Prognostic impact of metabolic syndrome in patients with primary endometrial cancer: a retrospective bicentric study.

PURPOSE: Endometrial cancer (EC) is the most common gynaecological cancer. Its incidence has been rising over the years with ageing and increased obesity of the high-income countries' populations. Metabolic syndrome (MetS) has been suggested to be associated with EC. The aim of this study was to assess whether MetS has a significant impact on oncological outcome in patients with EC. METHODS: This retrospective study included patients treated for EC between January 2010 and December 2020 in two referral oncological centers. Obesity, arterial hypertension (AH) and diabetes mellitus (DM) were criteria for the definition of MetS. The impact of MetS on progression free survival (PFS) and overall survival (OS) was assessed with log-rank test and Cox regression analyses. RESULTS: Among the 415 patients with a median age of 64, 38 (9.2%) fulfilled the criteria for MetS. The median follow-up time was 43 months. Patients suffering from MetS did not show any significant differences regarding PFS (36.0 vs. 40.0 months, HR: 1.49, 95% CI 0.79-2.80 P = 0.210) and OS (38.0 vs. 43.0 months, HR: 1.66, 95% CI 0.97-2.87, P = 0.063) compared to patients without MetS. Patients with obesity alone had a significantly shorter median PFS compared to patients without obesity (34.5 vs. 44.0 months, P = 0.029). AH and DM separately had no significant impact on PFS or OS (p > 0.05). CONCLUSION: In our analysis, MetS in patients with EC was not associated with impaired oncological outcome. However, our findings show that obesity itself is an important comorbidity associated with significantly reduced PFS.

The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates.

Thermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66°C) and with high growth rates on hydrogen (H2) plus carbon dioxide (CO2). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H2 + CO2. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H2 may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech's is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H2 + CO2. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H2-dependent Fd reduction during growth on sugar substrates, putatively the H2-dependent CO2 reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While ∆ech2 grew well on pyruvate after four transfers, ∆ech2 did not adapt to CO. Cell suspensions of ∆ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate. IMPORTANCE: Acetogens thrive by converting H2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G'<-20 kJ mol-1). CO2 serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H2 + CO2 is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold-energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H2 to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications.

K+ homeostasis is important for survival of Acinetobacter baumannii ATCC 19606 in the nosocomial environment.

Pathogenic bacteria have developed several mechanisms to thrive within the hostile environment of the human host, but it is often disregarded that their survival outside this niche is crucial for their successful transmission. Acinetobacter baumannii is very well adapted to both the human host and the hospital environment. The latter is facilitated by multifactorial mechanisms including its outstanding ability to survive on dry surfaces, its high metabolic diversity, and, of course, its remarkable osmotic resistance. As a first response to changing osmolarities, bacteria accumulate K+ in high amount to counterbalance the external ionic strength. Here, we addressed whether K+ uptake is involved in the challenges imposed by the harsh conditions outside its host and how K+ import influences the antibiotic resistance of A. baumannii. For this purpose, we used a strain lacking all major K+ importer ∆kup∆trk∆kdp. Survival of this mutant was strongly impaired under nutrient limitation in comparison to the wild type. Furthermore, we found that not only the resistance against copper but also against the disinfectant chlorhexidine was reduced in the triple mutant compared to the wild type. Finally, we revealed that the triple mutant is highly susceptible to a broad range of antibiotics and antimicrobial peptides. By studying mutants, in which the K+ transporter were deleted individually, we provide evidence that this effect is a consequence of the altered K+ uptake machinery. Conclusively, this study provides supporting information on the relevance of K+ homeostasis in the adaptation of A. baumannii to the nosocomial environment.

A novel hexameric NADP+ -reducing [FeFe] hydrogenase from Moorella thermoacetica.

Acetogenic bacteria such as the thermophilic anaerobic model organism Moorella thermoacetica reduce CO2 with H2 as a reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP of M. thermoacetica require NADH, NADPH, and reduced ferredoxin as reductants. Whereas an electron-bifurcating ferredoxin- and NAD+ -reducing hydrogenase HydABC had been described, the enzyme that reduces NADP+ remained to be identified. A likely candidate is the HydABCDEF hydrogenase from M. thermoacetica. Genes encoding for the HydABCDEF hydrogenase are expressed during growth on glucose and dimethyl sulfoxide (DMSO), an alternative electron acceptor in M. thermoacetica, whereas expression of the genes hydABC encoding for the electron-bifurcating hydrogenase is downregulated. Therefore, we have purified the hydrogenase from cells grown on glucose and DMSO to apparent homogeneity. The enzyme had six subunits encoded by hydABCDEF and contained 58 mol of iron and 1 mol of FMN. The enzyme reduced methyl viologen with H2 as reductant and of the physiological acceptors tested, only NADP+ was reduced. Electron bifurcation with pyridine nucleotides and ferredoxin was not observed. H2 -dependent NADP+ reduction was optimal at pH 8 and 60 °C; the specific activity was 8.5 U·mg-1 and the Km for NADP+ was 0.086 mm. Cell suspensions catalyzed H2 -dependent DMSO reduction, which is in line with the hypothesis that the NADP+ -reducing hydrogenase HydABCDEF is involved in electron transfer from H2 to DMSO.

DNA uptake from a laboratory environment drives unexpected adaptation of a thermophile to a minor medium component.

DNA uptake is widespread among microorganisms and considered a strategy for rapid adaptation to new conditions. While both DNA uptake and adaptation are referred to in the context of natural environments, they are often studied in laboratories under defined conditions. For example, a strain of the thermophile Thermoanaerobacter kivui had been adapted to growth on high concentrations of carbon monoxide (CO). Unusual phenotypes of the CO-adapted strain prompted us to examine it more closely, revealing a horizontal gene transfer (HGT) event from another thermophile, Thermoanaerobacter sp. strain X514, being cultured in the same laboratory. The transferred genes conferred on T. kivui the ability to utilize trehalose, a trace component of the yeast-extract added to the media during CO-adaptation. This same HGT event simultaneously deleted a native operon for thiamine biosynthesis, which likely explains why the CO-adapted strain grows poorly without added vitamins. Attempts to replicate this HGT by providing T. kivui with genomic DNA from Thermoanaerobacter sp. strain X514 revealed that it is easily reproducible in the lab. This subtle form of "genome contamination" is difficult to detect, since the genome remains predominantly T. kivui, and no living cells from the original contamination remain. Unexpected HGT between two microorganisms as well as simultaneous adaptation to several conditions may occur often and unrecognized in laboratory environments, requiring caution and careful monitoring of phenotype and genotype of microorganisms that are naturally-competent for DNA uptake.

GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Ethanologenesis from glycerol by the gut acetogen Blautia schinkii.

The human gut is an anoxic environment that harbours a multitude of microorganisms that not only contribute to food digestion. The microbiome is also involved in malfunctions such as diseases, inflammation processes or development of obesity, but it is also involved in processes that increase the human well-being. Both, the good and the bad, are mediated by fermentation end products of bacterial metabolism, among others. However, despite a steadily growing knowledge of 'who lives out there', little in known of 'what do they do out there'. The genus Blautia is commonly found in the gut and associated with human well-being, but the exploration of their metabolic potential has just started. We demonstrate that B. schinkii grows on glycerol by producing acetate and ethanol. Transcriptome studies and biochemical analyses revealed a glycerol dehydrogenase and dihydroxyacetone kinase that funnel the substrate into glycolysis. Consequently, cells also grew on dihydroxyacetone. Cells could be adapted to grow at high (up to 1.5 M) glycerol concentrations but then only ethanol was formed. Ethanol production from glycerol is not only of relevance for the human host but also for potential bioindustrial production of bioethanol from waste glycerol.

The VBNC state: a fundamental survival strategy of Acinetobacter baumannii.

IMPORTANCE: Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

A temperature dependent pilin promoter for production of thermostable enzymes in Thermus thermophilus.

BACKGROUND: Enzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited. RESULTS: In this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless ß-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol. CONCLUSIONS: Our results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

Molecular architecture and electron transfer pathway of the Stn family transhydrogenase.

The challenge of endergonic reduction of NADP+ using NADH is overcome by ferredoxin-dependent transhydrogenases that employ electron bifurcation for electron carrier adjustments in the ancient Wood-Ljungdahl pathway. Recently, an electron-bifurcating transhydrogenase with subunit compositions distinct from the well-characterized Nfn-type transhydrogenase was described: the Stn complex. Here, we present the single-particle cryo-EM structure of the Stn family transhydrogenase from the acetogenic bacterium Sporomusa ovata and functionally dissect its electron transfer pathway. Stn forms a tetramer consisting of functional heterotrimeric StnABC complexes. Our findings demonstrate that the StnAB subunits assume the structural and functional role of a bifurcating module, homologous to the HydBC core of the electron-bifurcating HydABC complex. Moreover, StnC contains a NuoG-like domain and a GltD-like NADPH binding domain that resembles the NfnB subunit of the NfnAB complex. However, in contrast to NfnB, StnC lost the ability to bifurcate electrons. Structural comparison allows us to describe how the same fold on one hand evolved bifurcation activity on its own while on the other hand combined with an associated bifurcating module, exemplifying modular evolution in anaerobic metabolism to produce activities critical for survival at the thermodynamic limit of life.

Identification and characterization of a novel pathway for aldopentose degradation in Acinetobacter baumannii.

The nosocomial pathogen Acinetobacter baumannii is well known for its extraordinary metabolic diversity. Recently, we demonstrated growth on L-arabinose, but the pathway remained elusive. Transcriptome analyses revealed two upregulated gene clusters that code for isoenzymes catalysing oxidation of a pentonate to α-ketoglutarate. Molecular, genetic, and biochemical experiments revealed one branch to be specific for L-arabonate oxidation, and the other for D-xylonate and D-ribonate. Both clusters also encode an uptake system and a regulator that acts as activator (L-arabonate) or repressor (D-xylonate and D-ribonate). Genes encoding the initial oxidation of pentose to pentonate were not part of the clusters, but our data are consistent with the hypothesis of a promiscous, pyrroloquinoline quinone (PQQ)-dependent, periplasmic pentose dehydrogenase, followed by the uptake of the pentonates and their degradation by specific pathways. However, there is a cross-talk between the two different pathways since the isoenzymes can replace each other. Growth on pentoses was found only in pathogenic Acinetobacter species but not in non-pathogenic such as Acinetobacter baylyi. However, mutants impaired in growth on pentoses were not affected in traits important for infection, but growth on L-arabinose was beneficial for long-term survival and desiccation resistance in A. baumannii ATCC 19606.

Lactate formation from fructose or C1 compounds in the acetogen Acetobacterium woodii by metabolic engineering.

Anaerobic, acetogenic bacteria are promising biocatalysts for a sustainable bioeconomy since they capture and convert carbon dioxide to acetic acid. Hydrogen is an intermediate in acetate formation from organic as well as C1 substrates. Here, we analyzed mutants of the model acetogen Acetobacterium woodii in which either one of the two hydrogenases or both together were genetically deleted. In resting cells of the double mutant, hydrogen formation from fructose was completely abolished and carbon was redirected largely to lactate. The lactate/fructose and lactate/acetate ratios were 1.24 and 2.76, respectively. We then tested for lactate formation from methyl groups (derived from glycine betaine) and carbon monoxide. Indeed, also under these conditions lactate and acetate were formed in equimolar amounts with a lactate/acetate ratio of 1.13. When the electron-bifurcating lactate dehydrogenase/ETF complex was genetically deleted, lactate formation was completely abolished. These experiments demonstrate the capability of A. woodii to produce lactate from fructose but also from promising C1 substrates, methyl groups and carbon monoxide. This adds an important milestone towards generation of a value chain leading from CO2 to value-added compounds. KEY POINTS: • Resting cells of the ΔhydBA/hdcr mutant of Acetobacterium woodii produced lactate from fructose or methyl groups + CO • Lactate formation from methyl groups + CO was completely abolished after deletion of lctBCD • Metabolic engineering of a homoacetogen to lactate formation gives a potential for industrial applications.

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :ß3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑßγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits ß and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal ß-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

A new metabolic trait in an acetogen: Mixed acid fermentation of fructose in a methylene-tetrahydrofolate reductase mutant of Acetobacterium woodii.

To inactivate the Wood-Ljungdahl pathway in the acetogenic model bacterium Acetobacterium woodii, the genes metVF encoding two of the subunits of the methylene-tetrahydrofolate reductase were deleted. As expected, the mutant did not grow on C1 compounds and also not on lactate, ethanol or butanediol. In contrast to a mutant in which the first enzyme of the pathway (hydrogen-dependent CO2 reductase) had been genetically deleted, cells were able to grow on fructose, albeit with lower rates and yields than the wild-type. Growth was restored by addition of an external electron sink, glycine betaine + CO2 or caffeate. Resting cells pre-grown on fructose plus an external electron acceptor fermented fructose to two acetate and four hydrogen, that is, performed hydrogenogenesis. Cells pre-grown under fermentative conditions on fructose alone redirected carbon and electrons to form lactate, formate, ethanol as well as hydrogen. Apparently, growth on fructose alone induced enzymes for mixed acid fermentation (MAF). Transcriptome analyses revealed enzymes potentially involved in MAF and a quantitative model for MAF from fructose in A. woodii is presented.

Reprogramming the metabolism of an acetogenic bacterium to homoformatogenesis.

Methyl groups are abundant in anoxic environments and their utilization as carbon and energy sources by microorganisms involves oxidation of the methyl groups to CO2, followed by transfer of the electrons to an acceptor. In acetogenic bacteria, the electron acceptor is CO2 that is reduced to enzyme bound carbon monoxide, the precursor of the carboxyl group in acetate. Here, we describe the generation of a mutant of the acetogen Acetobacterium woodii in which the last step in methyl group oxidation, formate oxidation to CO2 catalyzed by the HDCR enzyme, has been genetically deleted. The mutant grew on glycine betaine as methyl group donor, and in contrast to the wild type, formed formate alongside acetate, in a 1:2 ratio, demonstrating that methyl group oxidation stopped at the level of formate and reduced electron carriers were reoxidized by CO2 reduction to acetate. In the presence of the alternative electron acceptor caffeate, CO2 was no longer reduced to acetate, formate was the only product and all the carbon went to formate. Apparently, acetogenesis was not required to sustain formatogenic growth. This is the first demonstration of a genetic reprogramming of an acetogen into a formatogen that grows by homoformatogenesis from methyl groups. Formate production from methyl groups is not only of biotechnological interest but also for the mechanism of electron transfer in syntrophic interactions in anoxic environments.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Pyrrolizidine alkaloids in borage (Borago officinalis): Comprehensive profiling and development of a validated LC-MS/MS method for quantification.

Pyrrolizidine alkaloids (PA) from borage (Borago officinalis) consumed as herb and tea, may pose a food safety risk. Therefore, the European Union (EU) set maximum levels of PA in borage, among other foodstuffs, which are applicable since July 1st, 2022. Here, a comprehensive LC-MS/MS based profiling of PA and their N-oxides (PANO) in B. officinalis leaves is presented. Based on these results a targeted, quantitative LC-MS/MS method for the determination of individual PA/PANO present in borage was developed. Chromatographic separation was achieved for all PA/PANO detected in B. officinalis. An easy and fast extraction procedure was developed using a design of experiments approach (DOE). The most efficient extraction was achieved using 0.2% formic acid in 10% methanol at a temperature of 47.5 °C for 60 min. The final method was validated and showed good overall accuracy (recoveries 85-121%) and precision (RDS ≤11%). The method was applied to B. officinalis leave material, demonstrating its suitability for the intended purpose. In these borage samples, the acetylated forms, which are not regulated by EU, were among the quantitatively most relevant PA.

Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]-Hydrogenase Complex HydABC.

Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO2, but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H2). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P)+ and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P)+ binding affinity via reduction of a nearby iron-sulfur cluster, HydABC switches between the exergonic NAD(P)+ reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases.