Mechanism Of Electron Bifurcation Research Articles

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.

Looking for the mechanism of arsenate respiration of Fusibacter sp. strain 3D3, independent of ArrAB.

The literature has reported the isolation of arsenate-dependent growing microorganisms which lack a canonical homolog for respiratory arsenate reductase, ArrAB. We recently isolated an arsenate-dependent growing bacterium from volcanic arsenic-bearing environments in Northern Chile, Fusibacter sp. strain 3D3 (Fas) and studied the arsenic metabolism in this Gram-positive isolate. Features of Fas deduced from genome analysis and comparative analysis with other arsenate-reducing microorganisms revealed the lack of ArrAB coding genes and the occurrence of two arsC genes encoding for putative cytoplasmic arsenate reductases named ArsC-1 and ArsC-2. Interestingly, ArsC-1 and ArsC-2 belong to the thioredoxin-coupled family (because of the redox-active disulfide protein used as reductant), but they conferred differential arsenate resistance to the E. coli WC3110 ΔarsC strain. PCR experiments confirmed the absence of arrAB genes and results obtained using uncouplers revealed that Fas growth is linked to the proton gradient. In addition, Fas harbors ferredoxin-NAD+ oxidoreductase (Rnf) and electron transfer flavoprotein (etf) coding genes. These are key molecular markers of a recently discovered flavin-based electron bifurcation mechanism involved in energy conservation, mainly in anaerobic metabolisms regulated by the cellular redox state and mostly associated with cytoplasmic enzyme complexes. At least three electron-bifurcating flavoenzyme complexes were evidenced in Fas, some of them shared in conserved genomic regions by other members of the Fusibacter genus. These physiological and genomic findings permit us to hypothesize the existence of an uncharacterized arsenate-dependent growth metabolism regulated by the cellular redox state in the Fusibacter genus.

Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase.

Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.

An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism.

Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E′∼−280 mV, under physiological conditions) and reduce protons to H2 gas (E°′−414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E′∼−500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD+ and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP+, cofactor F420, formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

Molecular basis of a novel electron bifurcation mechanism in the HydABC complex

Molecular basis of a novel electron bifurcation mechanism in the HydABC complex

Impacts of food waste to sludge ratios on microbial dynamics and functional traits in thermophilic digesters

A self-stabilizing microbial community lays the foundation of the efficient biochemical reactions of the anaerobic digestion (AD) process. Despite extensive profiling of microbial community dynamics under varying operating parameters, the effects of food waste (FW) to feeding sewage sludge (FSS) ratios on the microbial assembly, functional traits, and syntrophic interspecies interactions in thermophilic microbial consortia remain poorly understood. Here, we investigated the long-term impacts of the FW: FSS ratio on the thermophilic AD microbiome using genome-centric metagenomics. Both the short reads (SRs) assembly, and the iterative hybrid assembly (IHA) of SRs and nanopore long reads (LRs) were used to reconstruct metagenome-assembled genomes (MAGs) and four microbial clusters were identified, demonstrating different microbial dynamics patterns in response to varying FW:FSS ratios. Cluster C1-C3 were comprised of full functional members with genetic potentials in fulfilling empirical AD biochemical reactions, wherein, syntrophic decarboxylating acetogens could interact with methanogens, and some microbes could be energized by the electron bifurcation mechanism to drive thermodynamics unfavorable reactions. We found the co-existence of both acetogenic and hydrogenotrophic methanogens in the AD microbiome, and they altered their trophic groups to scavenge the methanogenic substrates in ensuring the methane generation in digesters with different FW:FSS ratios. Another interesting observation was that two phylogenetically close Thermotogota species showed a possible strong competition on carbon source inferred by the nearly complete genetic overlap of their relevant pathways.

The electron-bifurcating FeFe-hydrogenase Hnd is involved in ethanol metabolism in Desulfovibrio fructosovorans grown on pyruvate.

Desulfovibrio fructosovorans, a sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of H2 into protons and electrons. Among them, Hnd is an electron-bifurcating hydrogenase, coupling the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 . It was previously hypothesized that its biological function involves the production of NADPH necessary for biosynthetic purposes. However, it was subsequently demonstrated that Hnd is instead a NAD+ -reducing enzyme, thus its specific function has yet to be established. To understand the physiological role of Hnd in D. fructosovorans, we compared the hnd deletion mutant with the wild-type strain grown on pyruvate. Growth, metabolite production and consumption, and gene expression were compared under three different growth conditions. Our results indicate that hnd is strongly regulated at the transcriptional level and that its deletion has a drastic effect on the expression of genes for two enzymes, an aldehyde ferredoxin oxidoreductase and an alcohol dehydrogenase. We demonstrated here that Hnd is involved in ethanol metabolism when bacteria grow fermentatively and proposed that Hnd might oxidize part of the H2 produced during fermentation generating both NADH and reduced ferredoxin for ethanol production via its electron bifurcation mechanism.

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol−1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ − 16 kJ mol−1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 μmol NADH min−1 mg−1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases.Graphic abstract

Energy Conservation and Hydrogenase Function in Methanogenic Archaea, in Particular the Genus Methanosarcina.

The biological production of methane is vital to the global carbon cycle and accounts for ca. 74% of total methane emissions. The organisms that facilitate this process, methanogenic archaea, belong to a large and phylogenetically diverse group that thrives in a wide range of anaerobic environments. Two main subgroups exist within methanogenic archaea: those with and those without cytochromes. Although a variety of metabolisms exist within this group, the reduction of growth substrates to methane using electrons from molecular hydrogen is, in a phylogenetic sense, the most widespread methanogenic pathway. Methanogens without cytochromes typically generate methane by the reduction of CO2 with electrons derived from H2, formate, or secondary alcohols, generating a transmembrane ion gradient for ATP production via an Na+-translocating methyltransferase (Mtr). These organisms also conserve energy with a novel flavin-based electron bifurcation mechanism, wherein the endergonic reduction of ferredoxin is facilitated by the exergonic reduction of a disulfide terminal electron acceptor coupled to either H2 or formate oxidation. Methanogens that utilize cytochromes have a broader substrate range, and can convert acetate and methylated compounds to methane, in addition to the ability to reduce CO2 Cytochrome-containing methanogens are able to supplement the ion motive force generated by Mtr with an H+-translocating electron transport system. In both groups, enzymes known as hydrogenases, which reversibly interconvert protons and electrons to molecular hydrogen, play a central role in the methanogenic process. This review discusses recent insight into methanogen metabolism and energy conservation mechanisms with a particular focus on the genus Methanosarcina.

Electron bifurcation mechanism and homoacetogenesis explain products yields in mixed culture anaerobic fermentations

Anaerobic fermentation of organic wastes using microbial mixed cultures is a promising avenue to treat residues and obtain added-value products. However, the process has some important limitations that prevented so far any industrial application. One of the main issues is that we are not able to predict reliably the product spectrum (i.e. the stoichiometry of the process) because the complex microbial community behaviour is not completely understood. To address this issue, in this work we propose a new metabolic network of glucose fermentation by microbial mixed cultures that incorporates electron bifurcation and homoacetogenesis. Our methodology uses NADH balances to analyse published experimental data and evaluate the new stoichiometry proposed. Our results prove for the first time the inclusion of electron bifurcation in the metabolic network as a better description of the experimental results. Homoacetogenesis has been used to explain the discrepancies between observed and theoretically predicted yields of gaseous H2 and CO2 and it appears as the best solution among other options studied. Overall, this work supports the consideration of electron bifurcation as an important biochemical mechanism in microbial mixed cultures fermentations and underlines the importance of considering homoacetogenesis when analysing anaerobic fermentations.

Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf)

Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 μm ferredoxin or 0.42 μm flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD(+) reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH + H(+) = butyryl-CoA + NAD(+) with Km = 1.4 μm ferredoxin or 2.0 μm flavodoxin. This reaction requires Na(+) (Km = 0.12 mm) or Li(+) (Km = 0.25 mm) for activity, indicating that Rnf acts as a Na(+) pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation.

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

The genome of Syntrophomonas wolfei: new insights into syntrophic metabolism and biohydrogen production

Syntrophomonas wolfei is a specialist, evolutionarily adapted for syntrophic growth with methanogens and other hydrogen- and/or formate-using microorganisms. This slow-growing anaerobe has three putative ribosome RNA operons, each of which has 16S rRNA and 23S rRNA genes of different length and multiple 5S rRNA genes. The genome also contains 10 RNA-directed, DNA polymerase genes. Genomic analysis shows that S. wolfei relies solely on the reduction of protons, bicarbonate or unsaturated fatty acids to re-oxidize reduced cofactors. Syntrophomonas wolfei lacks the genes needed for aerobic or anaerobic respiration and has an exceptionally limited ability to create ion gradients. An ATP synthase and a pyrophosphatase were the only systems detected capable of creating an ion gradient. Multiple homologues for β-oxidation genes were present even though S. wolfei uses a limited range of fatty acids from four to eight carbons in length.Syntrophomonas wolfei, other syntrophic metabolizers with completed genomic sequences, and thermophilic anaerobes known to produce high molar ratios of hydrogen from glucose have genes to produce H(2) from NADH by an electron bifurcation mechanism. Comparative genomic analysis also suggests that formate production from NADH may involve electron bifurcation. A membrane-bound, iron-sulfur oxidoreductase found in S. wolfei and Syntrophus aciditrophicus may be uniquely involved in reverse electron transport during syntrophic fatty acid metabolism. The genome sequence of S. wolfei reveals several core reactions that may be characteristic of syntrophic fatty acid metabolism and illustrates how biological systems produce hydrogen from thermodynamically difficult reactions.

Structural basis for the mechanism of electron bifurcation at the quinol oxidation site of the cytochrome bc 1 complex

At the heart of the Q cycle hypothesis, the cytochrome bc1 complex (bc1) is required to separate the two electrons from a quinol molecule at the quinol oxidation site. Recent studies have brought to light an intricate mechanism for this bifurcated electron transfer. A survey of the protein data bank shows 30 entries for the structures of bc1 and the homologous b6 f complex. These structures provide considerable insights into the structural organization of mitochondrial, bacterial, and plant enzymes. Crystallographic binding studies of bc1 with either quinone reduction (QN) and/or quinol oxidation (QP) site inhibitors offer atomic details on how these compounds interact with residues at their respective sites. Most importantly, the different locations and apparent flexibility observed in crystals for the extrinsic domain of the iron-sulfur protein (ISP) subunit suggest a mechanism for electron bifurcation at the QP site. Analyses of various inhibitor-bound structures revealed two classes of QP site inhibitors: Pm inhibitors that promote ISP mobility and Pf inhibitors that favor the fixation of the ISP conformation. Those analyses also shed light on a possible process by which the ISP motion switch is controlled. The first phase reduction of ISP is shown to be comparable to the reduction of the bL heme by pre-steady state kinetic analysis, whereas the second phase reduction of ISP share similar kinetics with the reduction of the bH heme. The reduction of cyt c1 is measured much slower, indicating that the reduced ISP remains bound at the QP site until the reduced heme bL is oxidized by the heme bH and supporting the existence of a control mechanism for the ISP motion switch.

Conformational change of the Rieske [2Fe-2S] protein in cytochrome bc1 complex.

Structures of mitochondrial bc1 complex have been reported based on four different crystal forms by three different groups. In these structures, the extrinsic domain of the Rieske [2Fe-2S] protein, surprisingly, appeared at three different positions: the "c1" position, where the [2Fe-2S] cluster exists in close proximity to the heme c1; the "b" position, where the [2Fe-2S] cluster exist in close proximity to the cytochrome b; and the "intermediate" position where the [2Fe-2S] cluster exists in-between "c1" and "b" positions. The conformational changes between these three positions can be explained by a combination of two rotations; (1) a rotation of the entire extrinsic domain and (2) a relative rotation between the cluster-binding fold and the base fold within the extrinsic domain. The hydroquinone oxidation and the electron bifurcation mechanism at the Q(P) binding pocket of the bc1 complex is well explained using these conformational changes of the Rieske [2Fe-2S] protein.