Respiratory Chain Research Articles

Mitochondria-Activated Wash-Free Fluorescent Probe for Visualizing Single-Cell Photodamage.

Cell viability assessment is essential in biological and medical research. There is a growing demand for fluorescent probes that can rapidly, reliably, and wash-free evaluate cell viability in complex scenarios. Electron transport chain (ETC) is one of the earliest indicators of cellular distress and holds great potential for assessing cell viability. In this study, we introduce Rhodalive, a mitochondria-targeted ETC-activated fluorescence turn-on probe for single-cell viability assessment. Rhodalive is specifically activated by free electrons leaked from the ETC and localized to the mitochondria, enabling wash-free, spatiotemporal, and super-resolution fluorescence imaging of active mitochondria. Moreover, Rhodalive can effectively distinguish live cells from fixed cells, quantify H2O2-induced cell damage, and visualize single-cell photodamage induced by localized blue light exposure and photodynamic therapy. Rhodalive provides a convenient and reliable tool for dynamically assessing single-cell viability with promising applications in evaluating early mitochondrial dysfunction and advancing drug discovery.

Exploiting metabolic vulnerability in glioblastoma using a brain-penetrant drug with a safe profile.

Glioblastoma is one of the most treatment-resistant and lethal cancers, with a subset of self-renewing brain tumour stem cells (BTSCs), driving therapy resistance and relapse. Here, we report that mubritinib effectively impairs BTSC stemness and growth. Mechanistically, bioenergetic assays and rescue experiments showed that mubritinib targets complex I of the electron transport chain, thereby impairing BTSC self-renewal and proliferation. Gene expression profiling and Western blot analysis revealed that mubritinib disrupts the AMPK/p27Kip1 pathway, leading to cell-cycle impairment. By employing in vivo pharmacokinetic assays, we established that mubritinib crosses the blood-brain barrier. Using preclinical patient-derived and syngeneic models, we demonstrated that mubritinib delays glioblastoma progression and extends animal survival. Moreover, combining mubritinib with radiotherapy or chemotherapy offers survival advantage to animals. Notably, we showed that mubritinib alleviates hypoxia, thereby enhancing ROS generation, DNA damage, and apoptosis in tumours when combined with radiotherapy. Encouragingly, toxicological and behavioural studies revealed that mubritinib is well tolerated and spares normal cells. Our findings underscore the promising therapeutic potential of mubritinib, warranting its further exploration in clinic for glioblastoma therapy.

Metformin Induces Apoptosis and Ferroptosis of Ovarian Cancer Cells Under Energy Stress Conditions

As ovarian cancer progresses, increased glucose use causes a glucose shortage in the tumor microenvironment. Therefore, it is crucial to find drugs that can effectively kill cancer cells in this energy stress setting. Here, we propose an effective therapeutic strategy that combines nutrient restriction with metformin to combat tumors. This study investigated the effects of metformin on ovarian cancer cells under energy stress conditions, mimicking the nutrient-deprived tumor microenvironment. We revealed that Metformin (10 mM) significantly reduced cell viability and proliferation under glucose deprivation conditions. Furthermore, it enhanced apoptosis and ferroptosis, as demonstrated by alterations in apoptotic protein expression and elevated levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA), lipid peroxidation (LPO), and Fe2+. Transcriptional profiling revealed significant alterations in genes related to iron homeostasis and oxidative phosphorylation. Moreover, Metformin was found to induce mitochondrial dysfunction without affecting mitochondrial DNA or the expression of enzymes in the tricarboxylic acid (TCA) cycle, resulting in decreased ATP production and compromised activities of the respiratory chain complexes. The direct interaction between metformin and the NDUFB4 subunit in mitochondrial complex I was corroborated through the application of cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays. In vivo, the combination of metformin and fasting cycles significantly inhibited SKOV3 cell-derived xenograft tumors in immunodeficient mice. Altogether, we have demonstrated that Metformin potentiates apoptosis and ferroptosis in ovarian cancer cells under energy stress conditions by targeting the NDUFB4 subunit of mitochondrial complex I, thus laying the groundwork for clinical testing. This study, though limited to cellular and animal levels, provides valuable insights into the therapeutic potential of metformin in ovarian cancer treatment.

Estimation of light utilisation and antioxidative protection in an alpine plant species (Soldanella alpina L.) during the leaf life cycle at high elevation.

Photosynthesis, electron transport to carbon assimilation, photorespiration and alternative electron transport, light absorption of the two photosystems, antioxidative protection and pigment contents were investigated in S. alpina leaves. S. alpina is an alpine snow-bed plant which can be found with green leaves after snowmelt. At least 24% of the leaves were formed at the beginning of the vegetation period in the previous year and survived two consecutive vegetation periods under contrasting environmental conditions. In leaves still covered by snow (SNOW), the parameters of antioxidative protection and carbon assimilation were lower than in leaves from the previous vegetation period (NEW) or several weeks after snowmelt (OLD). Directly after snowmelt, antioxidative protection was strongly but transitionally increased. The senescence of leaves did not depend on antioxidative scavenging capacity. Lower carbon assimilation was not related to increases in alternative electron flow (ETRalt) in SNOW leaves. In the second vegetation period, light absorption by PSII decreases in favour of PSI in OLD leaves. This allows OLD leaves to keep the electron transport chain more oxidised and to support photorespiration with increased ATP synthesis by cyclic electron transport around PSI. This study describes how the leaves of a unique plant can cope with contrasting environmental conditions.

Acetylation of proximal cysteine-lysine pairs by alcohol metabolism.

Alcohol consumption induces hepatocyte damage through complex processes involving oxidative stress and disrupted metabolism. These factors alter proteomic and epigenetic marks, including alcohol-induced protein acetylation, which is a key post-translational modification (PTM) that regulates hepatic metabolism and is associated with the pathogenesis of alcohol-associated liver disease (ALD). Recent evidence suggests lysine acetylation occurs when a proximal cysteine residue is within ∼15Å of a lysine residue, referred to as a cysteine-lysine (Cys-Lys) pair. Here, acetylation can occur through the transfer of an acetyl moiety via an S → N transfer reaction. Alcohol-mediated redox stress is known to occur coincidentally with lysine acetylation, yet the biochemical mechanisms related to cysteine and lysine crosstalk within ALD remain unexplored. A murine model of ALD was employed to quantify hepatic cysteine redox changes and lysine acetylation, revealing that alcohol metabolism significantly reduced the cysteine thiol proteome and increased protein acetylation. Interrogating both cysteine redox and lysine acetylation datasets, 1280 protein structures generated by AlphaFold2 represented by a 3D spatial matrix were used to quantify the distances between 557,815 cysteine and lysine residues. Our analysis revealed that alcohol metabolism induces redox changes and acetylation selectively on proximal Cys-Lys pairs with an odds ratio of 1.88 (p<0.0001). Key Cys-Lys redox signaling hubs were impacted in metabolic pathways associated with ALD, including lipid metabolism and the electron transport chain. Proximal Cys-Lys pairs exist as sets with four major motifs represented by the number of Cys and Lys residues that are pairing (Cys1:Lys1, Cysx:Lys1, Cys1:Lysx and Cysx:Lysx) each with a unique microenvironment. The motifs are composed of functionally relevant Cys-Ly altered within ALD, identifying potential therapeutic targets. Furthermore, these unique Cys-Lys redox signatures are translationally relevant as revealed by orthologous comparison with severe alcohol-associated hepatitis (SAH) explants, revealing numerous pathogenic thiol redox signals in these patients.

MDPAO1 peptide from human milk enhances brown adipose tissue thermogenesis and mitigates obesity.

The regulatory effect of breastfeeding on offspring metabolism has garnered significant attention as an effective strategy in combating childhood obesity. However, the underlying mechanism remains largely unknown. Through integrated analysis of multiple human milk peptide databases and functional screening, MDPAO1 (milk-derived peptide associated with obesity 1) was identified as having potential activity in promoting the expression of thermogenic genes. In lactating mice, intervention with MDPAO1 enhanced the thermogenic phenotype of brown adipose tissue (BAT) and overall metabolic activity. Moreover, MDPAO1 intervention led to reduced body weight gain, increased brown fat mass, and improved glucose tolerance and insulin sensitivity in a mouse model of high-fat diet (HFD)-induced obesity. RNA-seq analysis of BAT post-MDPAO1 intervention revealed close association with mitochondrial oxidative respiratory chain and mitophagy. Subsequent in vitro experiments conducted on primary brown adipocytes confirmed that MDPAO1 inhibited mitophagy, increased mitochondrial mass, and elevated levels of mitochondrial respiratory chain complexes. In conclusion, this study underscores the potential of MDPAO1, a peptide enriched in breast milk, in activating the thermogenic phenotype of brown adipose tissue and mitigating obesity, thus offering novel insights into the mechanisms underlying breastfeeding's role in preventing childhood obesity.

Celastrol ameliorates lipopolysaccharide (LPS)-induced acute lung injury by improving mitochondrial function through AMPK/PGC-1α/Nrf1-dependent mechanism

Acute lung injury (ALI) is a devastating clinical syndrome without effective therapy. Celastrol, as a natural anti-inflammatory compound, has showed therapeutic potential against inflammatory diseases. In this study, we have investigated the potential effect of Celastrol on lipopolysaccharide (LPS)-induced ALI. C57BL/6J mice, Nrf1-knockout mice and A549 (human alveolar epithelial cell line) cells were used to investigate the protective role of Celastrol in LPS-induced ALI. Our data showed that administration of Celastrol significantly alleviated lung pathologic injury and increased the survival rate, which was associated with the improvement of mitochondrial function in the injured lung. Moreover, Celastrol enhanced phosphorylation of AMP-activated protein kinase (AMPK) and expression of peroxisome proliferator-activated receptor coactivator protein-1α (PGC-1α), thereby increasing the nuclear translocation of nuclear respiratory factor 1 (Nrf1) and subsequent up-regulation of its downstream mitochondria electron transport chain complex I (NDUF) gene expression, which induced an increase in mitochondrial complex Ⅰ activity. The beneficial effects of Celastrol on regulation of Nrf1 were abolished by inhibition of AMPK and PGC-1α. Finally, in Nrf1 deficient mice, the protective effects of Celastrol on LPS-induced ALI were largely vanished. Our data indicated that Celastrol can prevent LPS-induced ALI by improving mitochondrial function through AMPK/PGC-1α/Nrf1-dependent mechanism, suggesting that Celastrol may represent a novel therapeutic potential for LPS-induced ALI.

Essential role of sulfide oxidation in brain health and neurological disorders.

Hydrogen sulfide (H2S) is an environmental hazard well known for its neurotoxicity. In mammalian cells, H2S is predominantly generated by transsulfuration pathway enzymes. In addition, H2S produced by gut microbiome significantly contributes to the total sulfide burden in the body. Although low levels of H2S is believed to exert various physiological functions such as neurotransmission and vasomotor control, elevated levels of H2S inhibit the activity of cytochrome c oxidase (i.e., mitochondrial complex IV), thereby impairing oxidative phosphorylation. To protect the electron transport chain from respiratory poisoning by H2S, the compound is actively oxidized to form persulfides and polysulfides by a mitochondrial resident sulfide oxidation pathway. The reaction, catalyzed by sulfide:quinone oxidoreductase (SQOR), is the initial and critical step in sulfide oxidation. The persulfide species are subsequently oxidized to sulfite, thiosulfate, and sulfate by persulfide dioxygenase (ETHE1 or SDO), thiosulfate sulfurtransferase (TST), and sulfite oxidase (SUOX). While SQOR is abundantly expressed in the colon, liver, lung, and skeletal muscle, its expression is notably low in the brains of most mammals. Consequently, the brain's limited capacity to oxidize H2S renders it particularly sensitive to the deleterious effects of H2S accumulation. Impaired sulfide oxidation can lead to fatal encephalopathy, and the overproduction of H2S has been implicated in the developmental delays observed in Down syndrome. Our recent findings indicate that the brain's limited capacity to oxidize sulfide exacerbates its sensitivity to oxygen deprivation. The beneficial effects of sulfide oxidation are likely to be mediated not only by the detoxification of H2S but also by the formation of persulfide, which exerts cytoprotective effects through multiple mechanisms. Therefore, pharmacological agents designed to scavenge H2S and/or enhance persulfide levels may offer therapeutic potential against neurological disorders characterized by impaired or insufficient sulfide oxidation or excessive H2S production.

Menaquinone-specific turnover by Mycobacterium tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond.

Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained interest as an antibiotic target because of its importance in survival under infectious conditions. Mtbd contains a characteristic disulfide bond that has been hypothesized to allow for Mtbd activity regulation at the enzymatic level, possibly helping M.tuberculosis to rapidly adapt to the hostile environment of the phagosome. Here, the role of the disulfide bond and quinone specificity have been determined by reconstitution of a minimal respiratory chain and the single-particle cryo-EM structure in the disulfide-reduced form. Mtbd was shown to be specific for menaquinone, while regulation by reduction of the Q-loop disulfide bond decreased oxidase activity up to 90%. Structural analysis shows that a salt bridge unique to Mtbd keeps the Q-loop partially structured in its disulfide-reduced form, which could facilitate the rapid activation of Mtbd upon exposure to reactive oxygen species. We signify Mtbd as the first redox sensory terminal oxidase and propose that this helps M.tuberculosis in the defense against reactive oxygen species encountered during infection.

Intra/extracellular transmembrane electron transfer for bioelectric enhancement mediated by in-situ bio-FeS and polypyrrole coating

This study involves the construction of an “inside-out” electronic network channel with in-situ formation of biogenic FeS (bio-FeS) nanoparticles in cells of Shewanella oneidensis MR-1 (S. oneidensis MR-1) and a coating of conductive polypyrrole (PPy) on the outer surface of entire cells. The microbial fuel cell driven by bio-FeS and PPy co-modified S. oneidensis MR-1 (FeS/PPy@MR-1) has a maximum power density 3.6 times higher than that of the native strain. Electrochemical analysis demonstrates that the modifications of bio-FeS and PPy markedly enhance the direct electron transfer and c-type cytochrome-bound flavin-mediated single-electron reaction pathways and expand the electron output flux. The electron-donating ability of FeS/PPy@MR-1 is 7 times greater than that of the native strain. Bio-FeS and PPy can mediate novel intra/extracellular transmembrane electron transport pathways independent of the traditional respiratory chain, bridge spatially discrete redox environments, and optimize electron transfer pathways to intracellular to extracellular transfer electrons rapidly and maximally. These findings provide insights into conductive nanomaterial-mediated intra/extracellular transmembrane electron transfer, which is critical for the environment and bioenergy fields.

Period3 modulates the NAD+-SIRT3 axis to alleviate depression-like behaviour by enhancing NAMPT activity in mice.

PER3 deficiency is associated with depression-like behaviors, but the underlying mechanisms remain unclear. This study aims to elucidate the role and mechanism of PER3 in regulating depression-like behaviors in mice. Depression-like behaviors were assessed using the sucrose preference test, tail suspension test, and forced swimming test. Metabolomic analysis was conducted on hippocampal tissues from Per3 knockout mice using chromatography-mass spectrometry. The regulatory role of PER3 on the expression of nicotinamide phosphoribosyltransferase (Nampt) was investigated through co-immunoprecipitation and chromatin immunoprecipitation assays. Metabolomic analysis revealed that Per3 deficiency disrupts mitochondrial function, as evidenced by reduced activities of key tricarboxylic acid (TCA) cycle enzymes (succinate dehydrogenase, citrate synthase, and α-ketoglutarate dehydrogenase), diminished expression of mitochondrial respiratory chain complexes I-V, and decreased nicotinamide adenine dinucleotide (NAD)+ levels in Per3 knockout mice. Supplementation with the NAD+ precursor nicotinamide (NAM) rescued mitochondrial function and alleviated depression-like behaviors in Per3 knockout mice. Similar effects were observed with intraperitoneal administration of the NAMPT activator P7C3-A20, while these effects were abolished by the NAMPT inhibitor FK866. Mechanistically, PER3 was found to regulate Nampt expression by binding to E-box elements within its intronic regions in conjunction with BMAL1. This interaction enhanced NAD+ production, activating SIRT3 to mitigate mitochondrial dysfunction in Per3 knockout mice. These findings uncover a novel mechanism by which PER3 ameliorates depressive behaviors through the regulation of NAMPT-controlled NAD+ levels and mitochondrial function, underscoring the critical role of PER3 in depression-related pathophysiology.

Electrical homeostasis of the inner mitochondrial membrane potential

The electric potential across the inner mitochondrial membrane must be maintained within certain bounds for the proper functioning of the cell. A feedback control mechanism for the homeostasis of this membrane potential is proposed whereby an increase in the electric field decreases the rate-limiting steps of the electron transport chain (ETC). An increase in trans-membrane electric field limits the rate of proton pumping to the inter-membrane gap by slowing the ETC reactions and by intrinsically induced electroporation that depolarizes the inner membrane. The proposed feedback mechanism is akin to a Le Chatelier's-type principle of trans-membrane potential feedback control.

Novel intronic variant in NDUFS7 gene results in mitochondrial complex I assembly defect with early basal ganglia and midbrain involvement with progressive neuroimaging findings.

Leigh syndrome is the most common phenotype of mitochondrial disorders in children. This study demonstrates clinical, neuroradiological, and molecular genetic findings in siblings with Leigh syndrome and isolated complex I assembly defect associated with intronic c.16 + 5G > A variant in the NDUFS7 gene. Whole exome sequencing was carried out to identify the causative variant. The gene and protein expression of NDUFS7 were studied using patient-derived fibroblasts. Assembly of mitochondrial respiratory chain enzymes was analyzed using Blue Native PAGE. This study shows that the NDUFS7 c.16 + 5G > A variant (rs375282422) has a causative role in Leigh syndrome. Evolution of neuroimaging findings related to this gene variant are demonstrated.

Mitochondrial respiration in white adipose tissue is dependent on body mass index and tissue location in patients undergoing oncological or parietal digestive surgery.

Adipose tissue (AT), is a major endocrine organ that plays a key role in health and disease. However, adipose dysfunctions, especially altered energy metabolism, have been under-investigated as white adipocytes have relatively low mitochondrial density. Nevertheless, recent studies suggest that mitochondria could play a major role in AT disorders and that AT mitochondrial activity could depend on adiposity level and location. This clinical study aimed to evaluate mitochondrial respiration and metabolism in human visceral (vAT) and subcutaneous (scAT) AT and their relationship with body mass index (BMI). This clinical study enrolled 67 patients (30 females/37 males) scheduled for digestive surgery without chemotherapy and parietal infection. BMI ranged from 15.4 to 51.9 kg·m-2 and body composition was estimated by computed tomographic images. Mitochondrial respiration was measured insitu in digitonin-permeabilized AT using high-resolution respirometry and a substrate/inhibitor titration approach. Protein levels of mitochondrial and lipid metabolism key elements were evaluated by Western blot. Maximal mitochondrial respiration correlated negatively with BMI (p < .01) and AT area (p < .001) regardless of the anatomical location. However, oxidative phosphorylation respiration was significantly higher in vAT (2.22 ± 0.15 pmol·sec-1·mg-1) than scAT (1.79 ± 0.17 pmol·sec-1·mg-1) (p < 0.001). In line with oxygraphy results, there were higher levels of mitochondrial respiratory chain complexes in low-BMI patients and vAT. Mitochondrial respiration decreased with increasing BMI in both scAT and vAT, without sex-associated difference. Mitochondrial respiration appeared to be higher in vAT than scAT. These differences were both qualitative and quantitative. Clinical Trials Registration IDNCT05417581.

Lactational high weight loss impairs follicular development by causing mitochondrial dysfunction of ovarian cells in sows and mitigated by butyrate supplement.

In modern sows, lactational high weight loss (HWL), caused by the large litter size and inadequate feed intake, has a negative effect on follicular development after weaning, resulting in poor reproductive performance in the subsequent parity. However, the underlying mechanism remains unclear. This research aimed to explore the mechanism that sows HWL during lactation damages follicular development and attempt to improve the reproductive function by treating with butyrate. Four multiparous sister sows were chosen to build a HWL model for lactating sows through feed restriction during the final week of a 21-day lactation. Spatially transcriptomics (ST) and tissue immunofluorescent staining were then utilized for the antral follicles in the ovarian surface to search for differentially expressed genes and proteins among different cell types. Subsequently, the mouse assay, including immunofluorescent staining, transmission electron microscopy, hormone detection and western blot, were conducted to verify the findings in sows and investigate the effect of butyrate on the follicular development in HWL mice. Based on the transcriptomic analysis, differentially expressed genes in granulosa cells, theca cells, and ovarian stromal cells were examined. The findings revealed that HWL disturbs the mitochondrial electron transport chain and steroidogenesis in all three cell types by downregulating the expression of NDUFB3, SDHB, CYCS, COX8A and CYP19A1, as well as upregulating the expression of STAR, CYP11A1 and CYP17A1. Furthermore, results from mouse assays demonstrated that HWL causes apoptosis and alters sex hormone secretion by impairing mitochondrial function and disordering the expression of steroidogenesis key enzymes in ovarian cells, while these effects were partially mitigated by butyrate. The mitochondrial dysfunction and abnormal steroidogenesis induced by HWL during lactation in ovarian cells harm the follicular development of weaning sows, which could be alleviated by butyrate.

Order-to-Disorder and Disorder-to-Order Transitions of Proteins upon Binding to Phospholipid Membranes: Common Ground and Dissimilarities

Cytochrome c is one of the most prominent representatives of peripheral membrane proteins. Besides functioning as an electron transfer carrier in the mitochondrial respiratory chain, it can acquire peroxidase capability, promote the self-assembly of α-synuclein, and function as a scavenger of superoxide. An understanding of its function requires knowledge of how the protein interacts with the inner membrane of mitochondria. The first part of this article provides an overview of a variety of experiments that were aimed at exploring the details of cytochrome c binding to anionic lipid liposomes, which serve as a model system for the inner membrane. While cytochrome c binding involves a conformational change from a folded into a partially disordered state, α-synuclein is intrinsically disordered in solution and subjected to a partial coil -&gt; helix transition on membranes. Depending on the solution conditions and the surface density of α-synuclein, the protein facilitates the self-assembly into oligomers and fibrils. As for cytochrome c, results of binding experiments are discussed. In addition, the article analyzes experiments that explored α-synuclein aggregation. Similarities and differences between cytochrome c and α-synuclein binding are highlighted. Finally, the article presents a brief account of the interplay between cytochrome c and α-synuclein and its biological relevance.

The new method for premature ovarian insufficiency treatment based on the effect of omaveloxolone (RTA 408) on the mitochondrial function of granulosa cells

Background: Premature ovarian insufficiency is a syndrome characterized by secondary hypergonadotropic ovarian insufficiency and a decrease in the ovarian function in women under 40 years of age, thereby leading to impaired reproductive function, metabolic changes, and a decrease in the quality of life. Increased production of reactive oxygen species inhibits the mitochondrial respiration chain and leads to mitochondrial dysfunction and oxidative stress, which are considered as the triggers of premature ovarian insufficiency. To date, there is no universal method of premature ovarian insufficiency prevention, and accepted treatment methods can only compensate for clinical symptoms, but not restore the lost ovarian reserve. Aim: The aim of this study was to develop a new method of premature ovarian insufficiency treatment based on an experimental ovarian granulosa cell model using omaveloxolone (RTA 408). Materials and methods: The cell model of premature ovarian insufficiency used in the study was realized according to the Patent RU 2 815 539 C1, 2023 (by M.I. Yarmolinskaya, K.A. Zakuraeva, A.Yu. Vinokurov, M.Yu. Pogonyalova). Ovarian granulosa cells isolated from three-month Wistar rats were subcultivated five times with subsequent seeding on coverslips. The cells on the coverslips were divided into three groups (three coverslip in each group). In Group 1 (comparison group — premature ovarian insufficiency model without treatment), cells were treated with cyclophosphamide (0.1 mg/ml) during 6 h. In Group 2 (main group — premature ovarian insufficiency model with experimental treatment) cells were pretreated with RTA 408 (2 µl/ml) during 1 h with subsequent addition of cyclophosphamide (0.1 mg/ml) and cultivation during 6 h. And in Group 3 (control group), cells were added no substances. Results: Compared to Groups 2 and 3, the use of omaveloxolone (RTA 408) led to an increase in reduced glutathione level and a decrease in reactive oxygen species production rate, which indicates the antioxidant and anti-inflammatory effects of the drug and may be considered as a perspective strategy of premature ovarian insufficiency treatment. The claimed method expands the number of premature ovarian insufficiency treatment strategies and avoids the use of hormonal medications and surgical procedures, thus reducing the risk of side effects and complications associated with their use.

MftG is crucial for ethanol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

Quinone extraction drives atmospheric carbon monoxide oxidation in bacteria.

Diverse bacteria and archaea use atmospheric CO as an energy source for long-term survival. Bacteria use [MoCu]-CO dehydrogenases (Mo-CODH) to convert atmospheric CO to carbon dioxide, transferring the obtained electrons to the aerobic respiratory chain. However, it is unknown how these enzymes oxidize CO at low concentrations and interact with the respiratory chain. Here, we use cryo-electron microscopy and structural modeling to show how Mo-CODHMs (CoxSML) from Mycobacterium smegmatis interacts with its partner, the membrane-bound menaquinone-binding protein CoxG. We provide electrochemical, biochemical and genetic evidence that Mo-CODH transfers CO-derived electrons to the aerobic respiratory chain through CoxG. Lastly, we show that Mo-CODH and CoxG genetically and structurally associate in diverse bacteria and archaea. These findings reveal the basis of the biogeochemically and ecologically important process of atmospheric CO oxidation, while demonstrating that long-range quinone transport is a general mechanism of energy conservation, which convergently evolved on multiple occasions.

MftG is crucial for ethanol metabolism of mycobacteria by linking mycofactocin oxidation to respiration.

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.