Biochimica et Biophysica Acta (BBA) - Bioenergetics

Thylakoid membranes (TM) in cyanobacteria and chloroplasts host the light-dependent reactions of oxygenic photosynthesis, which involve a linear electron transfer (LET) chain composed of multi-subunit complexes, including notably Photosystem II (PSII). Gloeobacterales, the earliest-diverging cyanobacterial lineage, lack TM and perform photosynthesis within specialized regions of the cytoplasmic membrane (CM), thereby representing an ancestral state with respect to other cyanobacteria, all equipped with TM and known as Phycobacteria. The emergence of TM, which increased the membrane surface available for oxygenic photosynthesis, was a key innovation that likely contributed to the Great Oxidation Event. This evolutionary transition involved the formation of a distinct membrane compartment, followed by the relocation of LET components from the CM to TM. Here, we present a phylogenomic analysis identifying three candidate proteins associated with membrane trafficking that may contribute to TM biogenesis, including the SPFH family member Slr1106, which we show was acquired via lateral gene transfer. Moreover, evolutionary analysis of 36 PSII assembly factors indicates key modifications in late-stage PSII assembly, notably in manganese homeostasis. We further highlight structural changes in the early-acting YidC translocase that may have facilitated the relocation of LET components from the CM to TM. Altogether, our phylogenetic and functional prediction analyses of proteins involved in membrane dynamics and PSII assembly factors bring new insights into the molecular innovations that led to the emergence of TM.

Mitochondrial Complex III dysfunction is frequently associated with pathogenic variants in the MT-CYB gene, yet the functional consequences of many missense substitutions remain unresolved because they are classified as variants of uncertain significance (VUS). One such variant, m.14849T>C (p.Ser35Pro), has been reported in patients with multisystem mitochondrial phenotypes, including septo-optic dysplasia, cardiomyopathy, and exercise intolerance, although its structural impact on Cytochrome b function remains unclear. In this study, we employed 300 ns all-atom molecular dynamics simulations to assess structural and energetic consequences of the S35P substitution in the Cytochrome b subunit of human mitochondrial Complex III. The S35P variant did not induce global destabilization of the protein scaffold but instead promoted localized perturbations within the heme bL microenvironment. The mutation was associated with loss of a heme-proximal hydrogen-bonding network involving Ser35 and a decrease in electrostatic interaction energy between the protein matrix and the heme bL cofactor. Radial distribution function analysis further supported loosening of local packing around the prosthetic group. Consistent with these local changes, dynamics analyses indicated increased flexibility in distal transmembrane helices that form the heme-pocket scaffold and greater variability in the inter-heme Fe(bL)-Fe(bH) distance. Together, our findings suggest that S35P may exert functional effects by reorganizing the heme bL microenvironment rather than by inducing large-scale structural destabilization, underscoring the value of structure- and dynamics-based evaluation for mitochondrial VUS and suggesting a plausible mechanistic link to the pathophysiology of multisystem mitochondrial diseases.

Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin (Erithacus rubecula) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a (ErCRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing ErCRY4a with the fbIC gene that encodes for 8-HDF synthase and thus for production of 8-HDF in E. coli. As a positive control for 8-HDF binding, we expressed Xenopus laevis 6-4 photolyase (Xl6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl6-4PL, but not to ErCRY4a. We studied the binding of MTHF using in vitro reconstitution followed by UV-Vis spectroscopy and isothermal titration calorimetry (ITC) assays. No interaction was observed between MTHF and ErCRY4a. To theoretically understand the binding of potential antenna chromophores to ErCRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of ErCRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that ErCRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

Sulfide:quinone oxidoreductase (SQR) is a critical enzyme that maintains sulfur metabolism by oxidizing sulfide to supersulfides, currently defined as sulfur metabolites with six valence electrons and no charge that are covalently catenated with other sulfur atoms and excludes disulfides. While SQR is known to contribute to mitochondrial electron transport, its physiological impact on systemic energy metabolism and longevity remains largely undefined. In this study, we investigated the role of SQR in mitochondrial bioenergetics and aging using SQR-deficient Schizosaccharomyces pombe ({Delta}hmt2) and a mitochondria-selective SQR-deficient (Sqrdl{Delta}N/{Delta}N) mice model. Functional analysis demonstrated that{Delta} hmt2 grew normally in glucose but not in glycerol, indicating impaired mitochondrial respiration. It showed reduced membrane potential, ATP, and lifespan. Consistent with the yeast findings, Sqrdl{Delta}N/{Delta}N mice exhibited accumulated levels of hydrogen sulfide and persulfides, and demonstrated impaired mitochondrial energy metabolism. Furthermore, supersulfide donor supplementation selectively conferred lifespan extension in wild-type yeast, but not in SQR-deficient strain, and similarly improved mitochondrial function exclusively in wild-type mouse embryonic fibroblasts, with no benefit observed in SQR-mutant counterparts. Together, our findings demonstrate that mitochondrial SQR plays an essential role in sulfur respiration, critically supporting mitochondrial function and organismal longevity across eukaryotes. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/716515v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@16d4da7org.highwire.dtl.DTLVardef@10514cdorg.highwire.dtl.DTLVardef@98b9ecorg.highwire.dtl.DTLVardef@d6667f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeveloped an SQR-deficient S. pombe ({Delta}hmt2) model that exhibits sulfur metabolism, mitochondrial dysfunction, and shortened chronological lifespan C_LIO_LISulfide and supersulfide donors prolong yeast lifespan in a SQR-dependent manner C_LIO_LIMitochondrial SQR is essential for membrane potential formation and ATP production in yeast and mammals C_LI

Lebers Hereditary Optic Neuropathy (LHON) is a rare genetic condition and severe neurological disorder characterized by dysfunctional mitochondria under extreme oxidative stress, resulting in retinal ganglion cell death and subsequent rapid bilateral loss of central vision. The m.14484T>C mutation in the ND6 subunit of mitochondrial complex I is known for inducing LHON, and is a prevalent LHON-associated mutation, yet its mechanism of impairment at the molecular level is currently unresolved. In this study, we explore the biophysical underpinnings of this mutation and its role in LHON through disruption of human complex I function. We consider, using atomistic simulations, the differential thermodynamics and kinetics of coenzyme Q10 binding between the mutant and wild-type forms, altered dynamics of the complex upon mutation, and key interactions between coenzyme Q10 and complex I binding sites. The hydrogen bond network present near and within the coenzyme Q10 binding domain, along with proper hydration of E-channel residues that couple redox chemistry to proton pumping, is found to be critical for complex I stability and quinone binding, which the ND6-centered mutation disrupts.

Autotrophic Fe(II) oxidation is a key microbial process linking iron cycling to global carbon and nitrogen turnover in anoxic and low-oxygen environments. While Fe(II) oxidation, and electron utilization by canonical denitrification or oxygen-reducing pathways have been proposed, the whole electron transport chain via the periplasm is unknown. To address this gap, we compared the transcriptome of Ferrigenium straubiae strain KS (= KCTC 25982; = DSM 118991) cultivated under autotrophic conditions using Fe(II) as the electron donor and either nitrate or oxygen as the terminal electron acceptor. Additionally, we reanalyzed the genome using bioinformatic tools (FeGenie, MHCScan, Foldseek, AlphaFold, COFACTOR, and InterProScan) to classify known, putative, and previously uncharacterized proteins. Using these analyses, we (i) identified a form II RubisCO-mediated, sedoheptulose-7-phosphate-forming transaldolase variant of the Calvin-Benson-Bassham cycle, (ii) revealed the metabolic possibility of a reversed oxidative TCA cycle and an alternative CO2 fixation route via 10-formyl-tetrahydrofolate, (iii) found novel structural features of the proposed Fe(II) oxidases (Cyc2 and MtoA/B: {beta}-barrel outer membrane cytochromes), (iv) observed two previously unrecognized redox-complexes specifically upregulated under nitrate-reducing Fe(II)-oxidizing conditions and (v) uncovered two sphaeroides heme protein homologues and one homologue of the closely related cytochrome c. RepositoriesGenBank whole genome sequence of strain KS: JAGRPI00000000 IMG Taxon ID of strain KS: 2878407288 NCBI BioProject ID of RNA-Seq data: PRJNA1399691 Strain availability: KCTC 25982 and DSM 118991 Normalized transcripts and Log2FoldChanges (Excel file), and supplementary information are available with the online version of this article. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/701106v1_ufig1.gif" ALT="Figure 1000"> View larger version (30K): org.highwire.dtl.DTLVardef@1d467bdorg.highwire.dtl.DTLVardef@1e6b270org.highwire.dtl.DTLVardef@ccdf37org.highwire.dtl.DTLVardef@52cec2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO2 fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH-III (CpBC1CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. CpBC1CODH-III shows moderate CO oxidation (150 U/mg) and CO2 reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal at g {approx} 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g {approx} 1.75, g {approx} 1.72), indicative of a bound CO2. Investigation of maturation effects showed that co-expression of CooCTJ stabilised CpBC1CODH-III production, but did not enhance maximum activity, which was primarily influenced by nickel availability. Comparative operon analysis with the well-studied clade F Rhodospirillum rubrum CODH (RrCODH) revealed high structural similarity in CODH and CooC, but significant divergence in CooJ, with conserved metal-binding regions identified via AlphaFold3 modelling and dot plot analysis. CpBC1CODH-III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. Here, we use site-directed mutagenesis to study the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Mutations at this position change the biochemical properties (KM for CO, product inhibition constant, catalytic bias...), and increase the resistance of the enzyme to the inhibitor O2, showing that isoleucine 563 indeed lines the gas channel. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far. We show that the size of the introduced amino acids is less important than their flexibility - along with the size of the cavity formed near the active site in the channel. We also conclude that O2 access to the active site cannot be slowed down without also affecting CO diffusion. This tradeoff will have to be considered in further attempts to use site-directed mutagenesis to make CODHs more O2 tolerant.

Proton pumping by respiratory Complex I is one essential element for generating the proton motive force that drives ATP synthesis in mitochondria. Although it is understood that electrons from NADH reduce ubiquinone at the peripheral arm and that four protons are transferred in the membrane domain, the mechanism by which this redox reaction initiates proton translocation remains unclear. A lateral pathway linking the quinone binding site to the membrane domain via ND1, ND3, and ND4L subunits has been proposed as the initial path of an excess proton. However, in experimental structures this region lacks a continuous water network between D66ND3 and E34ND4L, resulting in a hydration bottleneck that may regulate proton transfer. Using multiscale reactive molecular dynamics (MS-RMD) and a water wire connectivity metric, we directly simulate proton transport through this region as coupled the the hydration by water molecules. Our results reveal that proton transfer is thermodynamically feasible when transient hydration aligns with the presence of an excess proton, revealing the strong coupling between hydration and proton (PT) in this region of Complex I. These findings support a model where proton injection enhances local hydration, dynamically opening the pathway for proton transfer and regulating the onset of proton pumping in Complex I.

A method to measure telomere length in S. cerevisiae was developed based on bioluminescence resonance energy transfer (BRET). The system uses energy transfer between a luciferase-Rif2 fusion protein and fluorescently tagged Rap1. The study demonstrates that the BRET ratio correlates with the Rap1/Rif2 complex at the telomeres and thus the availability of telomeric Rap1 binding sites. This enables the measurement of telomere length in living cells. The system was able to reproduce reported deviations in telomere length in mutants lacking telomere length regulators, cells treated with telomere length modifying compounds and strains expressing inducible telomerase. The BRET ratio linearly correlated with the average number of telomeric nucleotides derived from long-read sequencing data using a novel algorithm for telomere length calculation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711003v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1850c4dorg.highwire.dtl.DTLVardef@1ead295org.highwire.dtl.DTLVardef@1a76358org.highwire.dtl.DTLVardef@6b3183_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lactate dehydrogenase (LDH) stands at the intersection of pyruvate metabolism. While it is believed that inhibition of LDH redirects pyruvate to mitochondrial metabolism, suppressing glycolysis and boosting oxidative phosphorylation, the mechanism remains largely unexplored. We found that individual LDH A or B knockouts had minimal impact on glycolysis, tricarboxylic acid cycle (TCA cycle), or oxidative phosphorylation (OXPHOS). However, combining LDH knockout with LDH inhibitor GNE-140 significantly suppressed these processes. Inhibition of LDH led to an increase in free NADH concentration and a decrease in free NAD+ concentration, the reduced free NAD+ concentration inhibited GAPDH, disrupting the balance of glycolytic intermediates, which were linked with thermodynamic shift of the Gibbs free energy of reactions between phosphofructokinase 1 (PFK1) and phosphoglycerate mutase (PGAM) in the glycolytic pathway, favoring their reverse direction. This disrupted glycolysis led to impaired TCA cycle and mitochondrial respiration due to reduced pyruvate and glutamine carbon influx into TCA cycle. Under hypoxia, LDH inhibition had a stronger effect, inducing energy crisis, redox imbalance, and cancer cell death. Our study reveals LDHs intricate control over glycolysis, TCA cycle, and mitochondrial respiration, highlighting the interplay of enzyme kinetics and thermodynamics in metabolic pathways, a crucial aspect for understanding metabolic regulation. Impact statementThis study elucidates a biochemical mechanism by which lactate dehydrogenase influences glycolytic flux in cancer cells, revealing a kinetic- thermodynamic interplay that contributes to metabolic regulation.

Long-range protein electron transfer (ET) often depends on tryptophan and tyrosine residues acting as radical relay sites. For example, cytochrome c peroxidase (CcP) generates a W191*+ radical to increase ET from cytochrome c (Cc) to the active center. W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base at position 232 (as Glu or His) recovers activity. E232 fluorination shifts the ET pH dependence to lower values, verifying that a hydrogen bond elevates the Y191* formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on activating Y191 with a basic residue, but through a different mechanism than for the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the basic residue and ZnP*+, respectively, facilitate Y191* formation. Replacing Cc with the irreversible oxidant [Co(NH3)5Cl]2+ isolates distinct protein radicals for characterization by Electron Paramagnetic Resonance (EPR) spectroscopy. Radical distributions reveal that W191*+ lies [~]15 mV in potential below ZnP*+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, ZnCcP Y,G191:E,H232 variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP*+/Trp*+ and the importance of a hydrogen bond to Y191* for maintaining a high potential to oxidize peripheral donors. These resolved reactivity patterns of CcP/ZnCcP have general relevance for engineering proton management to separate and migrate charge in proteins and potentially other molecular systems.

Synthetic 6-Br-Q0C10 has been shown to exhibit a partial electron transfer activity of native coenzyme Q in the isolated mitochondria. It reduces energy coupling efficiency by approximately 30%, suggesting that it may be useful in modulating cell growth in tissue culture. Whether or not it behaves in the same way in the whole cells, or animal, however, has not yet been fully examined. Recently we have investigated the effect of 6-Br-Q0C10 across multiple cell lines using three detection methods. Treatment with 6-Br-Q0C10 reduces cell proliferation in all cell lines tested, with different effectiveness. Obesity-related cell lines were the most susceptible, and a pronounced inhibitory effect was also observed in cancer cell lines. These results strengthen the idea of using 6-Br-Q0C10 to manage obesity or to retard the growth of rate cancer cells and thus prolonging life.

Oxidative phosphorylation is the most efficient way of generating ATP in respiring cells. As high energy electrons are the major source of reactive oxygen species their production needs to be carefully calibrated. In most organisms, NADH dehydrogenase serves as the primary source and gateway of electrons. This complex is responsible for oxidizing NADH to NAD+, which liberates two electrons that are then fed into the respiratory chain. In the Gram-positive model bacterium, Bacillus subtilis, a transcription factor (Rex) is utilized to monitor the rise in NADH level and subsequently increase the production of the NADH dehydrogenase Ndh. Thus, the generation of electrons through this pathway is tightly regulated. In this report, we reveal the presence of another independent mechanism to moderate Ndh activity involving a previously uncharacterized protein, YfhS. Additionally, we present the first experimental evidence showing that the functional NADH dehydrogenase is a two-protein complex comprised of a membrane-associated YjlC and the enzyme Ndh. We find that absence of YfhS leads to cell morphology and growth defects that are corrected by spontaneous mutations in ndh. We note that increased production of NADH dehydrogenase complex proteins by itself is not detrimental. However, strikingly, it is lethal in a strain lacking yfhS. These results reveal that YfhS is an important moderator of NADH dehydrogenase activity. We also demonstrate that YfhS and YjlC are interaction partners. A model developed based on our data indicates that YfhS is an important regulator of intracellular NADH concentration. Compounds that target specific microbial (Type II) NADH dehydrogenase, which is absent in human mitochondria, are considered promising drug candidates to help address the threat posed by antibiotic-resistant bacteria. Overall, our data unveiling the importance of YfhS and YjlC in controlling Ndh activity could be harnessed for the development of new therapeutics.

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.

Glucan-water-dikinase 1 (GWD1) plays an essential role in regulating starch metabolism in plants via O-6 phosphorylation of amylopectin. Here, we used biochemical characterization, AlphaFold2 modeling, X-ray crystallography and Small-Angle X-ray Scattering (SAXS) experiments to study its structure and catalytic mechanism. The protein is organized into five domains with two carbohydrate-binding modules (CBMs) at its N-terminal end followed by a central domain, whose structure was solved by X-ray crystallography in open and closed conformations. Next comes the domain carrying the catalytic histidine and the ATP-binding domain. We studied the spatial arrangement of the full enzyme and of several truncated forms by SAXS-driven modeling and identified a pivoting movement of the Histidine domain consistent with the enzymes autophosphorylation and subsequent phosphate transfer to a glucan. Our data suggest important residues at the domain interfaces that might assist catalysis and we hypothesize that the second CBM helps maintaining the catalytic domain close to the glucan chain for productive phosphate transfer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/704335v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1b860e5org.highwire.dtl.DTLVardef@1e172dcorg.highwire.dtl.DTLVardef@3c03edorg.highwire.dtl.DTLVardef@25c0d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Electrolytic hydrogen water (EHW) plays a critical role in modulating cellular metabolism; yet, the underlying molecular mechanisms remain unclear. This study utilized next-generation sequencing (NGS) to assess mRNA and miRNA expression in EHW-treated Caco-2 cells. Bioinformatics analysis identified differentially expressed genes (DEGs) and pathways influenced by EHW and highlighted its involvement in the oxidative stress response and tight junction formation. Protein-protein interaction (PPI) network analysis of the DEGs identified first-neighbor genes, supporting the role of EHW in suppressing oxidative stress-related genes while also enhancing the expression of the TCEB2-CUL5-COMMD8 (ECS complex) genes, both of which converged on the HIF-1 signaling pathway. We also constructed an mRNA-miRNA competing endogenous RNA (ceRNA) network, which revealed four hub genes, two non-coding RNAs (miR-429 and miR-200c-3p) and two protein-coding RNAs (CUL5 and GOLGA7). These genes co-target the transcription factor KLF4 in Caco-2 cells, forming a TF-miRNA-gene network (TMGN). EHW treatment significantly decreased the levels of miR-429 and miR-200c-3p and stabilized CUL5 and GOLGA7 transcripts post-transcriptionally as compared to ACW. Concurrently, reduced miRNA expression weakened their pre-transcriptional competition with mRNAs for KLF4 binding, further enhancing CUL5 and GOLGA7 expression. Phenotypic assays confirmed that continuous EHW treatment promotes Caco-2 cell differentiation. This study underscores the regulatory role of EHW in intestinal cells via feed-forward loops (FFLs), offering novel insights into the molecular mechanisms and functions of EHW. HighlightsO_LIIdentification of Novel Key Regulatory Genes Modulated by Electrolytic Hydrogen Water (EHW) Treatment: PPI network analysis demonstrated that EHW downregulates mitochondrial oxidative metabolism-related genes while upregulating TCEB2-CUL5-COMMD8 (ECS complex) expression within the HIF-1 axis. C_LIO_LIConstruction of a ceRNA Network: By integrating transcriptome and miRNA sequencing data from EHW-treated samples, we assembled an associated network and identified four hub genes in intestinal cells within the mRNA-miRNA ceRNA network: miR-429, miR-200c-3p, CUL5, and GOLGA7. C_LIO_LINovel Mechanistic Insights of Post- and Pre-Transcriptional Regulation by EHW: We identified KLF4 as a key transcription factor regulating EHW hub genes and constructed a TF-miRNA-gene (TMGN) feed-forward loop (FFL) network, offering new insights into EHW biomarkers. Our analysis revealed that EHW reduces miR-429 and miR-200c-3p levels, thereby enhancing CUL5 and GOLGA7 expression through both pre-transcriptional and post-transcriptional regulation. C_LIO_LIPhenotypic Confirmation: Continuous EHW treatment shortened the time required for Caco-2 cell differentiation. C_LI

Methanogenesis is a crucial component of Earths carbon cycle and a source of methane for biofuel production. The presence of higher energy electron acceptors, such as iron(III) oxides and quinones, is believed to significantly impact methanogenesis. This study investigated the physiological and proteomic responses of the type I Methanosarcina, M. barkeri, to the artificial quinone 9,10-anthraquinone-2,7-disulfonate disodium (2,7-AQDS), using H2/CO2 as substrates. Our findings revealed that during 2,7-AQDS reduction, cellular growth ceased. The lack of energy conservation was associated with direct inhibition of both methanogenesis and CO2 utilization, corroborated by a significant downregulation of the enzymes involved in this metabolic pathway. Furthermore, the significant upregulation of specific subunits of the reversible Ech hydrogenase suggests that this enzyme redirects electrons from H2 towards the most energetically favorable reaction (2,7-AQDS reduction), rather than the reduction of ferredoxin, which is a highly energy-demanding process, essential for initiating the CO2 reduction pathway. Additionally, it is conceivable that Ech homologues in other hydrogenotrophic methanogens also participate in the reduction of higher energy-yielding electron acceptors. These findings provide novel insights into how quinones, particularly in their oxidized state, directly impact methanogenesis, thereby influencing both artificial and natural methanogenic environments.

Experimental assessment and computational modeling were used to analyze substrate transport, tricarboxylic acid cycle kinetics, and oxidative phosphorylation in suspensions of purified cardiac mitochondria. The kinetics of ATP synthesis and carbohydrate oxidation, including during hypoxia and reoxygenation, were investigated using various substrate combinations and conditions. Model simulations fit to transient respiration and NAD(P)H measurements reveal novel insights into pyruvate dehydrogenase regulation, regulation of mitochondrial leak, and the clearance of oxaloacetate during respiration on succinate. High concentrations of succinate induced increased mitochondrial leak respiration driven in part by ROS-activated uncoupling. Oxidative phosphorylation under succinate-fueled respiration was inhibited by rapid buildup of oxaloacetate, inhibiting succinate dehydrogenase. Malic enzyme and oxaloacetate decarboxylase activities represent potenital pathways for removal of oxaloacetate, with glutamate further enhancing clearance. The developed model captures the observed transient behaviors as well as steady-state relationships between ATP synthesis rate and phosphate metabolite levels, lending a new systems-level understanding of mitochondrial energy metabolism. In sum, these findings offer a framework for simulating and interpreting mitochondrial function in vitro and in vivo. Key PointsThis study uses experiments and computer simulations to probe the interactions between substrate transport processes, TCA cycle kinetics, redox state, and oxidative ATP synthesis in cardiac mitochondria. The developed kinetic model simulates mitochondrial metabolism in vitro and represents a framework for integrative modeling of cardiac energy metabolism. Model-based analysis identifies a kinetic model of pyruvate dehydrogenase (PDH) deactivation during leak-state respiration and activation during oxidative phosphorylation. High levels of cation leak during respiration on succinate are explained by a ROS-dependent activation of uncoupling.