Hydrogenase Complex Research Articles

Isolation of an H2-dependent electron-bifurcating CO2-reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis.

In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2. Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin-based electron-bifurcating mechanism. [NiFe]-hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420, respectively. Recently, an Fdh-Hdr complex was purified as a 3-MDa megacomplex that contained Fmd, and its three-dimensional structure was elucidated by cryo-electron microscopy. In contrast, the Mvh-Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1-MDa Mvh-Hdr-Fmd megacomplex from Methanothermobacter marburgensis. After anion-exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1- and 0.5-MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1-MDa megacomplex was determined to be a dimeric Mvh-Hdr-Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe-4S]-clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh-Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh-Hdr and Fmd in the Mvh-Hdr-Fmd complex. The purified Mvh-Hdr-Fmd megacomplex catalyzed electron-bifurcating reduction of [13C]-CO2 to form [13C]-formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh-Hdr-Fmd megacomplex are electronically connected for the reduction of CO2, which likely involves MvhB polyferredoxin as an electron relay.

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.

An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A–E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Culexarchaeia, a novel archaeal class of anaerobic generalists inhabiting geothermal environments

Geothermal environments, including terrestrial hot springs and deep-sea hydrothermal sediments, often contain many poorly understood lineages of archaea. Here, we recovered ten metagenome-assembled genomes (MAGs) from geothermal sediments and propose that they constitute a new archaeal class within the TACK superphylum, “Candidatus Culexarchaeia”, named after the Culex Basin in Yellowstone National Park. Culexarchaeia harbor distinct sets of proteins involved in key cellular processes that are either phylogenetically divergent or are absent from other closely related TACK lineages, with a particular divergence in cell division and cytoskeletal proteins. Metabolic reconstruction revealed that Culexarchaeia have the capacity to metabolize a wide variety of organic and inorganic substrates. Notably, Culexarchaeia encode a unique modular, membrane associated, and energy conserving [NiFe]-hydrogenase complex that potentially interacts with heterodisulfide reductase (Hdr) subunits. Comparison of this [NiFe]-hydrogenase complex with similar complexes from other archaea suggests that interactions between membrane associated [NiFe]-hydrogenases and Hdr may be more widespread than previously appreciated in both methanogenic and non-methanogenic lifestyles. The analysis of Culexarchaeia further expands our understanding of the phylogenetic and functional diversity of lineages within the TACK superphylum and the ecology, physiology, and evolution of these organisms in extreme environments.

Expanding the phylogenetic distribution of cytochrome b-containing methanogenic archaea sheds light on the evolution of methanogenesis

Methane produced by methanogenic archaea has an important influence on Earth’s changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum “Ca. Halobacteriota” (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic “Ca. Methylarchaeum tengchongensis”, representing a novel archaeal order, designated the “Ca. Methylarchaeales”, of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus “Ca. Methanotowutia” of the “Ca. Methylarchaeales” encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the “Ca. Methylarchaeales” are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or “Ca. Halobacteriota” in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle.

Geometrical influence on the non-biomimetic heterolytic splitting of H2 by bio-inspired [FeFe]-hydrogenase complexes: a rare example of inverted frustrated Lewis pair based reactivity.

Despite the high levels of interest in the synthesis of bio-inspired [FeFe]-hydrogenase complexes, H2 oxidation, which is one specific aspect of hydrogenase enzymatic activity, is not observed for most reported complexes. To attempt H–H bond cleavage, two disubstituted diiron dithiolate complexes in the form of [Fe2(μ-pdt)L2(CO)4] (L: PMe3, dmpe) have been used to play the non-biomimetic role of a Lewis base, with frustrated Lewis pairs (FLPs) formed in the presence of B(C6F5)3 Lewis acid. These unprecedented FLPs, based on the bimetallic Lewis base partner, allow the heterolytic splitting of the H2 molecule, forming a protonated diiron cation and hydrido-borate anion. The substitution, symmetrical or asymmetrical, of two phosphine ligands at the diiron dithiolate core induces a strong difference in the H2 bond cleavage abilities, with the FLP based on the first complex being more efficient than the second. DFT investigations examined the different mechanistic pathways involving each accessible isomer and rationalized the experimental findings. One of the main DFT results highlights that the iron site acting as a Lewis base for the asymmetrical complex is the {Fe(CO)3} subunit, which is less electron-rich than the {FeL(CO)2} site of the symmetrical complex, diminishing the reactivity towards H2. Calculations relating to the different mechanistic pathways revealed the presence of a terminal hydride intermediate at the apical site of a rotated {Fe(CO)3} site, which is experimentally observed, and a semi-bridging hydride intermediate from H2 activation at the Fe–Fe site; these are responsible for a favourable back-reaction, reducing the conversion yield observed in the case of the asymmetrical complex. The use of two equivalents of Lewis acid allows for more complete and faster H2 bond cleavage due to the encapsulation of the hydrido-borate species by a second borane, favouring the reactivity of each FLP, in agreement with DFT calculations.

Mechanism of Diiron Hydrogenase Complexes Controlled by Nature of Bridging Dithiolate Ligand.

Bio‐inorganic complexes inspired by hydrogenase enzymes are designed to catalyze the hydrogen evolution reaction (HER). A series of new diiron hydrogenase mimic complexes with one or two terminal tris(4‐methoxyphenyl)phosphine and different μ‐bridging dithiolate ligands and show catalytic activity towards electrochemical proton reduction in the presence of weak and strong acids. A series of propane‐ and benzene‐dithiolato‐bridged complexes was synthesized, crystallized, and characterized by various spectroscopic techniques and quantum chemical calculations. Their electrochemical properties as well as the detailed reaction mechanisms of the HER are elucidated by density functional theory (DFT) methods. The nature of the μ‐bridging dithiolate is critically controlling the reaction and performance of the HER of the complexes. In contrast, terminal phosphine ligands have no significant effects on redox activities and mechanism. Mono‐ or di‐substituted propane‐dithiolate complexes afford a sequential reduction (electrochemical; E) and protonation (chemical; C) mechanism (ECEC), while the μ‐benzene dithiolate complexes follow a different reaction mechanism and are more efficient HER catalysts.

Diversity analysis of thermophilic hydrogenogenic carboxydotrophs by carbon monoxide dehydrogenase amplicon sequencing using new primers

The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

Time-Course Transcriptome of Parageobacillus thermoglucosidasius DSM 6285 Grown in the Presence of Carbon Monoxide and Air.

Parageobacillus thermoglucosidasius is a metabolically versatile, facultatively anaerobic thermophile belonging to the family Bacillaceae. Previous studies have shown that this bacterium harbours co-localised genes coding for a carbon monoxide (CO) dehydrogenase (CODH) and Ni-Fe hydrogenase (Phc) complex and oxidises CO and produces hydrogen (H2) gas via the water-gas shift (WGS) reaction. To elucidate the genetic events culminating in the WGS reaction, P. thermoglucosidasius DSM 6285 was cultivated under an initial gas atmosphere of 50% CO and 50% air and total RNA was extracted at ~8 (aerobic phase), 20 (anaerobic phase), 27 and 44 (early and late hydrogenogenic phases) hours post inoculation. The rRNA-depleted fraction was sequenced using Illumina NextSeq, v2.5, 1x75bp chemistry. Differential expression revealed that at 8 vs.. 20, 20 vs.. 27 and 27 vs.. 44 h post inoculation, 2190, 2118 and 231 transcripts were differentially (FDR < 0.05) expressed. Cluster analysis revealed 26 distinct gene expression trajectories across the four time points. Of these, two similar clusters, showing overexpression at 20 relative to 8 h and depletion at 27 and 44 h, harboured the CODH and Phc transcripts, suggesting possible regulation by O2. The transition between aerobic respiration and anaerobic growth was marked by initial metabolic deterioration, as reflected by up-regulation of transcripts linked to sporulation and down-regulation of transcripts linked to flagellar assembly and metabolism. However, the transcriptome and growth profiles revealed the reversal of this trend during the hydrogenogenic phase.

Anaerobic and hydrogenogenic carbon monoxide-oxidizing prokaryotes: Versatile microbial conversion of a toxic gas into an available energy.

Carbon monoxide (CO) is a gas that is toxic to various organisms including humans and even microbes; however, it has low redox potential, which can fuel certain microbes, namely, CO oxidizers. Hydrogenogenic CO oxidizers utilize an energy conservation system via a CO dehydrogenase/energy-converting hydrogenase complex to produce hydrogen gas, a zero emission fuel, by CO oxidation coupled with proton reduction. Biochemical and molecular biological studies using a few model organisms have revealed their enzymatic reactions and transcriptional response mechanisms using CO. Biotechnological studies for CO-dependent hydrogen production have also been carried out with these model organisms. In this chapter, we review recent advances in the studies of these microbes, which reveal their unique and versatile metabolic profiles and provides future perspectives on ecological roles and biotechnological applications. Over the past decade, the number of isolates has doubled (37 isolates in 5 phyla, 20 genera, and 32 species). Some of the recently isolated ones show broad specificity to electron acceptors. Moreover, accumulating genomic information predicts their unique physiologies and reveals their phylogenomic relationships with novel potential hydrogenogenic CO oxidizers. Combined with genomic database surveys, a molecular ecological study has unveiled the wide distribution and low abundance of these microbes. Finally, recent biotechnological applications of hydrogenogenic CO oxidizers have been achieved via diverse approaches (e.g., metabolic engineering and co-cultivation), and the identification of thermophilic facultative anaerobic CO oxidizers will promote industrial applications as oxygen-tolerant biocatalysts for efficient hydrogen production by genomic engineering.

Defining the roles of the hydrogenase 3 and 4 subunits in hydrogen production during glucose fermentation: A new model of a H2-producing hydrogenase complex

Hydrogen (H2)-producing hydrogenase (Hyd) activity of E. coli wild type and mutants with defects in subunits of Hyd-3 or Hyd-4 during fermentation at different glucose concentrations and pHs was studied. Hyd-3 was mainly responsible for H2 production but a significant contribution by Hyd-4 to total H2 production depended on the glucose concentration and pH. Surprisingly, not all Hyd-3 or Hyd-4 subunits contributed towards H2 production. Hyd-4 mainly exhibited H2-oxidizing activity in cells growing on 0.2% glucose at pH 7.5, while at pH 5.5 it had a significant impact on H2 production. Importantly, a hyfG mutant (lacking the large subunit of Hyd-4) had a ~2.2 fold decrease in H2 production when cells were grown with 0.2% glucose. A similar role of Hyd-4 was shown at pH 6.5 grown with 0.8% glucose. This study provides new information to allow improvements in H2 production yield and in our general understanding of H2 metabolism.

Tuning Reactivity of Bioinspired [NiFe]-Hydrogenase Models by Ligand Design and Modeling the CO Inhibition Process

Despite the report of several structural and functional models of the [NiFe]-hydrogenases, it is still unclear how the succession of electron and proton transfers during H2 production catalysis are controlled in terms of both sequence (order of the chemical or redox steps) and sites (metal and/or ligand). To address this issue, the structure of the previously described bioinspired [NiFe]-hydrogenase complex [LN2S2NiIIFeIICp(CO)]+ (LNiIIFeIICp, with LN2S2 = 2,2′-(2,2′-bipyridine-6,6′-diyl)bis(1,1′-diphenylethanethiolate) and Cp = cyclopentadienyl) has been fine-tuned by modifying exclusively the Fe site. In [LN2S2NiIIFeIICp*(CO)]+ (LNiIIFeIICp*, with Cp* = pentamethylcyclopentadienyl), the Cp– ligand has been replaced by Cp*– to change both the redox and structural properties of the overall complex as a consequence of the steric hindrance of Cp*–. The LNiIIFeIICp* complex acts as an efficient electrocatalyst to produce H2. Density functional theory (DFT) calculations support a CEEC cycle, following an init...

Electron Bifurcation and Confurcation in Methanogenesis and Reverse Methanogenesis

Reduction of the disulfide of coenzyme M and coenzyme B (CoMS–SCoB) by heterodisulfide reductases (HdrED and HdrABC) is the final step in all methanogenic pathways. Flavin-based electron bifurcation (FBEB) by soluble HdrABC homologs play additional roles in driving essential endergonic reactions at the expense of the exergonic reduction of CoMS–SCoM. In the first step of the CO2 reduction pathway, HdrABC complexed with hydrogenase or formate dehydrogenase generates reduced ferredoxin (Fdx2-) for the endergonic reduction of CO2 coupled to the exergonic reduction of CoMS–SCoB dependent on FBEB of electrons from H2 or formate. Roles for HdrABC:hydrogenase complexes are also proposed for pathways wherein the methyl group of methanol is reduced to methane with electrons from H2. The HdrABC complexes catalyze FBEB-dependent oxidation of H2 for the endergonic reduction of Fdx driven by the exergonic reduction of CoMS–SCoB. The Fdx2- supplies electrons for reduction of the methyl group to methane. In H2- independent pathways, three-fourths of the methyl groups are oxidized producing Fdx2- and reduced coenzyme F420 (F420H2). The F420H2 donates electrons for reduction of the remaining methyl groups to methane requiring transfer of electrons from Fdx2- to F420. HdrA1B1C1 is proposed to catalyze FBEB-dependent oxidation of Fdx2- for the endergonic reduction of F420 driven by the exergonic reduction of CoMS–SCoB. In H2- independent acetotrophic pathways, the methyl group of acetate is reduced to methane with electrons derived from oxidation of the carbonyl group mediated by Fdx. Electron transport involves a membrane-bound complex (Rnf) that oxidizes Fdx2- and generates a Na+ gradient driving ATP synthesis. It is postulated that F420 is reduced by Rnf requiring HdrA2B2C2 catalyzing FBEB-dependent oxidation of F420H2 for the endergonic reduction of Fdx driven by the exergonic reduction of CoMS–SCoB. The Fdx2- is recycled by Rnf and HdrA2B2C2 thereby conserving energy. The HdrA2B2C2 is also proposed to play a role in Fe(III)-dependent reverse methanogenesis. A flavin-based electron confurcating (FBEC) HdrABC complex is proposed for nitrate-dependent reverse methanogenesis in which the oxidation of CoM-SH/CoB-SH and Fdx2- is coupled to reduction of F420. The F420H2 donates electrons to a membrane complex that generates a proton gradient driving ATP synthesis.

Structure of an Ancient Respiratory System

Hydrogen gas-evolving membrane-bound hydrogenase (MBH) and quinone-reducing complex I are homologous respiratory complexes with a common ancestor, but a structural basis for their evolutionary relationship is lacking. Here, we report the cryo-EM structure of a 14-subunit MBH from the hyperthermophile Pyrococcus furiosus. MBH contains a membrane-anchored hydrogenase module that is highly similar structurally to the quinone-binding Q-module of complex I while its membrane-embedded ion-translocation module can be divided into a H+- and a Na+-translocating unit. The H+-translocating unit is rotated 180° in-membrane with respect to its counterpart in complex I, leading to distinctive architectures for the two respiratory systems despite their largely conserved proton-pumping mechanisms. The Na+-translocating unit, absent in complex I, resembles that found in the Mrp H+/Na+ antiporter and enables hydrogen gas evolution by MBH to establish a Na+ gradient for ATP synthesis near 100°C. MBH also provides insights into Mrp structure and evolution of MBH-based respiratory enzymes.

Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243T

ABSTRACTStrains of Eggerthella lenta are capable of oxidation-reduction reactions capable of oxidizing and epimerizing bile acid hydroxyl groups. Several genes encoding these enzymes, known as hydroxysteroid dehydrogenases (HSDH) have yet to be identified. It is also uncertain whether the products of E. lenta bile acid metabolism are further metabolized by other members of the gut microbiota. We characterized a novel human fecal isolate identified as E. lenta strain C592. The complete genome of E. lenta strain C592 was sequenced and comparative genomics with the type strain (DSM 2243) revealed high conservation, but some notable differences. E. lenta strain C592 falls into group III, possessing 3α, 3β, 7α, and 12α-hydroxysteroid dehydrogenase (HSDH) activity, as determined by mass spectrometry of thin layer chromatography (TLC) separated metabolites of primary and secondary bile acids. Incubation of E. lenta oxo-bile acid and iso-bile acid metabolites with whole-cells of the high-activity bile acid 7α-dehydroxylating bacterium, Clostridium scindens VPI 12708, resulted in minimal conversion of oxo-derivatives to lithocholic acid (LCA). Further, Iso-chenodeoxycholic acid (iso-CDCA; 3β,7α-dihydroxy-5β-cholan-24-oic acid) was not metabolized by C. scindens. We then located a gene encoding a novel 12α-HSDH in E. lenta DSM 2243, also encoded by strain C592, and the recombinant purified enzyme was characterized and substrate-specificity determined. Genomic analysis revealed genes encoding an Rnf complex (rnfABCDEG), an energy conserving hydrogenase (echABCDEF) complex, as well as what appears to be a complete Wood-Ljungdahl pathway. Our prediction that by changing the gas atmosphere from nitrogen to hydrogen, bile acid oxidation would be inhibited, was confirmed. These results suggest that E. lenta is an important bile acid metabolizing gut microbe and that the gas atmosphere may be an important and overlooked regulator of bile acid metabolism in the gut.

Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review.

Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0′ = −420 mV) with NADH (−320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (−10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms—in which they have been found—are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered.

Determination of the Genome and Primary Transcriptome of Syngas Fermenting Eubacterium limosum ATCC\xa08486

Autotrophic conversion of CO2 to value-added biochemicals has received considerable attention as a sustainable route to replace fossil fuels. Particularly, anaerobic acetogenic bacteria are naturally capable of reducing CO2 or CO to various metabolites. To fully utilize their biosynthetic potential, an understanding of acetogenesis-related genes and their regulatory elements is required. Here, we completed the genome sequence of the syngas fermenting Eubacterium limosum ATCC 8486 and determined its transcription start sites (TSS). We constructed a 4.4 Mb long circular genome with a GC content of 47.2% and 4,090 protein encoding genes. To understand the transcriptional and translational regulation, the primary transcriptome was augmented, identifying 1,458 TSSs containing a high pyrimidine (T/C) and purine nucleotide (A/G) content at the −1 and +1 position, respectively, along with 1,253 5′-untranslated regions, and principal promoter elements such as −10 (TATAAT) and −35 (TTGACA), and Shine-Dalgarno motifs (GGAGR). Further analysis revealed 93 non-coding RNAs, including one for potential transcriptional regulation of the hydrogenase complex via interaction with molybdenum or tungsten cofactors, which in turn controls formate dehydrogenase activity of the initial step of Wood-Ljungdahl pathway. Our results provide comprehensive genomic information for strain engineering to enhance the syngas fermenting capacity of acetogenic bacteria.

Quantum chemical approaches to [NiFe] hydrogenase.

The mechanism by which [NiFe] hydrogenase catalyses the oxidation of molecular hydrogen is a significant yet challenging topic in bioinorganic chemistry. With far-reaching applications in renewable energy and carbon mitigation, significant effort has been invested in the study of these complexes. In particular, computational approaches offer a unique perspective on how this enzyme functions at an electronic and atomistic level. In this article, we discuss state-of-the art quantum chemical methods and how they have helped deepen our comprehension of [NiFe] hydrogenase. We outline the key strategies that can be used to compute the (i) geometry, (ii) electronic structure, (iii) thermodynamics and (iv) kinetic properties associated with the enzymatic activity of [NiFe] hydrogenase and other bioinorganic complexes.

Contributing factors in the improvement of cellulosic H2 production in Clostridium thermocellum/Thermoanaerobacterium co-cultures

Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium.

Genomic, transcriptomic and proteomic analyses of Dehalobacter UNSWDHB in response to chloroform.

Organohalide respiring bacteria (ORB) are capable of utilising organohalides as electron acceptors for the generation of cellular energy and consequently play an important role in the turnover of natural and anthropogenically-derived organohalides. In this study, the response of a Dehalobacter sp. strain UNSWDHB to the addition of trichloromethane (TCM) after a 50 h period of its absence (suffocation) was evaluated from a transcriptomic and proteomic perspective. The up-regulation of TCM reductive dehalogenase genes (tmrABC) and their gene products (TmrABC) was confirmed at both transcriptional and proteomic levels. Other findings include the upregulation of various hydrogenases (membrane-associated Ni-Fe hydrogenase complexes and soluble Fe-Fe hydrogenases), formate dehydrogenases, complex I and a pyrophosphate-energized proton pump. The elevated expression of enzymes associated with carbon metabolism, including complete Wood Ljungdahl pathway, during TCM respiration raises interesting questions on possible fates of intracellular formate and its potential role in the physiology of this bacterium. Overall, the findings presented here provide a broader view on the bioenergetics and general physiology of Dehalobacter UNSWDHB cells actively respiring with TCM.