Correlating Conformational Equilibria with Catalysis in the Electron Bifurcating EtfABCX of Thermotoga maritima.

Electron bifurcation (BF) is an evolutionarily ancient energy coupling mechanism in anaerobes, whose associated enzymatic machinery remains enigmatic. In BF-flavoenzymes, a chemically high-potential electron forms in a thermodynamically favorable fashion by simultaneously dropping the potential of a second electron before its donation to physiological acceptors. The cryo-EM and spectroscopic analyses of the BF-enzyme Fix/EtfABCX from Thermotoga maritima suggest that the BF-site contains a special flavin-adenine dinucleotide and, upon its reduction with NADH, a low-potential electron transfers to ferredoxin and a high-potential electron reduces menaquinone. The transfer of energy from high-energy intermediates must be carefully orchestrated conformationally to avoid equilibration. Herein, anaerobic size exclusion-coupled small-angle X-ray scattering (SEC-SAXS) shows that the Fix/EtfAB heterodimer subcomplex, which houses BF- and electron transfer (ET)-flavins, exists in a conformational equilibrium of compacted and extended states between flavin-binding domains, the abundance of which is impacted by reduction and NAD(H) binding. The conformations identify dynamics associated with the T. maritima enzyme and also recapitulate states identified in static structures of homologous BF-flavoenzymes. Reduction of Fix/EtfABCX's flavins alone is insufficient to elicit domain movements conducive to ET but requires a structural "trigger" induced by NAD(H) binding. Models show that Fix/EtfABCX's superdimer exists in a combination of states with respect to its BF-subcomplexes, suggesting a cooperative mechanism between supermonomers for optimizing catalysis. The correlation of conformational states with pathway steps suggests a structural means with which Fix/EtfABCX may progress through its catalytic cycle. Collectively, these observations provide a structural framework for tracing Fix/EtfABCX's catalysis.

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima.

Electron bifurcation is an energy-conservation mechanism in which a single enzyme couples an exergonic reaction with an endergonic one. Heterotetrameric EtfABCX drives the reduction of low-potential ferredoxin (E°' ∼ -450 mV) by oxidation of the midpotential NADH (E°' = -320 mV) by simultaneously coupling the reaction to reduction of the high-potential menaquinone (E°' = -74 mV). Electron bifurcation occurs at the NADH-oxidizing bifurcating-flavin adenine dinucleotide (BF-FAD) in EtfA, which has extremely crossed half-potentials and passes the first, high-potential electron to an electron-transferring FAD and via two iron-sulfur clusters eventually to menaquinone. The low-potential electron on the BF-FAD semiquinone simultaneously reduces ferredoxin. We have expressed the genes encodingThermotoga maritimaEtfABCX in E. coli and purified the EtfABCX holoenzyme and the EtfAB subcomplex. The bifurcation activity of EtfABCX was demonstrated by using electron paramagnetic resonance (EPR) to follow accumulation of reduced ferredoxin. To elucidate structural factors that impart the bifurcating ability, EPR and NADH titrations monitored by visible spectroscopy and dye-linked enzyme assays have been employed to characterize four conserved residues, R38, P239, and V242 in EtfA and R140 in EtfB, in the immediate vicinity of the BF-FAD. The R38, P239, and V242 variants showed diminished but still significant bifurcation activity. Despite still being partially reduced by NADH, the R140 variant had no bifurcation activity, and electron transfer to its two [4Fe-4S] clusters was prevented. The role of R140 is discussed in terms of the bifurcation mechanism in EtfABCX and in the other three families of bifurcating enzymes.

Adaptations to high pressure of Nautilia sp. strain PV-1, a piezophilic Campylobacterium (aka Epsilonproteobacterium) isolated from a deep-sea hydrothermal vent.

Physiological and gene expression studies of deep-sea bacteria under pressure conditions similar to those experienced in their natural habitat are critical for understanding growth kinetics and metabolic adaptations to in situ conditions. The Campylobacterium (aka Epsilonproteobacterium) Nautilia sp. strain PV-1 was isolated from hydrothermal fluids released from an active deep-sea hydrothermal vent at 9° N on the East Pacific Rise. Strain PV-1 is a piezophilic, moderately thermophilic, chemolithoautotrophic anaerobe that conserves energy by coupling the oxidation of hydrogen to the reduction of nitrate or elemental sulfur. Using a high-pressure-high temperature continuous culture system, we established that strain PV-1 has the shortest generation time of all known piezophilic bacteria and we investigated its protein expression pattern in response to different hydrostatic pressure regimes. Proteogenomic analyses of strain PV-1 grown at 20 and 5 MPa showed that pressure adaptation is not restricted to stress response or homeoviscous adaptation but extends to enzymes involved in central metabolic pathways. Protein synthesis, motility, transport, and energy metabolism are all affected by pressure, although to different extents. In strain PV-1, low-pressure conditions induce the synthesis of phage-related proteins and an overexpression of enzymes involved in carbon fixation.

Obligately aerobic human gut microbe expresses an oxygen resistant tungsten-containing oxidoreductase for detoxifying gut aldehydes.

Brevibacillus massiliensis strain phR is an obligately aerobic microbe that was isolated from human feces. Here, we show that it readily takes up tungsten (W), a metal previously associated only with anaerobes. The W is incorporated into an oxidoreductase enzyme (BmWOR) that was purified from native biomass. BmWOR consists of a single 65 kDa subunit and contains a single W-pyranopterin cofactor and a single [4Fe-4S] cluster. It exhibited high aldehyde-oxidizing activity with very high affinities (apparent Km < 6 µM) for aldehydes common in the human gut and in cooked foods, including furfural, propionaldehyde, benzaldehyde and tolualdehyde, suggesting that BmWOR plays a key role in their detoxification. B. massiliensis converted added furfural to furoic acid when grown in the presence of W, but not in the presence of the analogous element molybdenum. B. massiliensis ferredoxin (BmFd) served as the electron acceptor (apparent Km < 5 µM) for BmWOR suggesting it is the physiological electron carrier. Genome analysis revealed a Fd-dependent rather than NADH-dependent Complex I, suggesting that WOR not only serves a detoxification role but its aldehyde substrates could also serve as a source of energy. BmWOR is the first tungstoenzyme and the first member of the WOR family to be obtained from a strictly aerobic microorganism. Remarkably, BmWOR oxidized furfural in the presence of air (21% O2, v/v) but only if BmFd was also present. BmWOR is the first characterized member of the Clade 83 WORs, which are predominantly found in extremely halophilic and aerobic archaea (Clade 83A), with many isolated from food sources, while the remaining bacterial members (Clade 83B) include both aerobes and anaerobes. The potential advantages for microbes found in foods and involved in human gut health that harbor O2-resistant WORs, including in Bacillus and Brevibacillus based-probiotics, are discussed.

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.

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes.

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅-, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH-, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.

Structure and electron transfer pathways of an electron-bifurcating NiFe-hydrogenase.

Electron bifurcation enables thermodynamically unfavorable biochemical reactions. Four groups of bifurcating flavoenzyme are known and three use FAD to bifurcate. FeFe-HydABC hydrogenase represents the fourth group, but its bifurcation site is unknown. We report cryo-EM structures of the related NiFe-HydABCSL hydrogenase that reversibly oxidizes H2 and couples endergonic reduction of ferredoxin with exergonic reduction of NAD. FMN surrounded by a unique arrangement of iron sulfur clusters forms the bifurcating center. NAD binds to FMN in HydB, and electrons from H2 via HydA to a HydB [4Fe-4S] cluster enable the FMN to reduce NAD. Low-potential electron transfer from FMN to the HydC [2Fe-2S] cluster and subsequent reduction of a uniquely penta-coordinated HydB [2Fe-2S] cluster require conformational changes, leading to ferredoxin binding and reduction by a [4Fe-4S] cluster in HydB. This work clarifies the electron transfer pathways for a large group of hydrogenases underlying many essential functions in anaerobic microorganisms.

Tungsten enzymes play a role in detoxifying food and antimicrobial aldehydes in the human gut microbiome.

Tungsten (W) is a metal that is generally thought to be seldom used in biology. We show here that a W-containing oxidoreductase (WOR) family is diverse and widespread in the microbial world. Surprisingly, WORs, along with the tungstate-specific transporter Tup, are abundant in the human gut microbiome, which contains 24 phylogenetically distinct WOR types. Two model gut microbes containing six types of WOR and Tup were shown to assimilate W. Two of the WORs were natively purified and found to contain W. The enzymes catalyzed the conversion of toxic aldehydes to the corresponding acid, with one WOR carrying out an electron bifurcation reaction coupling aldehyde oxidation to the simultaneous reduction of NAD+ and of the redox protein ferredoxin. Such aldehydes are present in cooked foods and are produced as antimicrobials by gut microbiome metabolism. This aldehyde detoxification strategy is dependent on the availability of W to the microbe. The functions of other WORs in the gut microbiome that do not oxidize aldehydes remain unknown. W is generally beyond detection (<6 parts per billion) in common foods and at picomolar concentrations in drinking water, suggesting that W availability could limit some gut microbial functions and might be an overlooked micronutrient.

Evolution of complex I-like respiratory complexes.

The modern-day respiratory complex I shares a common ancestor with the membrane-bound hydrogenase (MBH) and membrane-bound sulfane sulfur reductase (MBS). MBH and MBS use protons and sulfur as their respective electron sinks, which helped to conserve energy during early life in the Proterozoic era when the Earth's atmosphere was low in oxygen. MBH and MBS likely evolved from an integration of an ancestral, membrane-embedded, multiple resistance and pH antiporter and a soluble redox-active module encompassing a [NiFe] hydrogenase. In this review, we discuss how the structures of MBH, MBS, multiple resistance and pH, photosynthetic NADH dehydrogenase-like complex type-1, and complex I, which have been determined recently, thanks to the advent of high-resolution cryo-EM, have significantly improved our understanding of the catalytic reaction mechanisms and the evolutionary relationships of the respiratory complexes.

Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX.

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°' ∼-450 mV) using NADH (E°' -320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°' -80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.

Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S0)-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

Engineering the cellulolytic extreme thermophile Caldicellulosiruptor bescii to reduce carboxylic acids to alcohols using plant biomass as the energy source.

Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (Topt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR-Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.

Tuning Catalytic Bias of Hydrogen Gas Producing Hydrogenases.

Hydrogenases display a wide range of catalytic rates and biases in reversible hydrogen gas oxidation catalysis. The interactions of the iron-sulfur-containing catalytic site with the local protein environment are thought to contribute to differences in catalytic reactivity, but this has not been demonstrated. The microbe Clostridium pasteurianum produces three [FeFe]-hydrogenases that differ in "catalytic bias" by exerting a disproportionate rate acceleration in one direction or the other that spans a remarkable 6 orders of magnitude. The combination of high-resolution structural work, biochemical analyses, and computational modeling indicates that protein secondary interactions directly influence the relative stabilization/destabilization of different oxidation states of the active site metal cluster. This selective stabilization or destabilization of oxidation states can preferentially promote hydrogen oxidation or proton reduction and represents a simple yet elegant model by which a protein catalytic site can confer catalytic bias.

Integrated thermodynamic analysis of electron bifurcating [FeFe]-hydrogenase to inform anaerobic metabolism and H2 production.

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxinred- + NAD(P)H + 3 H+ = 2 ferredoxinox + NAD(P)+ + 2 H2). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH2 higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (~10 kJ mol-1 H2) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E°' largely determines the sensitivity of the bifurcating reaction to pH2, modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the 'Thauer limit' (4 H2 per glucose) as a function of temperature and pH2. For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 °C if pH 2 ≤ ~10 mbar. The results also predict Archaea, expressing a non-typical glycolysis pathway, would not benefit from a bifurcating [FeFe]-hydrogenase reaction; interestingly, no Archaea have been observed experimentally with a [FeFe]-hydrogenase enzyme.

The thermophilic biomass-degrading bacterium Caldicellulosiruptor bescii utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis.

Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C5 and C6 sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H2 concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD.

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes.

Hyperthermophilic archaea contain a hydrogen gas-evolving,respiratory membrane-bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates H2S. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered Pyrococcus furiosus (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable Km (∼490 µm) and Vmax values (12 µmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant P. furiosus strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd (Eo' = -480 mV) to reduce sulfane sulfur (Eo' -260 mV) and cleave organic (RS n R, n ≥ 3) and anionic polysulfides (S n2-, n ≥ 4) but that it does not produce H2S. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes.

A new era for electron bifurcation.

Electron bifurcation, or the coupling of exergonic and endergonic oxidation-reduction reactions, was discovered by Peter Mitchell and provides an elegant mechanism to rationalize and understand the logic that underpins the Q cycle of the respiratory chain. Thought to be a unique reaction of respiratory complex III for nearly 40 years, about a decade ago Wolfgang Buckel and Rudolf Thauer discovered that flavin-based electron bifurcation is also an important component of anaerobic microbial metabolism. Their discovery spawned a surge of research activity, providing a basis to understand flavin-based bifurcation, forging fundamental parallels with Mitchell's Q cycle and leading to the proposal of metal-based bifurcating enzymes. New insights into the mechanism of electron bifurcation provide a foundation to establish the unifying principles and essential elements of this fascinating biochemical phenomenon.