBs) and mobilizes during germination. Families of conglutins (β and γ) may play important roles as functional and signaling molecules, beyond the storage function, at intermediate steps of the seed germination process. In this regard, metabolic activities are closely associated with the regulation of oxidative homeostasis through AsA-GSH activities (γ-L-Glutamyl-L-cysteine synthetase, NOS, Catalase, Cu/Zn-SOD, GPx, GR, GS, GsT) after the imbibition of NLL mature seeds, metabolism activation, and dormancy breakage, which are key molecular and regulatory signaling pathways with particular importance in morphogenesis and developmental processes. (4) Conclusions: The knowledge generated in this study provides evidence for the functional changes and cellular tightly regulated events occurring in the NLL seed cotyledon, orchestrated by the oxidative-related metabolic machinery involved in seed germination advancement.

The Arabidopsis thioredoxin TRXh5regulates the S-nitrosylation pattern of the TIRK receptor being both proteins essential in the modulation of defences to Tetranychus urticae

The interaction between plants and phytophagous arthropods encompasses a complex network of molecules, signals, and pathways to overcome defences generated by each interacting organism. Although most of the elements and modulators involved in this interplay are still unidentified, plant redox homeostasis and signalling are essential for the establishment of defence responses. Here, focusing on the response of Arabidopsis thaliana to the spider mite Tetranychus urticae, we demonstrate the involvement in plant defence of the thioredoxin TRXh5, a small redox protein whose expression is induced by mite infestation. TRXh5 is localized in the cell membrane system and cytoplasm and is associated with alterations in the content of reactive oxygen and nitrogen species. Protein S-nitrosylation signal in TRXh5 over-expression lines is decreased and alteration in TRXh5 level produces changes in the JA/SA hormonal crosstalk of infested plants. Moreover, TRXh5 interacts and likely regulates the redox state of an uncharacterized receptor-like kinase, named THIOREDOXIN INTERACTING RECEPTOR KINASE (TIRK), also induced by mite herbivory. Feeding bioassays performed withTRXh5 over-expression plants result in lower leaf damage and reduced egg accumulation after T. urticae infestation than in wild-type (WT) plants. In contrast, mites cause a more severe injury in trxh5 mutant lines where a greater number of eggs accumulates. Likewise, analysis of TIRK-gain and -loss-of-function lines demonstrate the defence role of this receptor in Arabidopsis against T. urticae. Altogether, our findings demonstrate the interaction between TRXh5 and TIRK and highlight the importance of TRXh5 and TIRK in the establishment of effective Arabidopsis defences against spider mite herbivory.

CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

Redox Regulation of Salt Tolerance in Eutrema salsugineum by Proteomics.

Salt stress severely restricts plant growth and crop production, which is accompanied by accumulation of reactive oxygen species (ROS) that disturb cell redox homeostasis and oxidize redox-sensitive proteins. Eutrema salsugineum, a halophytic species closely related to Arabidopsis, shows a high level of tolerance to salinity and is increasingly used as a model plant in abiotic stress biology. To understand redox modifications and signaling pathways under salt stress, we used tandem mass tag (TMT)-based proteomics to quantify the salt-induced changes in protein redox modifications in E. salsugineum. Salt stress led to increased oxidative modification levels of 159 cysteine sites in 107 proteins, which play roles in carbohydrate and energy metabolism, transport, ROS homeostasis, cellular structure modulation, and folding and assembly. These lists of unknown redox reactive proteins in salt mustard lay the foundation for future research to understand the molecular mechanism of plant salt response. However, glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in plants. Our research indicates that EsGPX may be involved in regulating ROS levels and that plants with overexpressed EsGPX have much improved salt tolerance.

Unveiling the enigmatic traits of Corynebacterium glutamicum mycoredoxin-3: A tiny redox protein displaying swapped homodimer formation and DsbA-like oxidase activity

Mycoredoxins (Mrxs) are a group of small dithiol oxidoreductases that share a conserved CXXC active site sequence motif resembling glutaredoxins. They are commonly found in saprophytic microorganisms, including Actinobacteria such as Mycobacterium tuberculosis. Among the known mycoredoxins, Corynebacterium glutamicum (Cg) Mrx1, featuring a conserved CVQC active site, functions as a mycothiol-dependent monothiol oxidoreductase. On the other hand, Mrx2, also known as NrdH-redoxin and containing the same CVQC motif, exhibits dithiol oxidoreductase properties and receives electrons from thioredoxin reductase (TrxR). Recently, it has been reported that CgMrx3, featuring a CGSC motif, acts as a thioredoxin, although its structural and biophysical characteristics remain unexplored.In this study, we successfully determined the X-ray structure of CgMrx3 at a resolution of 1.7 Å, revealing a swapped dimer arrangement. We compared the structure of CgMrx3 with those of CgMrx1 and CgMrx2 and with the AlphaFold2 predicted structure of Mrx3 from Mycobaterium tuberculosis (MtMrx3). We correlated the number of hydrogen bonds accepted by the nucleophilic cysteines with the relatively low pKa's determined for MtMrx3 and CgMrx3. Finally, we showed that CgMrx3 has DsbA-like oxidase activity. Taken together, our results provide valuable insights into the structural and functional characteristics of Mrx3, thereby enhancing our understanding of mycoredoxin-dependent redox biology.

Electron flux is a key determinant of uranium isotope fractionation during bacterial reduction

Uranium isotopic signatures in the rock record are utilized as a proxy for past redox conditions on Earth. However, these signatures display significant variability that complicates the interpretation of specific redox conditions. Using the model uranium-reducing bacterium, Shewanella oneidensis MR-1, we show that the abundance of electron donors (e.g., labile organic carbon) controls uranium isotope fractionation, such that high electron fluxes suppress fractionation. Further, by purifying a key uranium-reducing enzyme, MtrC, we show that the magnitude of fractionation is explicitly controlled by the protein redox state. Finally, using a mathematical framework, we demonstrate that these differences in fractionation arise from the propensity for back-reaction throughout the multi-step reduction of hexavalent uranium. To improve interpretations of observed fractionations in natural environments, these findings suggest that a variable intrinsic fractionation factor should be incorporated into models of uranium isotope systematics to account for differences in electron flux caused by organic carbon availability.

Genetically Encoded Fluorescent Probe for Detection of Heme-Induced Conformational Changes in Cytochrome c.

Cytochrome c (Cytc) is a key redox protein for energy metabolism and apoptosis in cells. The activation of Cytc is composed of several steps, including its transfer to the mitochondrial membrane, binding to cytochrome c heme lyase (CCHL) and covalent attachment to heme. The spectroscopic methods are often applied to study the structural changes of Cytc. However, they require the isolation of Cytc from cells and have limited availability under physiological conditions. Despite recent studies to elucidate the tightly regulated folding mechanism of Cytc, the role of these events and their association with different conformational states remain elusive. Here, we provide a genetically encoded fluorescence method that allows monitoring of the conformational changes of Cytc upon binding to heme and CCHL. Cerulean and Venus fluorescent proteins attached at the N and C terminals of Cytc can be used to determine its unfolded, intermediate, and native states by measuring FRET amplitude. We found that the noncovalent interaction of heme in the absence of CCHL induced a shift in the FRET signal, indicating the formation of a partially folded state. The higher concentration of heme and coexpression of CCHL gave rise to the recovery of Cytc native structure. We also found that Cytc was weakly associated with CCHL in the absence of heme. As a result, a FRET-based fluorescence approach was demonstrated to elucidate the mechanism of heme-induced Cytc conformational changes with spatiotemporal resolution and can be applied to study its interaction with small molecules and other protein partners in living cells.

Identification and adaptive evolution analysis of glutaredoxin genes in Populus spp.

Glutaredoxin (GRX) is a class of small redox proteins widely involved in cellular redox homeostasis and the regulation of various cellular processes. The role of GRX gene in the differentiation of Populus spp. is rarely reported. We compared the similarities and differences of GRX genes among four sections of poplar using bioinformatics, corrected the annotations of some GRX genes, and focused on analysing their transcript profiling and adaptive evolution in Populus spp. A total of 219 GRX genes were identified in four sections of poplar, among which annotations for 13 genes were corrected. Differences in GRX genes were found between sect. Turanga, represented by P. euphratica, and other poplar sections. Most notably, P. euphratica had the smallest number of duplication events for GRX genes (n = 9) and no tandem duplications, whereas there were >25 duplication events for all other poplars. Furthermore, we detected 18 pairs of GRX genes under positive selection pressure in various sections of poplar, and identified two groups of GRX genes in the Salicaceae that potentially underwent positive selection. Expression profiling results showed that the PtrGRX34 and its orthologous genes were upregulated under stress treatments. In summary, the GRX gene family underwent expansion during poplar differentiation, and some genes underwent rapid evolution during this process, which may be beneficial for Populus spp. to adapt to environmental changes. This study may provide more insights into the molecular mechanisms of Populus spp. adaptation to environmental changes and the adaptive evolution of GRX genes.