Flavin-Based Electron Bifurcation, A New Mechanism of Biological

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

pubs.acs.org/CR

Flavin-Based Electron Bifurcation, A New Mechanism of Biological Energy Coupling Wolfgang Buckel*,†,‡ and Rudolf K. Thauer†,‡ †

Fachbereich Biologie, Philipps-Universität, 35032 Marburg, Germany Max-Planck-Institut für Terrestrische Mikrobiologie, 35043 Marburg, Germany

‡

ABSTRACT: There are two types of electron bifurcation (EB), either quinone- or flavin-based (QBEB/FBEB), that involve reduction of a quinone or flavin by a twoelectron transfer and two reoxidations by a high- and low-potential one-electron acceptor with a reactive semiquinone intermediate. In QBEB, the reduced low-potential acceptor (cytochrome b) is exclusively used to generate ΔμH+. In FBEB, the “energyrich” low-potential reduced ferredoxin or flavodoxin has dual function. It can give rise to ΔμH+/Na+ via a ferredoxin:NAD reductase (Rnf) or ferredoxin:proton reductase (Ech) or conducts difficult reductions such as CO2 to CO. The QBEB membrane complexes are similar in structure and function and occur in all domains of life. In contrast, FBEB complexes are soluble and occur only in strictly anaerobic bacteria and archaea (FixABCX being an exception). The FBEB complexes constitute a group consisting of four unrelated families that contain (1) electron-transferring flavoproteins (EtfAB), (2) NAD(P)H dehydrogenase (NuoF homologues), (3) heterodisulfide reductase (HdrABC) or HdrABC homologues, and (4) NADH-dependent ferredoxin:NADP reductase (NfnAB). The crystal structures and electron transport of EtfAB-butyryl-CoA dehydrogenase and NfnAB are compared with those of complex III of the respiratory chain (cytochrome bc1), whereby unexpected common features have become apparent.

CONTENTS 1. Introduction 2. Quinone-Based Electron Bifurcation (QBEB) 2.1. Ubiquinol Dehydrogenases (Cytochrome bc1 Complexes) 2.2. Menaquinol Dehydrogenases (Cytochrome bc1 Complexes) 2.3. Plastoquinol Dehydrogenases (Cytochrome b6 f Complexes) 2.4. Alternative Complex III (ACIII) 3. Flavin-Based Electron Bifurcation (FBEB) 3.1. Discovery of FBEB in Butyrate-Forming Anaerobes 3.2. Electron-Transferring Flavoprotein (EtfAB)Containing Complexes 3.2.1. EtfAB-Butyryl-CoA Dehydrogenase 3.2.2. EtfAB(CarED)-Caffeyl-CoA Reductase 3.2.3. EtfAB(LctCB)-Lactate Dehydrogenase 3.2.4. EtfAB(FixBA)-Ubiquinone Reductase 3.2.5. Nonbifurcating EtfAB-Propionyl-CoA Dehydrogenase 3.2.6. Comparison of EtfAB Sequences 3.3. NAD(P)H Dehydrogenase (NuoF Homologues)-Containing Complexes 3.3.1. NADH Dehydrogenase-[FeFe]-Hydrogenase 3.3.2. NADPH Dehydrogenase-[FeFe]-Hydrogenase © XXXX American Chemical Society

3.3.3. NADH-Dehydrogenase-[W/Se]-Formate Dehydrogenase 3.4. Heterodisulfide Reductase (HdrABC)-Containing Complexes 3.4.1. HdrABC-[NiFe]-Hydrogenase 3.4.2. HdrABC-[W/Se]-Formate Dehydrogenase 3.4.3. HdrABC-F420H2 Dehydrogenase 3.4.4. Other Putatively Bifurcating HdrABCContaining Complexes 3.5. NADH-Dependent Ferredoxin: NADP Reductase (NfnAB) 4. Electron Transfer in Electron-Bifurcating Complexes 4.1. Cytochrome bc1 Complex 4.2. NfnAB Complex 4.3. EtfAB-Bcd Complex 4.4. Comparison of the Three Well-Characterized Electron-Bifurcating Complexes Author Information Corresponding Author ORCID Notes Biographies Acknowledgments

B B C C D D D D E E H I J K L M

O O O P P P Q R S S T U V V V V V V

M Received: November 24, 2017

N A

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews References

Review

The systems with benzo- and naphthoquinones, also called quinone-based electron bifurcation (QBEB), occur in mitochondria, chloroplasts, phototrophic bacteria, and respiring bacteria and archaea (Figure 3). The hydroquinone (QH2) bound at the outer Q binding site (Qo) is the start point of bifurcation. The high-potential one-electron acceptor is the Rieske iron sulfur protein (ISP). The low-potential acceptor is cytochrome bL (see below). Flavin-based electron bifurcation (FBEB) has been discovered in strictly anaerobic bacteria and archaea.2,3,5 The twoelectron donors for FBEB are NAD(P)H, F420H2, formate, or molecular hydrogen. High-potential terminal one-electron acceptors are NAD, crotonyl-CoA, caffeyl-CoA, pyruvate, the heterodisulfide (CoM-S-S-CoB, the oxidized form of HScoenzyme M + HS-coenzyme B in methanogens),5 menaquinone, or ubiquinone (Figure 1). Ferredoxins and flavodoxins act as low-potential terminal acceptors that upon reoxidation drive electrochemical H+ and Na+ pumps such as ferredoxin-NAD reductase (Rnf)9−11 and ferredoxin-proton reductase (Ech).12 Reduced ferredoxins or flavodoxins also enable difficult reductions such as protons to hydrogen, nitrogen to ammonia, CO2 to CO or formyl groups, CO2 + acyl-CoA to 2-oxoacids, carboxylates to alcohols, benzoyl-CoA to cyclohexadienecarboxyl-CoA, and the activation of 2-hydroxyacyl-CoA dehydratases.5,13 Among the 11 experimentally verified bifurcation systems, 5 are reversible in vivo. The back reaction is called electron confurcation in which the two one-electron accepting cofactors act together as donors to reduce the bifurcating cofactor (Figure 1).

V

1. INTRODUCTION Whereas quinone-based electron bifurcation (QBEB) was discovered over 40 years ago,1 the first papers on flavin-based electron bifurcation (FBEB) were only published in 2008.2,3 In the last 10 years, FBEB has changed our view on how most anaerobic microorganisms conserve energy, and we have probably only seen the tip of the iceberg. In the meantime, various aspects of FBEB have been reviewed.4−8 The following treatise focuses mainly on structural and mechanistic aspects. In electron bifurcation, the flow of two electrons branches toward two different one-electron acceptors: one with a higher reduction potential and the other with a lower reduction potential than that of the donating electron pair (Figure 1).

2. QUINONE-BASED ELECTRON BIFURCATION (QBEB) Early research on mitochondrial respiration with double beam spectrophotometers revealed a puzzling result. In respiring mitochondria with succinate as donor and oxygen as acceptor, cytochrome b became reduced.21 After having established the theory of the proton motive force, this unexpected finding led to the proposal of the quinone (Q) cycle by Peter Mitchell in 1975.1 As a mechanism for the generation of an electrochemical proton gradient at the inner mitochondrial membrane, Mitchell postulated the reduction of ubiquinone (UQ, 2,3-dimethoxy-5decaprenyl-1,4-benzoquinone, Figure 2) at the inside of the membrane and the oxidation of ubihydroquinone (UQH2), also called ubiquinol, at the outside (Figure 3). Because reduction of UQ consumes 2 protons at the inside and oxidation of UQH2 releases 2 protons at the outside, a gradient of 2H+/2e− is formed. Mitchell proposed further that the oxidation of UQH2 at the outside of the membrane (Qo site) by the high-potential cytochrome c removes only one electron, whereas the other reduces the low-potential cytochrome bL, which in turn transfers the electron to the high-potential cytochrome bH and further to UQ sitting at the inside of the membrane (Qi site). Repetition of this bifurcation yields UQH2, which diffuses to the outside to be oxidized in the same manner. Thus, the bifurcating flow of two electrons through the Q-cycle results in an additional gradient of 2H+/2e−, which doubles the amount of energy conserved. This seminal discovery stimulated intensive research that led to the biochemical and structural characterization of the mitochondrial cytochrome bc1 complexes22−29 as well as of the cytochrome b6 f complexes from chloroplasts and their bacterial counterparts,30 all of which catalyze quinone-based electron bifurcation.

Figure 1. Summary of all established electron bifurcation systems. Numbers in brackets are midpoint reduction potentials (Em at pH 7). Flavin-based electron bifurcation (FEBEB) in yellow; flavin: FAD or FMN (for structures, see Figure 2). Quinone-based electron bifurcation (QBEB) in dark red; quinone: benzo- or naphthoquinone (Figure 2). Em of the flavin couples are almost 400 mV more negative than that of the quinone couples. Whereas the flavin-based “bifurcases” are mainly cytoplasmic, the quinone-based “bifurcases” are integral membrane proteins. References of the midpoint reduction potentials: NAD(P), H+/H2, CO2/formate, and pyruvate/lactate;14 caffeyl-CoA/dihydrocaffeyl-CoA was assumed to be slightly more negative than crotonylCoA/butyryl-CoA;15 F420/F420H2;16 heterodisulfide/(CoMSH + CoBSH);17 flavodoxin18 and ferredoxin19 were taken as 420 mV; cytochrome c (see section 2.1); plastocyanin, MK/MKH2, UQ/UQH2, and plastoquinone (PQ/PQH2).20

Electron bifurcation amplifies the reducing power of one electron at the energetic cost of the other electron. In nature, two cofactor families with bifurcating properties are known: ubiquinone, plastoquinone, and menaquinones (referred to as quinones) as well as FAD and FMN (referred to as flavins) (Figure 2). All have three consecutive oxidation states (ox): the quinone state (ox = 0), the semiquinone state (ox = −1), and the hydroquinone state (ox = −2). Other putative candidates could be transition metal ions with three readily accessible oxidation states under in vivo conditions, such as cobalt and nickel (ox = +1, +2, +3) as well as molybdenum and tungsten (ox = +4, +5, +6).4 B

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 2. Bifurcating cofactors. (top) Three important quinones involved in quinone-based electron bifurcation (QBEB), whose length of isoprenoid side chains may vary. The three oxidation states of quinones are shown with the non-natural benzoquinone as an example. (bottom) FMN and FAD are the bifurcating cofactors of flavin-based electron bifurcation (FBEB); R = ribitol-5′-phosphate (FMN) or ribitol-5′-ADP (FAD). An inherent property of the quinones and the flavins in the nonprotein bound state is that they have crossed-over redox potentials: the quinone/semiquinone (Q/ SQ) couple has a more negative midpoint potential than the SQ/hydroquinone (SQ/HQ) couple. (In the “normal” uncrossed case, upon oxidation of the hydroquinone form, the first electron would have a more negative redox potential than the second one).4 Therefore, the SQ states do not accumulate because two molecules disproportionate to the Q and HQ states. This is the basis for the electron-bifurcation mechanism proposed in section 4 “Electron transfer in bifurcation systems”.

2.1. Ubiquinol Dehydrogenases (Cytochrome bc1 Complexes)

on which the cluster swings between the Qo site and cytochrome c1 carrying one electron from UQH2 to cytochrome c. The remaining electron at the semiquinone (UQ•−) immediately reduces cytochrome bL, which transfers the electron further via cytochrome bH to UQ at the Qi site. The resulting UQ•− at the Qi site is stable and “waits” for the next round until it becomes reduced and protonated to UQH2. Finally, the reduced cytochrome c carries single electrons further to cytochrome c oxidase (Figure 3).

The mitochondrial cytochrome bc1 or complex III catalyzes the oxidation of UQH2 (Em = +90 mV) by cytochrome c (Em = +250 mV, from horse heart).31 Complex III is a dimer composed of 2 × 11 subunits, three of which are catalytically essential: cytochrome b, Rieske iron−sulfur protein (ISP), and cytochrome c1. Cytochrome b is an integral membrane protein that contains the two UQ binding sites and two hemes b, the low-potential heme bL (Em = −60 mV), and the high-potential heme bH (Em = +82 mV) (Figure 3). Cytochrome c1 with cytochrome c as cofactor and the Rieske iron−sulfur protein (Em = +285 mV) are located at the outside of cytochrome b near the Qo site (all Em from yeast complex III).20 The Rieske iron− sulfur protein comprises a high-potential [2Fe-2S] cluster of which one Fe is coordinated by a pair of cysteines and the other by a pair of histidines. The cluster is located on a flexible domain

2.2. Menaquinol Dehydrogenases (Cytochrome bc1 Complexes)

Whereas the Gram-negative aerobic proteobacteria take UQ as electron carrier similar to mitochondria, the Gram-negative anaerobic proteobacteria, the Gram-positive aerobic bacilli, and aerobic archaea use menaquinone (2-methyl-3-heptafarnesyl1,4-naphtoquinone, MK, Figure 2) with a 160 mV lower reduction potential (Em = −70 mV). Correspondingly, the C

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

glucose, −76 kJ is required to form one mol ATP. Using this quotient, the approximate yield of ATP from any catabolic pathway can be calculated just by dividing the free energy change under standard conditions by −76.14 This has been verified for many aerobic pathways, but with anaerobes, much lower ATP yields have often been estimated than those obtained by thermodynamic calculations. An example is the fermentation of ethanol and acetate to butyrate, caproate, and hydrogen by Clostridium kluyveri discovered by Horace Albert Barker in the late 1930s.33 Today, this reaction is called “chain elongation” and is used to convert organic waste from alcoholic fermentations to butyrate (C4), caproate (C6), and small amounts of caprylate (C8).34 Chain elongation follows the balanced reaction 1, but for the sake of simplicity, caproate and caprylate productions are omitted. Figure 3. Quinone-based electron bifurcation (QBEB) in the cytochrome bc1 complex (light blue). The arrows show electron flows in the membrane (red), proton translocation (blue), quinone (Q, QH2), and Rieske movements (white). Qi and Qo are the inner and outer binding sites of Q and QH2, respectively. The cytochromes are in red. The two positions of the Rieske iron sulfur protein (ISP) are shown with solid and dashed ovals.

6 ethanol + 4 acetate− → 5 butyrate− + H+ + 2H 2 + 4H 2O;

ΔG°′ = −180 kJ mol−1 H+

(1)

Using the quotient mentioned above, the pathway should yield −180/−76 ≙ 2.5 ATP under standard conditions (all concentrations 1 M with the exception of that of protons = 10−7 M). Under conditions in the environment (concentration of ethanol, acetate, and butyrate ≈ 1 mM), the free energy change is reduced to −95 kJ mol−1 ≙ 1.25 ATP. Biochemical investigations in the 1970s revealed that 6 ethanol were oxidized by 12 NAD to 6 acetyl-CoA. One acetyl-CoA was used for substrate-level phosphorylation (SLP), and 2 NADH were used for 2 H2 production. For the synthesis of 5 butyrate, 5 acetyl-CoA, 5 acetate (4 + 1 from SLP) and 10 NADH were required.35,36 Hence, two problems arose, synthesis of 1.0 ATP rather than 2.5 ATP (standard conditions) or 1.25 ATP (environmental conditions), and the endergonic production of H2 (Em = −414 mV) from the remaining 2 NADH (Em = −320 mV). Because of this result and work by others, it was assumed that anaerobes were inefficient energy converters because they were only able to conduct SLP but not electron-transport phosphorylation (ETP). Furthermore, the production of H2 at pressures up to 100 kPa remained elusive. Inspections of the individual reactions of the pathway of butyrate synthesis showed that the reduction of crotonyl-CoA to butyryl-CoA (Em = −10 mV) by NADH was highly exergonic, ΔG°′ = −60 kJ mol−1, whereas the free energies of the others were ∼0. Although cell extracts of butyrate producing clostridia catalyzed the reduction of crotonyl-CoA by NADH, this activity was lost during purification. However, butyryl-CoA dehydrogenase was present as assayed with butyryl-CoA and ferricenium hexafluorophosphate as an artificial electron acceptor.37 Using this assay, the enzyme could be purified to homogeneity, which was composed of three different subunits: one was identified as butyryl-CoA dehydrogenase (Bcd) and the others as the heterodimeric electron transferring flavoprotein EtfAB. This unexpected quaternary structure helped to put forward the hypothesis that the exergonic reduction of crotonyl-CoA by NADH could drive the endergonic reduction of ferredoxin by NADH.2 This hypothesis could be verified with an assay composed of pure EtfAB-Bcd from C. kluyveri, NADH, crotonylCoA, and ferredoxin from Clostridium tetanomorphum, Clostridium pasteurianum, or Acidaminococcus fermentans. Thereby, ferredoxin was completely reduced to E′ ≈ −500 mV as monitored by its absorption maximum at 410 nm but only if all ingredients of the assay were present in excess. Hence, EtfABBcd bifurcated as expected leading to the consumption of

reduction potentials of the two heme groups in cytochrome b and of the Rieske iron−sulfur protein (ISP) are also lower.20 2.3. Plastoquinol Dehydrogenases (Cytochrome b6 f Complexes)

The cytochrome b6 f complex from chloroplasts of plants, algae, and cyanobacteria is functionally almost identical to the cytochrome bc1 complex. It is composed of 2 × 9 subunits, three of which are catalytically active, cytochrome b6, Rieske iron−sulfur protein, and the c-type cytochrome f. Plastoquinol (PQ, Em = +115 mV) replaces UQH2 as the electron donor, and plastocyanin (or cytochrome c6) instead of cytochrome c works as acceptor. Plastoquinone (2,3-dimethyl-nonaprenyl-1,4-benzoquinone, Figure 2) obtains two electrons from photosystem II; after bifurcation, each electron is transferred by plastocyanin, a small copper protein (Em = +370 mV), or by cytochrome c6 to photosystem I.30 2.4. Alternative Complex III (ACIII)

In the photosynthetic bacterium Chlorof lexus aurantiacus, no cytochrome bc1 or b6 f complex has been detected. Biochemical and bioinformatic studies revealed that these complexes were replaced by an alternative complex III, which is composed of subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. To date, quinone reductase and quinol oxidase activities but no electron bifurcation could be detected yet with the alternative complex III.32 Though the complex contains no molybdenum and the conserved molybdenum binding amino acids are absent, it is tempting to speculate that this homology is reminiscent of a lost bifurcating function of molybdenum.

3. FLAVIN-BASED ELECTRON BIFURCATION (FBEB) 3.1. Discovery of FBEB in Butyrate-Forming Anaerobes

The mechanism of energy conservation in aerobes is well established. The catabolism of glucose to CO2 and H2O via the Embden−Meyerhof pathway, the Krebs cycle, and the mitochondrial respiratory chain results in the synthesis of up to 38 ATP from ADP and inorganic phosphate. Because the free energy of this reaction amounts to ΔG°′ = −2870 kJ mol−1 D

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

CoA to 2-enoyl-CoA, the first step of β-oxidation of fatty acids, where they transfer one electron from the acyl-CoA dehydrogenase to ubiquinone of the respiratory chain via the Etf-quinone oxidoreductase. Etfs are composed of three domains, the A or α-subunit consists of domains I and II, and the B or β-subunit forms domain III. Domains I+III are the rigid part or base of the molecule, whereas domain II is flexible and can rotate versus the base. Domain II is connected to domains I +III via several bendable contacts without secondary structures. α-FAD is located at the flavodoxin-like domain II in a stretched conformation, whereby the N1−C2=O group of the pyrimidine ring is intramolecularly hydrogen bonded to the 4′OH of the ribityl side chain. This interaction and further hydrogen bonds to conserved amino acids are well conserved in all Etfs, which are considered as a major structural feature to neutralize the negatively charged FAD•− and FADH− species reflected in the high reduction potentials of the α-FAD/FAD•− couple (Em = +81 mV for EtfMe) and of the α-FAD•−/FADH− couple (Em = −130 mV for EtfMe).45 Other interactions with the isoalloxazine ring are a hydrogen bond from the protonated N5 to Ser-A270-OG and a salt bridge to the guanidinium group of Arg-A253. As a result, the one-electron reduction potentials of the α-FAD are not crossed-over (see legend to Figure 2 and section 4). β-FAD is arranged in a compressed S-shaped conformation and binds with its AMP moiety at the same site where AMP is bound in nonbifurcating EtfABs, namely between domains I and III (Figure 4). Apparently, the nonbifurcating Etfs have lost the

NADH and crotonyl-CoA at a ratio of 2:1, showing that both reductions were tightly coupled (reaction 2a). 2NADH + crotonyl‐CoA − CoA + 2Fdox → 2NAD+ + butyryl‐CoA + 2Fd red−;

ΔG°′ = −41 kJ mol−1 (2a)

Reduced ferredoxin (Em = −420 mV) was reoxidized with [FeFe]-hydrogenase from Clostridium pasteurianum,38 which demonstrated how the oxidation of NADH led to the exergonic formation of hydrogen (reaction 2b).3 2NADH + crotonyl‐CoA + 2H+ → 2NAD+ + butyryl‐CoA + H 2 ;

ΔG°′ = −42 kJ mol−1

(2b)

The free energies of reactions 2a and 2b were calculated by adding up the reduction potentials of the redox pairs, which are defined as Em: oxidant + e− = reductant or −(reductant = oxidant + e−). ΔEm = Σ Em with Em = −10 mV for crotonyl-CoA + 2 e− = butyryl-CoA, Em = −420 mV for Fdox + 1e− = Fdred−, Em = −414 mV for 2 H+ + 2e− = H2, Em = −(−320 mV for NADH = NAD+ + H+ + 2 e−); ΔG°′ = −nFΔEm, where n is the number of electrons and F is the Faraday constant (96.5 kJ mol−1 V−1). Most of the subsequent ΔG°′ values of redox reactions were calculated via this procedure provided that all of the reduction potentials were known. The discovery of flavin-based electron bifurcation and how the prediscovery of electron transport phosphorylation with protons (Ech) or NAD (Rnf) as electron acceptors paved the way is described in more detail in a parallel historical review in Frontiers in Microbiology.39 3.2. Electron-Transferring Flavoprotein (EtfAB)-Containing Complexes

3.2.1. EtfAB-Butyryl-CoA Dehydrogenase. Early attempts failed to crystallize the EtfAB-Bcd complexes from the Gram-positive Firmicutes C. kluyveri3 and C. tetanomorphum.40 Therefore, EtfABAf from the Gram-negative Firmicute Acidaminococcus fermentans VR4 was isolated from cell-free extracts, which together with homotetrameric BcdAf (4 × 42 kDa) from the same organism catalyzed the bifurcation as did the EtfABBcd complexes.41 Using information obtained by peptide mapping, the correct etfAB genes (Acfer 0556 and 0555) could be identified in the genome of A. fermentans.41 Later, it turned out that the second pair of etf genes present in this organism encoded a nonbifurcating Etf.42 All subsequent experiments were performed with recombinant EtfABAf with a C-terminal His-tag at the α-subunit produced in Escherichia coli. The “as purified” EtfABAf contained 0.7 mol FAD per mol protein (37.6 kDa + 28.4 kDa), which could be increased to 2.0 mol FAD/mol by 15 h incubation with excess FAD. Besides the two FAD, αFAD and β-FAD, other cofactors such as riboflavin, riboflavin5′-phosphate (FMN), hydroxy-FAD or AMP or iron−sulfur clusters could not be detected in EtfABAf, which catalyzed the NADH-dependent reduction of iodotetrazolium chloride to the red formazane. This so-called diaphorase activity demonstrated that EtfABAf was able to interact with NADH, in contrast to the AMP and α-FAD-containing EtfABs, which are involved in the β-oxidation of fatty acids. EtfABAf crystallized readily, and its structure could be solved41 because it closely resembled the established structures of nonbifurcating Etfs from human origin43 and aerobic bacteria.44 These Etfs contain α-FAD and AMP (at the place of β-FAD) as prosthetic groups and are involved in the β-oxidation of acyl-

Figure 4. Partial structure of the electron-transferring flavoprotein (EtfAB) from A. fermentans in the presence of NAD. Domains I and II in light blue belong to subunit EtfA, and the gray domain III represents subunit EtfB. For the whole structure of EtfAB, see Figure 6 and ref 41. The distance between the 8-methyl group of α-FAD and the 8-methyl group of β-FAD amounts to 18 Å. Upon rotation of domain II by 10° to the left, the distance shortens to 14 Å.

riboflavin-5′-phosphate part of β-FAD without movement of the remaining AMP from its original place. The guanidinium group of Arg-A146 is projected toward the bottom of the isoalloxazine ring and interacts with N5 of FAD. Interestingly, this arginine is conserved in all Etfs, though in the nonbifurcating Etfs the place of the isoalloxazine ring is empty. EtfABAf crystallizes in a conformation in which the distance between α-FAD and β-FAD amounts to 18 Å, too long for an efficient electron transfer. E

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 5. Proposed scheme of electron bifurcation in the cytoplasmic EtfAB-Bcd complex. Adapted with permission from ref 47. Copyright 2017, Nature Publishing Group.

however remained almost unchanged for more than 400 ms. As expected, the formation of α-FAD•− and its reduction to αFADH− are slower processes than the reduction of β-FAD to βFADH− by NADH. Together with BcdAf, recombinant EtfABAf catalyzed the bifurcation with the same activity as that of the one isolated from A. fermentans. Under optimum conditions, an oxidation rate of 3 μmol NADH min−1 mg−1 Etf was achieved with the hydrogenase assay (reaction 2) at an EtfABAf/BcdAf (tetramer) ratio of 2:1, suggesting that 1 Etf interacted with 2 subunits of Bcd (see below). The dependence of the bifurcation rate on the concentration of ferredoxin from C. tetanomorphum saturated at 2 ferredoxin/EtfAB with an apparent Km = 0.2 μM. When NADH instead of ferredoxin was varied, the activity reached a maximum at ∼100 μM and decreased linearly by 30% at 300 μM NADH. This inhibition by high NADH concentration was probably due to the complete reduction of EtfAB. Although the amount of formed hydrogen (reaction 2) was not measured, the tight coupling between the reductions of ferredoxin and crotonyl-CoA was demonstrated by the consumption of 2.1 ± 0.1 NADH/crotonyl-CoA and by the close to 100% reduction of 15 μM ferredoxin as revealed by the identical absorbencies at 410 nm of ferredoxin reduced by bifurcation or by dithionite. Binding of NADH to β-FAD likely induces a conformational change, which allows the reduction of β-FAD by hydride transfer and shortens the distance to the α-FADs by 4 Å. The hydroquinone form of β-FADH− bifurcates; one electron goes to α-FAD and the other to ferredoxin, which was modeled at 6 Å distance. The reduced α-FAD is thought to take the electron to the FAD of Bcd, named δ-FAD, which after repetition of the bifurcation obtains a second electron and transfers a hydride to crotonyl-CoA affording butyryl-CoA (Figure 5).41 The high reduction potentials of α-FAD raise the question as to whether α-FAD or α-FAD•− acts as a high-potential acceptor. In vivo, where NADH is present, α-FAD certainly exists in the α-FAD•− or α-FADH− state. Hence, α-FAD•− could accept the highpotential electron and bring it as α-FADH− to δ-FAD of Bcd. Because of its reduction potential (Em = −10 mV), δ-FAD can take only one electron, and α-FAD•− returns ready for the next one-electron uptake. Recently, the crystal structure of the whole recombinant EtfAB-BcdCd complex from Clostridium dif f icile could be solved.47 The complex was found in a heterododecameric (EtfAB-Bcd)4 state with a molecular mass of 440 kDa. The tetrameric Bcd with one δ-FAD in each subunit forms the core

Modeling revealed that the distance could be shortened to 14 Å, where an electron transfer is possible without disturbing the tertiary structure. In the presence of NAD, the crystal structure of EtfABAf showed that this cofactor was bound close to β-FAD, though only the ADP part of NAD gave good electron density, whereas the nicotinamide-ribose part was completely disordered (Figure 4).41 EtfABAf exhibits the UV−visible spectrum of a flavin with an absorbance maximum at 450 nm and a smaller one at 380 nm, though the trough between the maxima is almost absent. A similar spectrum was reported by Sato et al.45 with EtfABMe isolated from the related anaerobic bacterium Megasphaera elsdenii, which belongs together with A. fermentans to the Gramnegative Firmicutes.46 Addition of increasing concentrations of NADH reduced EtfAB; at 0.5 NADH/EtfAB (2 FAD), the 450 nm peak partially disappeared, and the stable red anionic semiquinone (λmax = 375 nm) of α-FAD became visible (Em Q/ SQ = +81 mV; SQ/HQ = −136 mV, measured with EtfABMe).45 At 1 NADH/EtfAB, only the spectrum of β-FAD (Em Q/HQ = −279 mV)45 remained, which looked similar to that of free FAD with the deep trough between the two peaks. Upon further stepwise reduction, both peaks together disappeared continuously without formation of a second semiquinone. At 2 NADH/ EtfAB, the molecule was fully reduced, and only the featureless spectrum of the hydroquinone of FAD was obtained. Interestingly, the difference spectrum between the oxidized Etf and the half reduced Etf, which can be assigned to α-FAD, exhibits a maximum at 400 nm and a shoulder at 450 nm. This very unusual spectrum of a flavin could be caused by the intramolecular hydrogen bond in α-FAD mentioned above. The additional presence of Bcd increased the anionic semiquinone peak of α-FAD, which was also obtained if NADH was replaced by butyryl-CoA. Hence, butyryl-CoA (Em = −10 mV) also reduces α-FAD via δ-FAD to the anionic semiquinone, but because of the low reduction potential of the α-FAD•−/FADH− couple (Em = −136 mV), further reduction to the hydroquinone is not possible. Preliminary stopped flow experiments at 6 °C showed that mixing of 50 μM EtfAB (100 μM FAD) with 100 μM NADH caused the disappearance of the 450 nm peak of EtfAB within the dead time of the instrument (1.5 ms), whereas a peak at around 370 nm assigned as anionic semiquinone reached its maximum at 10 ms. At 100 ms, the Etf was completely reduced. Mixing of 50 μM EtfAB with 50 μM NADH again led to the formation of the anionic semiquinone within 1.5 ms, which F

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 6. Structures of the EtfAB-Bcd complex. Only one EtfAB (EtfA green, EtfB brown) attached to two Bcd subunits (Bcd1 yellow, Bcd2 pink) is shown. The red arrow indicates the direction of the C-terminal helix in domain II of EtfA.47

Flavodoxin (Fld) from A. fermentans is an FMN-containing protein (14.54 kDa), which replaces ferredoxin at low iron concentrations in the growth medium.48 Because of its high first redox potential (Em Q/SQ = −60 mV; Em SQ/Q = −420 mV),18 it occurs predominantly as blue protonated semiquinone in the cell. For the following experiments, an oxidized yellow recombinant Fld (Q-form) with a C-terminal His-tag produced in E. coli was used.49 A bifurcating reaction with 0.5 μM EtfAB, 1 μM Bcd, 250 μM NADH, and 50 μM Fld (29 μM FMN) was started with 75 μM crotonyl-CoA and measured simultaneously at three wavelengths: 340 nm (NADH), 450 nm (Q), and 578 nm (SQ). The reaction proceeded with a short lag phase as indicated by an initially accelerated decrease in absorbance at 340 and 450 nm, and the concomitant increase at 578 nm showed the formation of SQ. Interestingly, when Q was reduced completely (450 nm), SQ reached a maximum, and the further reduction to HQ continued 3-times faster. Hence SQ rather than Q provides the better substrate for bifurcation by which Fld is completely reduced to HQ, reaching E′ ≈ −500 mV. In the presence of hydrogenase, hydrogen is produced, whereby catalytic amounts of Fld (1 μM) cycle between SQ and HQ. Thus, the apparent Km of flavodoxin (0.4 μM), as determined in this coupled system, refers to the SQ form. Ferredoxin from A. fermentans with two [4Fe-4S] clusters also exhibits two reduction potentials, though not so far apart (Em1 = −340 mV; Em2 = −405 mV).48 Therefore, under these conditions ferredoxin also cycles between half reduced Fd− and fully reduced Fd2−.41 In the thermodynamic calculations, for the sake of simplicity we use ferredoxin (Fdox) for the oxidized or half reduced state and ferredoxin− (Fdred−) for the fully reduced state with the same second reduction potential of Em = −420 mV as flavodoxin. Electron bifurcation with EtfAf-BcdAf works with flavodoxin from A. fermentans as well as with ferredoxin from C. tetanomorphum. Because flavodoxin and ferredoxin are structurally unrelated, there seems to be no specificity with respect to the low-high potential acceptor. Even cytochrome c and, as shown below, oxygen can replace ferredoxin. Over 50 years ago, experiments suggested that both EtfMe and BcdMe from Peptostreptococcus elsdenii, renamed as Megasphaera elsdenii, catalyzed together the reduction of crotonyl-CoA by NADH, but no ferredoxin was required.50 Apparently, the experiments were performed under aerobic conditions. By repetition of this experiment under anaerobic conditions, no reaction could be observed. The absence of oxygen and the presence of ferredoxin

to which four EtfAB are peripherally attached. Each Etf interacts with two Bcd subunits; one contact connects domains I+III to subunit Bcd2 and the other domain II to subunit Bcd1 (Figure 6). Surprisingly, domain II has rotated by 80° to a position in which α-FAD is only 8 Å apart from δ-FAD, whereas the distance to β-FAD has been enlarged to 37 Å. Because in this state an electron can be transferred from α-FAD to δ-FAD, it is called “dehydratase state” (D-state). A superposition of EtfABAf and BcdAf onto EtfAB-BcdCd revealed domains I+III and Bcd well aligned, whereas domain II is oriented as in EtfAf, where αFAD and β-FAD were 18 Å apart, which is called “bifurcationlike state”. Subsequent rotation of domain II by 10° brings the FADs 4 Å closer together, which enables bifurcation (“bifurcation state” or B-state). As shown by the crystal structure, the D-state most likely represents the resting state of the EtfAB-BcdCd complex. It has been proposed that binding of NADH to EtfAB-BcdCd or reduction of β-FAD to the hydroquinone induces the conversion of the D- to the B-state. When ferredoxin or flavodoxin is present, bifurcation occurs. One electron goes, most likely together with a proton, to αFAD•−, and the other electron goes to to ferredoxin or flavodoxin. Domain II with α-FADH− swings over to δ-FAD of Bcd (D-state), which receives the first electron. Repetition of the process yields a second reduced ferredoxin or flavodoxin and adds the second electron to the semiquinone of δ-FAD. Finally, the resulting δ-FADH− reduces crotonyl-CoA to butyryl-CoA (Figure 5). For more insight into the rotation or swinging of domain II to be obtained, three groups of variants of EtfABCd were prepared. The first group includes mutations at the Bcd1/Bcd2-domain II interface formed in the D-state. Salt bridges are found between Bcd E198−EtfA R243 and Bcd D345−EtfA R243, which upon removing the negative charges by changes to glutamine/alanine or asparagine, respectively, abolished the activities. The second group of mutations addresses the bendable covalent linkers between domain III and the EtfB arm (EtfB F233 and V237) and between domain I and II (EtfA R255). Whereas V237Q also abolished the activity, the F233A, R255Q, and R255A mutations surprisingly enhanced the activities up to 3-fold, probably due to an increased flexibility. The third group comprises residues that influence the rotation of domain II. EtfB I125 and E165 are located in loop regions contacting domain II. T18 and P40 belong to the EtfB protrusion. Mutation of each one of the four residues (E165A/D, I125D/F, T18E and P40L) caused loss of activities.47 G

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 7. Proposed scheme of electron bifurcation in the cytoplasmic EtfAB-caffeyl-CoA reductase system catalyzing 1/2 reaction 4b. CarED is homologous to EtfAB, respectively.

plex catalyzes the actual reduction of caffeyl-CoA to dihydrocaffeyl-CoA by NADH with ferredoxin serving as a low-potential acceptor similar to EtfAB-Bcd (reaction 4b and Figure 7).55 The reduction potential of caffeyl-CoA/dihydrocaffeyl-CoA is estimated to be slightly lower (Em ≈ −30 mV) than that of crotonyl-CoA/butyryl-CoA (Em = −10 mV) due to the electron-donating aromatic ring with the p-hydroxy group. The bifurcating hydrogenase produces NADH and additional reduced ferredoxin (see section 3.3.1 and reaction 4c).56 Three reduced ferredoxins are reoxidized by NAD mediated by Rnf (reaction 4d), which leads to the synthesis of ∼0.9 ATP via an electrochemical Na+ gradient (see above and reaction 4e). In summary, 1 H2 reduces 1 caffeate, whereby in addition 0.9 ATP is conserved via ETP (reaction 4).

caused electron bifurcation at similar rates as those observed with EtfAf and BcdAf. Under air in the presence of catalytic amounts of crotonyl-CoA (apparent Km = 2.6 ± 0.5 μM) or butyryl-CoA (!) (apparent Km = 1.0 ± 0.2 μM), NADH reduced O2 to H2O2.51 Closer inspection revealed a normal bifurcation of NADH to crotonyl-CoA, but ferredoxin was replaced by O2, which was reduced with one electron to superoxide (O2•−, E = −330 mV).52 Addition of epinephrine to the reaction identified superoxide by the formation of the colored adenochrome (λmax = 480 nm), which was not observed in the presence of superoxide dismutase. Crotonyl-CoA was indeed reduced to butyryl-CoA, but BcdMe also catalyzed the O2-dependent reoxidation of butyryl-CoA to crotonyl-CoA leading to H2O2. In summary, EtfMe and BcdMe together with either crotonyl-CoA or butyryl-CoA acted as NADH oxidase (reaction 3).

CoA‐transferase: dihydrocaffeyl‐CoA + caffeate−

EtfAB‐Bcd: 2NADH + 2O2 + crotonyl‐CoA +

→ 2NAD + 2O2

•−

+ butyryl‐CoA

⇋ dihydrocaffeate− + caffeyl‐CoA

bifurcating caffeyl‐CoA reductase: caffeyl‐CoA + 2NADH

spontaneous or superoxide dismutase: 2O2•− + 2H+ → H 2O2 + O2

(4a)

(3a)

+ 2Fdox → dihydrocaffeyl‐CoA + 2NAD+ + 2Fd red−; (3b)

Bcd: butyryl‐CoA + O2 → crotonyl‐CoA + H 2O2

(3c)

sum: 2NADH + 2O2 + 2H+ → 2NAD+ + 2H 2O2

(3)

ΔG°′ = −37 kJ mol−1 caffeyl‐CoA

(4b)

bifurcating hydrogenase: H 2 + 0.5NAD+ + Fdox ⇋ 0.5NADH + 1.5H+ + Fd red−

3.2.2. EtfAB(CarED)-Caffeyl-CoA Reductase. The acetogenic anaerobic bacterium Acetobacterium woodii is able to thrive with hydrogen and CO2. Cultures of this organism with H2 and CO2 grew to higher cell densities when caffeate (3,4dihydroxycinnamic acid) was also present. Caffeate was reduced to dihydrocaffeate (3,4-dihydroxyphenylpropionic acid), and less acetate was formed. Hence, the reduction of caffeate to dihydrocaffeate by H2 conserves energy.53 Recently, it has been elucidated that this energy conservation proceeds via two electron bifurcating complexes, EtfAB-caffeyl-CoA reductase (reaction 4b)54 and NADH dehydrogenase-[FeFe]-hydrogenase (see section 3.3.1) (reaction 4c), a sodium iontranslocating ferredoxin:NAD reductase complex (Rnf) (reaction 4d)11 and a sodium ion-translocating F-ATP-synthase complex (3.3 ΔμNa+ per ATP) (reaction 4e).55 Initially, caffeic acid is activated to caffeyl-CoA by an ATP-dependent synthetase or by CoA transfer from the reduced product dihydrocaffeylCoA affording dihydrocaffeate (reaction 4a), which is excreted.54 The bifurcating EtfAB-caffeyl-CoA reductase com-

(4c)

Rnf: 3Fd red− + 1.5NAD+ + 1.5H+ ⇋ 3Fdox + 1.5NADH + [3Δμ Na +]

(4d)

F1Fo synthase: [3ΔμNa +] + 0.9[ADP + P] i ⇋ 0.9[ATP + H 2O]; (3.3Δμ Na + per ATP)

(4e)

sum: caffeate− + H 2 + 0.9[ADP + P] i → dihydrocaffeate− + 2H+ + 0.9[ATP + H 2O]

(4)

The purified caffeyl-CoA reductase complex is composed of three subunits that are encoded by the carCDE genes. CarC (42.5 kDa) represents the actual caffeyl-CoA reductase, and the CarDE proteins (28.2 and 41.4 kDa) are homologous to EtfAB. The molecular mass of CarE is 4 kDa larger than that of EtfAAf (37.6 kDa), which results from the presence of a ferredoxin-like extension with two [4Fe-4S] clusters at the N-terminus. The H

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 8. Proposed scheme of electron confurcation in the cytoplasmic EtfAB-lactate dehydrogenase system. LctCB is homologous to EtfAB.

bacteria to humans. Because the reduction potential of pyruvate/lactate (Em = −190 mV) is much higher than that of NAD/NADH (Em = −320 mV), the reaction cannot proceed under standard conditions. Though the NAD/NADH ratio in aerobic E. coli amounts to 31:1, the resulting effective reduction potential is still too low (E′ = −286 mV).57 Aerobic bacteria circumvent this problem with FAD or FMN-containing lactate dehydrogenases, which transfer the electrons further to menaquinone (Em = −70 mV) or ubiquinone (Em = +90 mV) of the respiratory chains, e.g., D-Ldh from E. coli.58 Mammals use the NAD-dependent L-lactate dehydrogenase (Ldh isoenzyme band 5 or M4), which is located at the outer side of the inner mitochondrial membrane and associated with a monocarboxylate permease and cytochrome c oxidase, forming the “lactate oxidation complex”.59,60 During oxidation of lactate with NAD, the permease removes pyruvate to be oxidized to acetyl-CoA or carboxylated to oxaloacetate in the matrix, whereas the cytochrome c oxidase stimulates respiration, resulting in faster NADH uptake and oxidation. Because of the removal of the products pyruvate and NADH, the Ldh equilibrium is shifted toward lactate oxidation. A. woodii, devoid of quinones, drives lactate oxidation with NAD by electron confurcation with reduced ferredoxin, which decreases ΔG°′ of the reaction from +25 to +6 kJ mol−1 (reaction 5 and Figure 8). In the reverse direction, 2 NADH bifurcate to the higher potential pyruvate and the lower potential ferredoxin.61

whole complex appears to form a homotrimer of CDE subunits (3 × 112 = 336 kDa) as approximately confirmed by size exclusion chromatography (350 kDa). The FAD content was estimated as (4.0 ± 0.2)/112 kDa, though only 3 were expected, α-FAD and β-FAD of CarDE and δ-FAD of CarC. The iron and sulfur contents were estimated as 9 Fe and 9 S/122 kDa, whereas 8 atoms of each element were required for two [4Fe4S] clusters. This is surprising because most flavin- and ironsulfur proteins lose some of the cofactors during purification, especially if the oxidized state α-FAD of EtfA is not tightly bound.45 During purification, the activity of CarCDE was measured routinely via the oxidation of dithionite reduced methylviologen by caffeyl-CoA. Electron bifurcation was measured with the purified complex using NADH as donor and caffeyl-CoA as high-potential acceptor. Oxidation of NADH did not start before addition of clostridial ferredoxin or high amounts of FAD or FMN. In contrast to size exclusion chromatography, the justpublished crystal structure of caffeyl-CoA reductase revealed a heterododecameric Car(CDE)4 complex. The core consists of a CarC tetramer to which four CarDE dimers are peripherally attached. Each CarDE dimer is bound via two contact points to two CarC subunits, like in the very similar structure of the (EtfAB-Bcd)4 complex (section 3.2.1 and Figure 6). The flexible domain II of CarD, however, occupies a position similar to that of domain II of EtfA in free EtfAB in which the bifurcating eFAD of CarE is 19 Å apart from d-FAD in domain II of CarD. Modeling experiments show that, similar to (EtfAB-Bcd)4, domain II of CarD is able to rotate between the bifurcating Bstate (e-FAD and d-FAD 14 Å apart) and the dehydrogenase Dstate (d-FAD and c-FAD on CarC 8 Å apart). A remarkable difference between Car(CDE)4 and (EtfAB-Bcd)4 is the presence of a clostridial-type ferredoxin with two [4Fe-4S] clusters as N-terminal extension of CarE. Though this ferredoxin occupies a position near the bifurcating e-FAD, removal of this did not affect the structure, stability, or bifurcating activity of Car(CDE)4. The authors speculate that this ferredoxin might act as low-potential electron acceptor though the distance between e-FAD and the proximal [4Fe-4S] cluster is 19 Å, too long for an efficient e-transfer. However, in the middle between e-FAD and the proximal [4Fe-4S] cluster is located Trp96, which could act as relay station for the electron. The reduced ferredoxin of the N-terminal extension may give the electron further to a soluble ferredoxin or to Rnf in the cytoplasmic membrane. 3.2.3. EtfAB(LctCB)-Lactate Dehydrogenase. The oxidation of lactate to pyruvate by NAD provides a severe thermodynamic problem for all organisms from anaerobic

2NAD+ + D‐lactate− + 2Fd red− ⇋ 2NADH + pyruvate− + 2Fdox ;

ΔG°′ = + 6 kJ mol−1 lactate

(5)

A. woodii grows on 80 mM DL-lactate as carbon and energy source at 30 °C with a doubling time of 5 h to an optical density of 1.4. Ferredoxin for lactate oxidation is reduced by the oxidative decarboxylation of pyruvate to acetyl-CoA mediated by pyruvate-ferredoxin oxidoreductase (PFOR). Half of the NADH from lactate oxidation is used to synthesize more acetylCoA from CO2 for which ATP and additional reduced ferredoxin are required. The other half of NADH reduces ferredoxin mediated by Rnf and driven by ΔμNa+ generated by the F1Fo-ATPase (3.3 ΔμNa+ per ATP). This pathway exclusively conserves ATP via SLP from acetyl-CoA, whereas reverse electron transport phosphorylation (reverse ETP) supplies reduced ferredoxin. In summary, 4 lactate are converted to 6 acetate concomitant with the formation of only 1.6 ATP. For a complete scheme, see Weghoff et al.61 I

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 9. Proposed scheme of the membrane-associated FixABCX system from Azotobacter vinelandii. Fld stands for flavodoxin; FldH• stands for for the blue neutral semiquinone, and FldH− stands for the hydroquinone. FixAB and FixX are peripheral membrane proteins; FixC is an integral membrane protein.

woodii. Using LctD as query, the M. elsdenii genome revealed four hits with 50−60% sequence identities, three of which were annotated as glycolate oxidase and one as FAD-binding oxidoreductase, which is the most likely candidate for the putative confurcating Ldh. 3.2.4. EtfAB(FixBA)-Ubiquinone Reductase. The electron donor for the reduction of nitrogen to ammonia (nitrogen fixation) is either reduced ferredoxin or the hydroquinone form of flavodoxin. Whereas reduced ferredoxin and flavodoxin are readily available in anaerobes, aerobes and purple bacteria need a special system to achieve such a low reduction potential. There has been indirect evidence that the f ix gene products, forming the FixABCX complex from the phototroph Rhodospirillum rubrum, are responsible for the supply of electrons for nitrogen fixation.64 Because FixBA is homologous to EtfAB, it has been proposed that electron bifurcation from NADH to a quinone (rhodoquinone in R. rubrum; ubiquinone in Azotobacter vinelandii)65,66 could lead to reduced ferredoxin.2 This idea is supported by the homology of FixC to the electron transferring flavoprotein ubiquinone oxidoreductase (EtfQO). Hence, NADH could reduce a-FAD on FixA to a-FADH−, which could bifurcate to b-FAD•− on FixB and via FixX to ferredoxin or flavodoxin. b-FADH− on FixB would then swing over to FixC to reduce a quinone of the respiratory chain (Figure 9). Recently a paper by the BETCy group appeared with the aim to elucidate the mechanism of FixABCX from the diazotrophic bacterium Azotobacter vinelandii.66 BETCy is the abbreviation of “Biological Electron Transfer and Catalysis”, an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE). Though a strict aerobe, A. vinelandii is able to fix nitrogen and uses Rnf and FixABCX to provide reduced ferredoxin or flavodoxin as shown by single and double mutants of these complexes. The f ixABCX genes were homologously overexpressed in A. vinelandii, and the produced membrane proteins were solubilized with dodecylmaltoside. Purification by ion exchange and size exclusion chromatography yielded the whole complex as a heterotetramer. Analysis by SDS-PAGE revealed four bands with the expected molecular masses of 48.7 kDa (FixC), 39.2 kDa (Fix B), 30.7 kDa (Fix A), and 10.7 kDa (FixX) at a 1:1:1:1 ratio. Upon mass spectrometry, the whole complex fragmented into FixAB with 2 FAD and FixCX with 1

In cell extracts, the confurcating lactate dehydrogenase was assayed with ferricyanide as electron acceptor and purified to homogeneity with a specific activity of 30 U mg−1. This high activity is obtained in the presence of 50 mM CaCl2 and 5 μM FAD, which is also required for stability of the enzyme. When the preparation was analyzed by SDS-PAGE, three distinct bands were detected, which were identified by peptide mass fingerprinting as EtfB (29 kDa), EtfA (46 kDa), and a lactate dehydrogenase (Ldh, 51 kDa). The three proteins form a tight complex with an apparent stoichiometry of 1:1:1. Most likely, the complex contains three FADs and, as predicted from comparison with other Etf-containing complexes, one [4Fe-4S] cluster due to the ferredoxin-like sequence with four cysteines in EtfA. The confurcating reaction with NAD and D-lactate was dependent on ferredoxin (reaction 5), which was kept in the reduced form with purified CO-dehydrogenase under an atmosphere of 100% CO. The apparent Km values are 31 μM reduced ferredoxin, 430 μM NADH, and 3.6 mM D-lactate. The reaction with L-lactate revealed a high Km = 112 mM that was probably due to a contamination of L-lactate with D-lactate or more likely of the enzyme preparation with a lactate racemase. In the reverse direction, as expected, the bifurcating reaction required NADH, pyruvate, and oxidized ferredoxin with an NADH:ferredoxin stoichiometry close to 2:1. It proceeded at a specific activity of 1.2 U mg−1 protein, less than half the rate of the confurcating reaction (2.9 U mg−1 protein) in the physiological direction. The genes of the confurcating Ldh are located in an operon of the genome of A. woodii in the order lctB (encoding EtfB), lctC (EtfA), lctD (Ldh), lcdE (lactate permease), and lcdF (lactate racemase). Similar gene arrangements, especially LctBCD, are present in the genomes of many strict anaerobes. Hence, confurcation with reduced ferredoxin appears to be a general mode of lactate oxidation by NAD in anaerobic bacteria. Over 40 years ago, a D-Ldh in M. elsdenii was described, which like LctBCD catalyzed the oxidation of D-lactate by ferricyanide.62 Furthermore, it was shown that the free EtfABMe (see section 3.2) transferred electrons from D-lactate via the D-Ldh to butyryl-CoA dehydrogenase without the participation of ferredoxin.63 Although this result was probably due to an artifact caused by exposure to air, it indicated an interaction of the D-Ldh with EtfAB similar to the tight LctBCD complex in A. J

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

grown on L-alanine. Only a 3-fold purification (0.28−0.79 U mg−1) was necessary to obtain a pure preparation. Hence, EtfAB-Pcd represents ∼30% of the soluble protein in a C. propoinicum cell. Butyryl-CoA (Km = 100 ± 10 μM) was also oxidized (Vmax = 1.4 ± 0.1 U mg−1) by EtfAB-Pcd in the presence of the artificial electron acceptor. Surprisingly, EtfABPcd catalyzed the reduction of acrylyl-CoA (Km = 2 ± 1 μM) with NADH (Vmax = 1.8 ± 0.2 U mg−1) in the absence of ferredoxin (reaction 7), but crotonyl-CoA was not accepted either in the absence or presence of ferredoxin. The acrylyl-CoA analogue vinylmethylketone (3-buten-2-one) was also reduced by NADH even at a faster rate (Vmax = 12 U mg−1) but with a much higher apparent Km = 1800 ± 50 μM. EtfAB-Pcd also exhibited diaphorase and NADH-oxidase activities.67 The absence of bifurcation with EtfAB-Pcd is especially astonishing because the reduction potential of acrylyl-CoA/propionyl-CoA (Em = +69 mV) is even 79 mV higher than that of crotonylCoA/butyryl-CoA (Em = −10 mV; reaction 7).15 SDS-PAGE of pure EtfAB-Pcd and staining with Coomassie revealed three bands with molecular masses of 41, 38, and 28 kDa at a ratio of 2.0:1.0:0.8, respectively. These results could be confirmed by separation of the three subunits with reversedphase HPLC in 0.1% trichloroacetic acid at a ratio of 1.7:1.0:1.0. N-terminal sequencing identified the subunits as homologues of a homotetrameric butyryl-CoA dehydrogenase (Pcd) and EtfAB, respectively. Hence, the quaternary structure of the whole complex EtfAB(Pcd)2 is similar to the bifurcating EtfAB(Bcd)2 complexes. The molecular mass of the complex determined by gel filtration (600 ± 50 kDa) indicated a composition of four EtfAB(Pcd)2 subcomplexes (592 kDa), whereas the molecular mass of (EtfAB-Pcd)4 (520 kDa), similar to (EtfAB-Bcd)4, would be too low. The flavin content (90% FAD and 10% FMN) was determined as 2.4 mol per mol EtfAB(Pcd)2. The genome of C. propionicum comprises three acrABC gene clusters, each of which contains the three genes coding for Pcd, EtfB, and EtfA (gene numbers 3161-3159, 23232325, and 2517-2515).68 The N-terminal sequences of Pcd, EtfB, and EtfA matched best to the proteins encoded by cluster 3159−3161 in this order (pcd-etf B-etfA), which was confirmed by peptide mapping. In the two other clusters, the three genes have the reverse order (etfA-etf B-pcd). EtfA 3159 differs from EtfA 2325 by only 3 amino acids, whereas the other proteins exhibit significant differences. None of the clusters are located in operons with adjacent identified genes of metabolically related enzymes. To try to explain why the EtfAB-Pcd complex has evolved to be not electron bifurcating, the energy metabolism of alanineand lactate-fermenting anaerobes that form propionic acid has to be looked at in detail: Clostridium propionicum ferments alanine and serine to acetate and propionate (Figure 10).69,70

FAD, 2 [4Fe-4S] clusters, and an additional mass of 377 Da, probably riboflavin (m = 376.37 Da). Because the crystal structure of FixABCX could not be obtained, cross-linking experiments and molecular modeling lead to a putative Fix prototype in which the bifurcating pathways could be inserted (Figure 9). From comparison with EtfABAf the bifurcating β-FAD was placed on FixA as a-FAD. The low-potential electron was proposed to flow via the two [4Fe-4S] clusters in FixX to flavodoxin semiquinone (FldH•), which was used in the activity experiments, yielding the hydroquinone (FldH−). The high-potential electron probably took the path via b-FAD in FixB and c-FAD in FixC to ubiquinone (UQ, reaction 6). EPR spectroscopy showed that NADH or dithionite reduced the [4Fe-4S]2+ clusters to [4Fe4S]+ and most likely b-FAD to the semiquinone. Because the dithionite-reduced isolated subunit FixX from Roseif lexus castenholzii exhibited a similar EPR spectrum, it was concluded that FixX from A. vinelandii also contained the clusters. Apparently, both clusters are comparable to those in the bifurcating NfnAB, though this enzyme is not related to FixABCX (see section 3.5). 2NADH + 2FldH· + UQ 1 → 2NAD+ + 2FldH− + UQ 1H 2 ; ΔG°′ = − 60 kJ mol−1 UQ 1

(6)

The spectrophotometric bifurcation assay, recorded simultaneously at three wavelengths (340, 450, and 580 nm), contained 200 μM NADH and 300 μM ubiquinone (due to better water solubility UQ1 taken instead of the natural UQ8). After adding 0.8 μM FixABCX, a specific NADH oxidation rate of 396 ± 44 nmol min−1 mg−1 protein was already observed at 340 nm, which after addition of 85 μM Fld SQ only increased by 32% to 524 ± 21 nmol min−1 mg−1 with a turnover number of 68 ± 3 min−1. Most likely in the absence of FldH•, UQ1 not only served as high-potential but also as low-potential acceptor because due to its water solubility it can interact with FixC as well as with FixX. Furthermore, the measurements at 580 nm in the absence of FixABCX revealed that FldH• reduced UQ1, which was confirmed by the formation of oxidized Fld at 450 nm. In the presence of all components, the disappearance of FldH• (578 nm) and the appearance of Fld (450 nm) came to halt because the formation of FldH− by electron bifurcation was apparently balanced by its oxidation back to FldH• by UQ1. Although this experiment suggests that FixABC indeed bifurcates, an unequivocal demonstration requires another approach, the direct identification of FldH− that can only be formed by bifurcation. Coupling with hydrogenase and detection of hydrogen by GC would be a direct demonstration of bifurcation by FixABCX, which has been done with the reduced ferredoxin generated by EtfAB-Bcd from C. kluyveri.3 3.2.5. Nonbifurcating EtfAB-Propionyl-CoA Dehydrogenase. Although all EtfAB-butyryl-CoA dehydrogenases assayed in this respect have been shown to be electron bifurcating, the very closely related Etf-propionyl-CoA dehydrogenase appears not to be. +

3 alanine + 2H 2O → 3NH4 + + CO2 + acetate− + 2 propionate−;

(8)

The fermentation of three L-alanine branches at pyruvate, with one oxidized to acetate, results in ATP and reduced ferredoxin (Figure 10). Ferredoxin-NAD reductase (Rnf) regenerates NADH and the thereby formed ΔμNa+ may drive the uptake of alanine. The other two pyruvates are reduced via D-lactate to propionate, which involves the nonbifurcating EtfAB-Pcd (reaction 8).67 Hence, because of the lack of bifurcation, only one ATP is conserved in the fermentation of three alanines, which thermodynamically would allow at least

+

NADH + acrylyl‐CoA + H → NAD + propionyl‐CoA; ΔG°′ = − 75 kJ mol−1 acrylyl‐CoA

ΔG°′ = − 162 kJ mol−1 acetate

(7)

Using an assay with propionyl-CoA as substrate (Km = 50 ± 5 μM) and ferricenium as electron acceptor, EtfAB-Pcd (also called acrylyl-CoA reductase, Acr) could be purified to homogeneity from cell extracts prepared from C. propionicum K

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

ent pyruvate dehydrogenase. Oxidation of NADH by menaquinone (MK) catalyzed by quinone NADH dehydrogenase (Nuo) gives rise to 0.5 ATP via ΔμH+, and reoxidation of the reduced menaquinone results in the formation of another 0.5 mol ATP via ΔμH.73,74 Thus, via the methylmalonyl-CoA pathway, twice the amount of ATP is formed per mol of acetate than in the acrylate pathway. This agrees well with the different growth yields per mol lactate: 2.6 g mol −1 with C. homopropionicum and 6 g mol−1 with P. f reudenreichii.75 Clostridia using the acrylate pathway (Figure 10) compensates this lower cell yield by an almost 3-times faster growth rate and an over 6-times faster maximal substrate turnover rate. This growth type “fast but less efficient” could be an adaptation to the environment; alternatively, it could be a tribute to the toxicity of acrylyl-CoA. This intermediate is a potent electrophile that reacts with DNA. Reducing the steady state concentration of acrylyl-CoA by a fast turnover rate and a more irreversible reaction should be advantageous for the cell. Contrary to first expectations, the saturation constants for lactate (Ks, equivalent to Km of enzymes) was estimated as 560 ± 210 μM for C. homopropionicum and 140 ± 30 μM for P. f reudenreichii.75 This difference likely reflects the different lactate dehydrogenases, confurcating Ldh and FAD-Ldh, respectively. 3.2.6. Comparison of EtfAB Sequences. The sequence comparison51 includes the known bifurcating EtfAB from A. fermentans (EtfAB-1)41 and M. elsdenii,51 the Etf-Bcd complexes of C. kluyveri3 and C. dif f icile,76 the Etf-caffeyl-CoA reductase,55 and Etf-lactate dehydrogenase61 complexes of A. woodii as well as FixAB of R. rubrum77 and A. vinelandii.66 Nonbifurcating Etfs were from C. propionicum,67,68 A. fermentans (EtfAB-2),42 Methylophilus methylotrophus,78 Paracoccus denitrif icans,44 and Homo sapiens43 in addition to an uncharacterized second Etf from R. rubrum79 (Figure 12). The almost completely conserved sequence in EtfB comprising 18 amino acids from G117 to G134 (A. fermentans numbering), especially the cluster of 8−10 amino acids (I121)DGDTAQVG(P130) appears to be unique to the bifurcating EtfAB. The crystal structure41 and modeling experiments80 suggest that the DGDTAQVGP sequence is responsible for binding of β-FAD, which is absent in nonbifurcating Etfs. Hence, EtfAB-2 from A. fermentans should not bifurcate, which was verified experimentally.41 Surprisingly, EtfB in all three EtfAB-propionyl-CoA dehydrogenase gene clusters from C. propionicum contained the IDGDTAQVGP sequence indicating that they belong to the family of bifurcating Etfs but apparently lost this ability (see section 3.2.5). The reason for this could be the 15 amino acid-comprising sequence TEEEFAKEAAMGCED inserted into the EtfAs 3159 and 2325 between K128 and P144 of two of the three EtfAB-propionylCoA dehydrogenases (Figure 12). This sequence with six

Figure 10. Proposed fermentation pathways of alanine by C. propionicum and of lactate by C. homopropionicum. Red and blue arrows indicate reactions specific for alanine and lactate, respectively. Black arrows stand for reactions common to both pathways. The reaction catalyzed by EtfAB-propionyl-CoA dehydrogenase is shown on top of the figure. Whereas oxidation of lactate to pyruvate requires a confurcating lactate dehydrogenase, the oxidation of alanine is driven by the in vivo low ammonia concentrations (∼1 mM). For further details, see text.

two ATP (−162/−77 = 2.1; reaction 8). The closely related C. homopropionicum ferments lactate but not alanine (reaction 9 and Figure 10).71 3 lactate− → CO2 + acetate− + 2 propionate− + H 2O; ΔG°′ = −168 kJ mol−1 acetate

(9)

C. homopropionicum contains a confurcating EtfAB-Ldh, which catalyzes the oxidation of lactate with 2 NAD and 2 Fdred− (see Figure 10 and section 3.2.3).40,50 This therefore required 2 Fdred− stemming from the oxidation of pyruvate to acetyl-CoA, which gives rise to 1 ATP by SLP. The 2 NADH formed are used to reduce 2 lactate via acrylyl-CoA to 2 propionate catalyzed by the EtfAB-Pcd complex, which is not bifurcating and therefore not coupled with the formation of ATP. Propionibacterium f reudenreichii also ferments three lactate to one acetate and two propionate (reaction 9) but synthesizes propionate via the coenzyme B12-dependent methylmalonylCoA mutase (Figure 11). The bacterium uses in its fermentation an FAD-lactate-malate dehydrogenase72 and an NAD-depend-

Figure 11. Proposed pathway of lactate fermentation by P. f reudenreichii. MK, menaquinone. L

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 12. Partial sequences of EtfAB. Experimentally verified or very likely bifurcating Etfs are shown in bold letters. Afer, Acidaminococcus fermentans; Mels, Megasphaera elsdenii; Ckluy, Clostridium kluyveri; Awood, Acetobacterium woodii; Cprop, Clostridium propionicum; Rru, Rhodspirillum rubrum; Azv, Azotobacter vinelandii; Methylo, Methylophilus methylotrophus; Para, Paracoccus denitrif icans; Human, Homo sapiens. EtfB, consensus sequence of electron bifurcating Etf (red). EtfA, apparently inserted sequences into EtfA of C. propionicum (in cyan). Gene numbers follow the abbreviations. Car, caffeyl-CoA reductase; Lct, bifurcating lactate dehydrogenase. The number at the end of a partial sequence indicates the position of the last amino acid in the sequence of the whole protein. Three further Etfs, two from Rhodospirillum rubrum (Rru) and one from A. fermentans (Afer0986), are included. Rru2266 is proposed to bifurcate,2 whereas Afer0986 did not bifurcate with BcdAf.42 Adapted with permission from ref 51. Copyright 2015, John Wiley and Sons.

negative and one positive charges forms a loop in the vicinity of β-FAD. It possibly prevents binding of the negatively charged ferredoxin. A nucleotide sequence encoding completely different 22 amino acids is inserted at the equivalent place into the third EtfAB-propionyl-CoA dehydrogenase gene cluster, which is not expressed in C. propionicum when grown on α-alanine.51 However, the absence of ferredoxin alone does not uncouple the bifurcation. Experiments with EtfAB-Bcd demonstrated that omission of ferredoxin in a bifurcation reaction (reaction 2b in section 3.1) resulted in no NADH oxidation (see also section 3.5).

C6H12O6 + 2H 2O → 2CO2 + 2 acetate− + 2H+ + 4H 2 ; ΔG°′ = −216 kJmol−1 glucose

(10)

Mechanistically, 4 ATP can be conserved by substrate level phosphorylation from 1 glucose, 2 ATP via the NAD-dependent Embden−Meyerhof pathwa,y and 2 ATP via acetyl-CoA derived by ferredoxin-dependent oxidation of 2 pyruvate (for a metabolic scheme, see Figure 12A in Buckel and Thauer 2013).5 Thermodynamically, however, only 3 ATP appear to be possible, but ΔG°′ is defined at 25 °C (298 K); at 80 °C (353 K), the growth temperature of T. maritima, ΔG′ drops to −274 kJ mol−1, where 4 ATP/glucose become feasible. Another thermodynamic problem remains because 4 H2 have to be produced from 4 Fdred− and 2 NADH. The organism elegantly solved this problem by producing a confurcating [FeFe]hydrogenase catalyzing reaction 11 in the indicated stoichiometry81

3.3. NAD(P)H Dehydrogenase (NuoF Homologues)-Containing Complexes

3.3.1. NADH Dehydrogenase-[FeFe]-Hydrogenase. The anaerobic bacterium Thermotoga maritima ferments glucose to 2 CO2, 2 acetate, and 4 H2 (reaction 10) M

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

including the acetogens A. woodii and Moorella thermoacetica in which the physiological function of the enzyme is to catalyze the reduction of NAD and ferredoxin with 2 H2 (reverse of reaction 11). Acetogenic bacteria have to tackle the difficult reduction of 2 CO2 by 4 H2 via the Wood−Ljungdahl pathway (for metabolic schemes, see Figure 16A in Buckel and Thauer 2013).5 One CO2 is converted to a methyl group and the other to CO, which together with CoA are combined to acetyl-CoA. The main problem of this pathway appears to be the very low reduction potential for the reduction of CO2 to CO (Em = −520 mV), which cannot be reached with H2 even at 105 Pa. A bifurcating hydrogenase with NAD as high-potential acceptor (E′ = −280 mV) could reach −550 mV. Acetogens, however, thrive at ≥100 Pa H2 equal to a potential of E′ ≤ − 326 mV, which could be amplified by bifurcation only to a potential of −370 mV. A possible solution to this problem came from Arren Bar-Even, who argued that E′ = −400 mV (300 Pa H2) would be sufficient for the reduction of CO2 to CO because this endergonic reaction is coupled to the exergonic synthesis of acetyl-CoA from CO, methyltetrahydrofolate, and CoA.88 The bifurcating hydrogenases of acetogens are similar to those of T. maritima. The Moorella thermoacetica89 and Acetocbacterium woodii56 enzymes contain almost the same but not identical sets of FeS clusters in their three different subunits (HydABC) as the enzyme from T. maritima (Figure 13). The enzyme of A. woodii comprises a forth small subunit (HydD, 14 kDa) without the FeS cluster. Surprisingly, after purification of the electronbifurcating hydrogenase from A. woodii, the preparation did not contain flavin, but the NAD- and ferredoxin-dependent hydrogenase activity required added FMN. In the electronbifurcating hydrogenase from M. thermoacetica, 0.8 FMN/ heterotrimer was detected, most likely due to the addition of FMN to all buffers used for purification. Both enzymes were assayed in both directions, hydrogen formation and consumption, in the presence of 10−50 μM FMN. Whether FMN was reduced during the assays was not analyzed. Apparently, the amounts of FMN had no influence on the measured 1:1 stoichiometry between NADH and 2 Fdred−. 3.3.2. NADPH Dehydrogenase-[FeFe]-Hydrogenase. The acetogenic bacterium Clostridium autoethanogenum that grows on CO, H2, and CO2 (syngas) contains an NADP-specific bifurcating hydrogenase/formate dehydrogenase, which has been purified and characterized.90,91 It is composed of six different subunits and catalyzed reaction 12 in the given stoichiometry

NADH + 2Fd red− + 3H+ ⇋ NAD+ + 2Fdox + 2H 2 ; ΔG°′ = +17 kJ mol−1 NADH

(11)

Fdred−

Though any clostridial (E′ = −500 mV) alone can give rise to H2 (Em = −414 mV), the extra energy of ΔE = 86 mV is used to lower the potential of the electrons of NADH (Em = −320 mV) to the level of hydrogen resulting in ΔG′ = +3 kJ mol−1 NADH in reaction 11. In 1999, the corresponding enzyme had already been purified under anoxic conditions as a soluble, viologen reducing hydrogenase.82 It is composed of three subunits, α (73 kDa), β (68 kDa), and γ (19 kDa), each of which contains FeS clusters as predicted from the amino acid sequences (HydABC). The α-subunit (HydA) is very similar to the one-subunit [FeFe]-hydrogenase from C. pasteurianum with 30% sequence identity and contains the active site H-cluster and 2 [2Fe-2S] and 3 [4Fe-4S] clusters. The β-subunit (HydB) is related to the NADH-binding NuoF subunit of NADH:quinone oxidoreductase from E. coli and contains FMN, 1 [2Fe-2S], and 3 [4Fe-4S] clusters. Ferredoxin might interact with the [2Fe-2S] cluster in the γ-subunit (HydC), which is related to NuoE.81 A second flavin suitable for electron bifurcation could not be detected. Electron bifurcation at FMN of the β-subunit56 appears unlikely because binding of NAD, the electron acceptor/donor with the highest potential, and bifurcation at the same cofactor is not imaginable (Figure 13). A crystal structure is urgently needed to solve this problem. However, all attempts to obtain diffracting crystals have failed until now.

Figure 13. Proposed scheme for electron bifurcation catalyzed by NADH dehydrogenase-[FeFe]-hydrogenase. Adapted with permission from ref 5. Copyright 2013, Elsevier.

NADPH + 2Fd red− + 3H+ ⇋ NADP+ + 2Fd ox + 2H 2 ; ΔG°′ = + 17 kJ mol−1 NADPH

In the Nuo complex of the respiratory chain, NADH reduces FMN to FMNH−, which has been proposed to bifurcate.83,84 The low-potential electron is thought to reduce the [2Fe-2S] cluster N1a on NuoE (12.3 Å apart, edge to edge), whereas the high-potential electron goes to the [4Fe-4S] cluster N3 on NuoF (7.6 Å apart) and further via six additional FeS clusters to ubiquinone. As soon as N3 has been reoxidized, the electron on N1a returns to FMN and flows to the formed semiquinone of ubiquinone. This transient storage of the electron on N1a avoids a long-living FMN semiquinone, which could react with oxygen to superoxide. However, recent studies do not support this elegant view.85−87 Furthermore, the distances between FMN and the iron−sulfur clusters do not match those of the established bifurcating systems (see section 4.4 and Table 1). The electron-bifurcating NADH dehydrogenase-[FeFe]hydrogenase complex has been found in many other anaerobes

(12)

As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8 kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase; the 65.5 kDa subunit (HytB) is an iron−sulfur FMN protein harboring the NADP binding site (homologous to NuoF); the 51.4 kDa subunit (HytA) is the proper [FeFe]-hydrogenase, and the 18.1 kDa (HytC), 28.6 kDa (HytD), 19.9 kDa (HytE1), and 20.1 kDa (HytE2) subunits are iron−sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP with H2 or formate (see reaction 13, in which NADP is replaced by NAD) and the reversible formation of H2 and CO2 from formate. The former names of NAD and NADP were diphosphopyridine nucleotide (DPN) and triphosphopyridine N

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

nucleotide (TPN). Thus, the NAD (DPN)-specific hydrogenase is abbreviated as Hyd and the NADP (TPN)-specific enzyme as Hyt. 3.3.3. NADH-Dehydrogenase-[W/Se]-Formate Dehydrogenase. Clostridium acidurici ferments uric acid to ammonia, CO2, and acetate. In the first step, ferredoxin reduces uric acid to xanthine, which is further degraded via formimidoglycine to formate and pyruvate. Oxidation of pyruvate yields part of the reduced ferredoxin required for the first step and acetyl-CoA for substrate-level phosphorylation. The other part of reduced ferredoxin stems from the electron bifurcating formate dehydrogenase (reaction 13).92

CoM‐S‐S‐CoB + 2H 2 + 2Fdox → CoM‐SH + CoB‐SH + 2H+ + 2Fd red−;

ΔG°′ = −24 kJ mol−1 NAD

(13)

The enzyme is composed of four subunits: FdhF2 (99 kDa), HylB (68.0 kDa), HylA (35.9 kDa), and HylC (18.4 kDa). FdhF2 is the actual formate dehydrogenase with a molybdopterin cofactor, selenocysteine, 1 [2Fe-2S] and 4 [4Fe-4S] clusters. HylB, Hyl for hydrogenase-like, contains FMN to which NADH is suggested to bind, because it is homologous to NuoF with 1 [2Fe-2S] and 3 [4Fe-4S] clusters. HylA holds 1 [2Fe-2S] and 4 [4Fe-4S] clusters, and HylC has 1 [2Fe-2S]. HylB shows high sequence identities to HydB and HytB of the electron bifurcating [FeFe]-hydrogenases but lacks the H-cluster. Apparently, the electron bifurcating module HylABC can be combined with both formate dehydrogenases and [FeFe]hydrogenases just as the EtfAB module can be combined with butyryl-CoA dehydrogenase, lactate dehydrogenase, and caffeylCoA reductase. As with the bifurcating hydrogenases, the role of FMN has to be clarified. It has to be established whether HylB and HydB contain a second FMN, one for NAD binding and the other for bifurcation. In the Introduction, it was mentioned that metals such as molybdenum could also be the site of electron bifurcation. One example is the molybdenum in arsenite oxidase that has crossedover reduction potentials. Upon one-electron reduction of the fully oxidized Mo(VI), only the Mo(IV) state can be detected because the reduction potential of the Mo(VI)/Mo(V) couple is more positive than that of the Mo(V)/Mo(IV) couple.93 Thus, it has to be considered that the molybdenum in the formate dehydrogenase subunit of the NADH dehydrogenase-formate dehydrogenase rather than a flavin could be the site of electron bifurcation.4

Figure 14. Crystal structure and proposed pathways of electron flow of the bifurcating heterodisulfide reductase/hydrogenase MvhADG− HdrABC from the thermophilic methanogenic archaeon Methanothermococcus thermolithotrophicus. Adapted with permission from ref 96. Copyright 2017, The American Association for the Advancement of Science.

The multisubunit enzyme complex is composed of a dimer of two HdrABC-MvhAGD heterohexamers with an FAD-containing HdrA dimer in the center. Two catalytic arms, MvhAGD and HdrBC, are attached to each subunit. Hydrogen is oxidized at the [NiFe] center in MvhA, and the electrons flow through 3 [4Fe-4S] clusters (MG1−3) in the MvhG subunit, 2 [4Fe-4S] clusters (HA1−2) in the HdrA subunit, to MvhD, a [2Fe-2S] cluster subunit, 30 Å apart from the bifurcating FAD. The authors propose three different scenarios for how this large gap is bridged and how the FAD is reduced by an electron pair. In one scenario, favored by the authors of this review, the disulfide bridge C541−C326 of HdrA located in the middle between MvhD and FAD could be reduced by two single electron transfers and oxidized by the bifurcating FAD by withdrawal of a hydride. The high-potential electron goes from FADH− to the [4Fe-4S] cluster HA4 and further via the [4Fe-4S] clusters HC1 and HC2 on HdrC to the noncubane cluster pair, the proximal HB1 and the distal HB2 on HdrB. Each of these novel [4Fe-4S] clusters appears to be composed of a [2Fe-2S] and a [3Fe-4S] cluster, which share one iron and one sulfur. One sulfur of the [3Fe-4S] cluster is replaced by a bridging cysteine ligand. From

3.4. Heterodisulfide Reductase (HdrABC)-Containing Complexes

3.4.1. HdrABC-[NiFe]-Hydrogenase. Most methanogens are able to produce methane by reduction of CO2 with hydrogen (reaction 14) CO2 + 4H 2 ⇋ CH4 + 2H 2O;

(15)

In nature, where the methanogens thrive, the partial pressure of H2 is much lower than 105 Pa, which shifts the effective reduction potential of H2 to a more positive region. The lowest partial pressure of H2, at which methanogens can grow, is ∼8 Pa, resulting in E′ near −290 mV.95 Starting with H2 at this potential, bifurcation to heterodisulfide would lead to a reduced ferredoxin with E′ near −450 mV, just low enough to reduce CO2 to formylmethanofurane. In contrast, acetogens use NAD as a high-potential acceptor in a bifurcation of H2 to ferredoxin. Because the intracellular reduction potential of NAD (E′ = −280 mV, see Nfn below) is 140 mV more negative than that of the heterodisulfide (Em′ = −140 mV), acetogens would require at least 2000 Pa H2 (near −360 mV) to obtain a reduced ferredoxin with E′ near −450 mV. During the writing of this review, the crystal structure of the MvhADG−HdrABC complex with 2 × 14 iron−sulfur clusters, 2 FAD and 2 [NiFe] centers has been published (Figure 14).96

2HCOO− + 2Fdox + NAD+ ⇋ 2CO2 + 2Fd red− + NADH + H+;

ΔG°′ = −52 kJ/mol

ΔG°′ = −131 kJ/mol (14)

The first step in methanogenesis is the reduction of CO2 with ferredoxin to the formyl group of formylmethanofurane (formyl-MFR). In methanogens without cytochromes, the required reduced ferredoxin is regenerated by electron bifurcation in reaction 15 catalyzed by the soluble hydrogenase−heterodisulfide reductase complex (MvhADG− HdrABC; Mvh stands for methylviologen hydrogenase; for a metabolic scheme see Figure 15 in ref 5). The electrons from H2 (Em = −414 mV) bifurcate to the heterodisulfide (Em = −140 mV) and to ferredoxin.94 O

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

methyltetrahydrosarcinapterin is transferred to CoM-SH, forming methyl-S-CoM, which reacts with CoB-SH to form methane and the heterodisulfide. If finally reduced ferredoxin regenerates CoM-SH and CoB-SH, a balanced pathway could be constructed. However, the formation of acetyl-CoA requires 1.0 ATP and the methyltransfer from methyltetrahydrosarcinapterin to CoM-SH only gains 0.5 ATP via 2 × ΔμNa+. Recently, a bifurcating heterodisulfide reductase HdrA2B2C2 from M. acetivorans has been characterized and shown to catalyze reaction 18.100

soaking experiments with heterodisulfide, the following mechanism has been deduced: The heterodisulfide binds between HB1 and HB2 and accepts one electron from each cluster, which causes homolysis of the S−S bond and binding of CoB-S• and CoM-S• to one tetracoordinated iron of each cluster. A proton-coupled one-electron transfer releases CoBSH and another electron from the second bifurcation round liberates CoM-SH from the transiently pentacoordinated irons. The low-potential electron most likely leaves the FAD semiquinone via the remaining 2 [4Fe-4S] clusters (HA5 and HA6) to end up in ferredoxin. Whether there are conformational rearrangements involved remains to be established. It should be noted here that methanogens with cytochromes have solved the problem of ferredoxin reduction differently. For the reduction of the heterodisulfide, they use the enzyme complex VhoACG-HdrDE of which the C- and E-subunits contain cytochrome b.95 Vho stands for viologen hydrogen oxidizing, and Hdr stands for heterodisulfide reductase. VhoACG is a [NiFe]-hydrogenase located at the outside of the cytoplasmic membrane and catalyzes the reduction of methanophenazine by H2. HdrDE sits at the inside and catalyzes the reduction of the heterodisulfide with the dihydro form of this electron carrier. Methanophenazine consists of 2-hydroxyphenazine that is linked via an ether bridge to five isoprene units (Em = −165 mV). The free energy of these combined reactions translocates 4 H+, of which two are used by the energyconverting [NiFe]-hydrogenase EchABCDEF to drive the reduction of 2 ferredoxin required for the first step of methanogenesis; with the remaining 2 H+, ATP is synthesized.95 It should be mentioned that the amount of translocated protons or sodium ions, as in case of some Rnfs, has not been measured. It is based solely on thermodynamic calculations. 3.4.2. HdrABC-[W/Se]-Formate Dehydrogenase. Methanococcus maripaludis reduces CO2 to methane not only with hydrogen but also with formate as electron donor. In the presence of hydrogen, the organism produces a heterodisulfide reductase/hydrogenase VhuAUDG−HdrABC similar to that from M. thermolithoautotrophicus MvhADG−HdrABC. In M. maripaludis, the hydrogenase proteins are called VhuAUDG [Vhu for viologen reducing hydrogenase containing selenocysteine (U)]. Upon switching to formate, the VhuAUG proteins are exchanged by formate dehydrogenase FdhAB, whereas VhuD is identical in both versions.97 This makes sense because VhuD has the same function as MvhD, which contains the [2Fe2S] cluster 30 Å apart from the bifurcating FAD in HdrA. The FdhAB VhuG−HdrABC complex has been proposed to catalyze reaction 16.98 A stoichiometry was not determined.

CoM‐S‐S‐CoB + 2F420H 2 + 2Fdox = CoM‐SH + CoB‐SH + 2F420 + 2Fd red− + 2H+; ΔG°′ = −36 kJ mol−1

(18)

The enzyme complex is related to the bifurcating heterodisufide reductase MvhADG−HdrABC from CO 2 reducing methanogens without cytochromes, but MvhADG is absent. It has been proposed that the NADH-like F420H2 directly binds to HdrA2 and reduces the bifurcating FAD by hydride transfer. Hence, F420H2 would take the role of H2 or formate in this system. However, this electron bifurcating enzyme does not solve the ATP problem of M. acetivorans because F420 is not involved in the pathway from acetate to methane. A solution to this problem could be the Na+ pump Rnf, present in M. acetivorans, which mediates the oxidation of the reduced ferredoxin coupled to the generation of 2 × ΔμNa+. The discovery of the ferredoxin-dependent bifurcating heterodisulfide reductase with F420H2 as donor indicates that F420 could replace NAD in Rnf.100 Hence, the ferredoxin reduced by this heterodisulfide reductase could pass again through Rnf. Overall, 3 × 2 ΔμNa+, equivalent to 1.5 ADP, would be generated, and 0.5 ATP would remain for growth, which is predicted from the overall thermodynamics (see section 3.1). In the paper, in which the electron bifurcating HdrA2B2C2 is described, alternative solutions are proposed.100 3.4.4. Other Putatively Bifurcating HdrABC-Containing Complexes. The hdrABC genes are widespread in the anaerobic archaea and bacteria, especially in sulfate-reducing organisms (SRO), where they are proposed to play the central role in energy conservation. Sulfate is reduced to sulfide (H2S or HS−) in two steps: adenosinephosphosulfate (APS) to bisulfite (HSO3−) and bisulfite to sulfide. Hydrogen or various organic compounds, such as lactate or ethanol, serve as reductants (reaction 19). SO4 2 − + 2 ethanol → H 2S + 2 acetate− + 2H 2O;

CoM‐S‐S‐CoB + 2HCOO− + 2Fdox → CoM‐SH

ΔG°′ = −139 kJ mol−1

+ CoB‐SH + CO2 + 2Fd red−; ΔG°′ = −58 kJ/mol (16)

(19)

In both reductive steps, energy is conserved, probably involving electron bifurcation. It has been shown that Desulfovibrio vulgaris strain Hildenborough produces the FlxABCD−HdrABC proteins when grown on ethanol and sulfate. Most likely, a FlxABCD−HdrABC complex exhibits a function very similar to that of the heterodisulfide reductase MvhADG−HdrABC from methanogens. It has been proposed that the FlxABCD proteins take NADH, obtained from oxidation of ethanol, and transfer the electron pair to HdrABC, where it bifurcates. The high-potential electron goes to the disulfide DsrC, which is involved in the reduction of bisulfite, and the low-potential electron reduces ferredoxin.101

3.4.3. HdrABC-F420H2 Dehydrogenase. Methanosarcina acetivorans thrives on the overall decarboxylation of acetate to methane and CO2.99 acetate− + H+ → CH4 + CO2 ; ΔG°′ = −36 kJ mol−1 (17)

Though the reaction appears to be a very simple decarboxylation (reaction 17), it summarizes a whole pathway composed of many steps. In this pathway, acetyl-CoA is cleaved to methyltetrahydrosarcinapterin, CoA and CO, which is oxidized with ferredoxin to CO2. The methyl group of P

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

In the first reductive step, the QmoABC proteins of Desulfovibrio sp. interact with AprAB, the adenosinephosphosulfate (APS) reductase; QmoA is homologous to HdrA.102 Presumably, the membrane protein QmoC with two cytochromes b accepts electrons from menaquinol (MKH2, Em = −70 mV), whereas QmoB catalyzes the oxidation of lactate to pyruvate (Em = −190 mV). The electrons from QmoC and QmoB confurcate at the flavin of QmoA and flow further to AprAB, where APS is reduced to sulfite and AMP (Em = −60 mV) (reaction 20). As the oxidation of menaquinol releases two protons, the whole process generates a proton motive force composed mainly of the electrostatic component of 150 mV against which the electrons have to be pulled through the membrane. Thus, the effective reduction potential of MK increases from −70 to +80 mV.

3.5. NADH-Dependent Ferredoxin: NADP Reductase (NfnAB)

In most organisms, NAD occurs mainly in the oxidized form, whereas in the case of NADP, the reduced form predominates. The oxidized/reduced ratios in aerobically glucose-fed exponentially growing E. coli cells are 31 for NAD/NADH and 0.018 for NADP/NADPH.57 Hence, the effective reduction potential of NAD inside the cell amounts to E′ = −280 mV and that of NADP to E′ = −370 mV rather than Em = −320 mV. For these different potentials to be achieved, the respiratory chain keeps NADH at a low level, whereas NADPH is generated in almost irreversible reactions, such as the oxidative decarboxylation of 6-phosphogluconate to ribulose-5-phosphate. In addition, a membrane-bound transhydrogenase catalyzes the endergonic reduction of NADP by NADH, driven by the proton motive force. All available evidence indicates that reduction potentials of NADP and NAD are also different in anaerobic bacteria, though in E. coli under anoxic conditions the NAD/NADH ratio is shifted to values lower than 31.105 The discovery of confurcating NADP reductases in C. kluyveri106 and M. thermoacetica107 confirms this view. This NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) catalyzes the reversible concomitant reduction of 2 NADP by 1 NADH and 2 reduced ferredoxin (reaction 23).

2APS2 − + MKH 2 + lactate− = 2AMP2 − + 2HSO3− + MK + pyruvate− + 2H+; ΔG°′ = +2 kJ mol−1 (20)

Another HdrABC-containing complex catalyzes the reduction of benzoyl-CoA to cyclohexadienecarboxyl-CoA, the most difficult step in the anaerobic degradation of benzoate, which requires extremely low potential electrons (Em = −622 mV). This is achieved by either an ATP-driven electron transfer (class I benzoyl-CoA reductase) or by a putative electron bifurcation with 4 reduced ferredoxins as donor and NAD as a highpotential acceptor (class II benzoyl-CoA reductase)13 (reaction 21). The class II complex from Geobacter metallireducens is composed of 8 subunits (BamBCDEFGHI) of which BamB together with BamCF forms the tungsten-containing benzoylCoA reducing module. The putative bifurcating module BamED is homologous to HdrAC, whereas HdrB is replaced by BamGHI, which is related to Nuo (NADH quinone oxidoreductase). It has been proposed that NAD binds to the FAD-containing BamH, whereas the ferredoxins deliver electrons to the 2 FAD-containing BamE.103 However, reaction 21 is only exergonic with a special ferredoxin having a standard reduction potential of Em ≤ −485 mV as recently found in Hydrogenobacter thermophilus.19 With the common ferredoxin (Em = −420 mV), the reaction would become endergonic by ΔG°′ = +21 kJ mol−1.

2NADP+ + NADH + 2Fd red− + H+ ⇋ 2NADPH + NAD+ + 2Fdox ; ΔG°′ = +20 kJ mol−1

Textbooks assign to NAD a sole catabolic function, whereas NADPH appears only to be involved in anabolic reductive pathways such as fatty acid biosynthesis. The catabolic bifurcating synthesis of butyrate from acetyl-CoA in C. kluyveri represents a nice exception to this rule (section 3.1). Two acetyl-CoA condense to acetoacetyl-CoA, which is reduced to 3hydroxybutyryl-CoA. Dehydration yields crotonyl-CoA, the substrate for the bifurcating EtfAB-butyryl-CoA dehydrogenase complex (section 3.2.1). The endergonic condensation of 2 acetyl-CoA needs to be pulled by 3-hydroxybutyryl-CoA dehydrogenase, which occurs in two different forms, NAD or NADP dependent. In cells fermentating high concentrations of ethanol and acetate (≤1 M), only the NAD-dependent form is made, whereas under environmental conditions (∼1 mM ethanol and acetate), both forms are present. With NADH (E′ = −280 mV), the 3-hydroxybutyryl-CoA dehydrogenase (Em = −240 mV) works close to the equilibrium, but NADPH (E′ = −370 mV) shifts the reaction close to completion. Because the production of NADPH requires reduced ferredoxin (reaction 23), less of this reductant is available for energy conservation via the H+-dependent ferredoxin-NAD reductase (Rnf). Hence, under standard conditions 1.5 ATP should be theoretically conserved via Rnf, whereas at 1 mM substrate concentration, only 0.25 ATP is obtained (section 3.2.1). Nfn has been purified or obtained as a recombinant enzyme from C. kluyveri,106 M. thermoacetica,107 Thermotoga maritima,108 and Pyrococcus f uriosus.109 The latter organism contains two different Nfn, NfnI and NfnII, of which only NfnI bifurcates.110 All of these enzymes are composed of two subunits, NfnA and NfnB, with molecular masses of 30 and 50 kDa, respectively. The crystal structure of Thermotoga NfnAB revealed the position of the bifurcating b-FAD on NfnB close to the center of the whole complex (Figure 15). NfnA contains one [2Fe-2S] cluster, which is coordinated by three cysteines and one aspartate. The cluster is located at a distance of 15 Å from b-

benzoyl‐CoA + 4Fd red− + NAD+ + 3H+ ⇋ cyclohexadienecarboxyl‐CoA + 4Fdox + NADH; ΔG°′ = −3 kJ mol−1

(21)

A third example is the MetFV HdrABCMvhD complex from the acetogenic Moorella thermoacetica that has been purified and shown to catalyze the reduction of methylene-tetrahydrofolate (methylene-H4F) to methyl-tetrahydrofolate (methyl-H4F) (Em = −200 mV) with reduced benzyl viologen and the reduction of benzyl viologen with NADH.104 The results were interpreted to indicate that the FAD- and FMN-containing complex couples the endergonic reduction of a still to be identified specific ferredoxin with NADH to the exergonic reduction of methylene-H4F with NADH (reaction 22). The subunit HdrA was shown to harbor 2 FAD, one of which was proposed to be the electron bifurcating flavin.104 2NADH + methylene‐H4F + 2Fdox ⇋ 2NAD+ + methyl‐H4F + 2Fd red−; ΔG°′ ≈ −5 kJ mol−1

(23)

(22) Q

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

electron reduction potential of a-FAD was determined at pH 8 as Em8 = −276 mV by the same experiment. The extremely low reduction potentials of the proximal and distal [4Fe-4S] clusters on NfnB (NfnIL) were estimated by square wave voltammetry as Em8 = −718 and −513 mV, respectively. By this method, the average two-electron reduction potentials of both FAD were also determined as Em8 = −275 ± 10 mV. Hence, both FAD exhibit the same two-electron reduction potential. The oneelectron reduction potentials of b-FAD cannot be determined by the methods used above. Therefore, transient absorption spectroscopy (TAS) was applied as described in section 4. As mentioned above, NfnII of P. f uriosus is unable to bifurcate but catalyzes the reversible reduction of ferredoxin by NADPH. In addition, NfnII does not catalyze the NADH-dependent dye reduction, which is observed with NfnI and agrees with a blocked NAD(H) binding site in the crystal structure and absence of key binding residues.110 Surprisingly, both NfnII and EtfAB-Pcd each exhibit an extra loop of 15 amino acids, which could interfere with the respective binding site (see section 3.2.6) but share no sequence identity. However, the absence of these binding sites alone does not explain the lack of bifurcation because omission of one of the two one-electron acceptors leads to zero activity. Hence, in addition to the absence of either oneelectron acceptor, a short cut must lead both electrons from the “bifurcating FAD” to the other one-electron acceptor.

Figure 15. Proposed electron transfer routes in the NADH-dependent ferredoxin:NADP reductase (NfnAB). The scheme catalyzing 1/2 reaction 23 is based on crystal structures and spectroscopic data from T. maritima, C. kluyveri, and P. f uriosus.

4. ELECTRON TRANSFER IN ELECTRON-BIFURCATING COMPLEXES Electron bifurcation requires a cofactor that can be reduced by a two-electron step and oxidized by two one-electron steps or vice versa. This bifurcating cofactor, a quinone or a flavin, must exhibit “crossed-over potentials”, i.e., the potentials of the two oxidation states of the cofactor must be inverted from those one would expect by “normal” one-electron oxidations of a hydroquinone (HQH2). In the normal mode, the first electron to leave has a lower reduction potential or a higher reductive power than the second one. Hence, both electrons could reduce a high-potential acceptor, which would disfavor electron bifurcation. Inversion of this order ensures that the first electron can only be removed by a high-potential acceptor, which gives the remaining semiquinone a very low reduction potential able to reduce any nearby low-potential acceptor. Whether the potentials are normal or crossed-over is a function of the stability constant Ks of the semiquinone (eq 24). The difference ΔEm of the one-electron reduction potentials of the bifurcating cofactor can be calculated from Ks (eq 25).

FAD from which the NAD-binding a-FAD is another 7.5 Å apart. Two [4Fe-4S] clusters lie on NfnB at the opposite side of bFAD, the proximal cluster at a distance of 7.3 Å and the distal cluster at an additional 9.4 Å. Actually the distal [4Fe-4S] cluster, the proximal [4Fe-4S] cluster, b-FAD, the [2Fe-2S] cluster, and a-FAD form one straight line. It has been proposed that NADPH reduces b-FAD to b-FADH−, which bifurcates; the high-potential electron goes to the [2Fe-2S] cluster and further to a-FAD, which interacts with NAD. The low-potential electron jumps via the proximal [4Fe-4S] cluster to the distal [4Fe-4S] cluster, where it reduces ferredoxin. Modeling and H/D exchange studies indicated a concerted rigid body movement of NfnA toward NfnB, which shortens the distance between bFAD and the [2Fe-2S] cluster from 15 to 13 Å to enable an efficient electron transfer.111 Using the same method together with statistical coupling analysis (SCA), specific pathways of communication were identified in NfnI from P. furiosus and revealed allosteric coupling across protein subunits upon nucleotide and ferredoxin binding.112 These results, however, were not correlated with the proposed electron transfer (Figure 15). The smaller subunit A of Pyrococcus NfnI has been called NfnIS and the larger subunit B NfnIL.109 NfnI has been purified as sulfide dehydrogenase already 23 years ago.113 Later, the unusual high reduction potential of the [2Fe-2S] cluster that is coordinated by two aspartate and two cysteine residues was measured as Em8 = +80 mV at pH 8.114 Recently, the enzyme has been characterized as NfnI and has become the subject of more intensive physiochemical investigations.109 Titration of the enzyme with NADPH yielded a stable neutral blue semiquinone of a-FAD (S-FAD) as seen by a broad peak at 620 nm, though the absorbance was very low, ∼10-times lower than that of the main FAD peak at 452 nm (ε = 11.3 mM−1 cm−1). The two-

K s = [SQ]2 × ([Q] × [HQ])−1

(24)

ΔEm = Em1 − Em2 = 2.3RT (F )−1log K s = 0.059log K s (at 298 K)

(25)

According to the Nernst equation, the reduction potential of the first oxidation step decreases linearly with increasing log Ks and that of the second step increases in the same way (Figure 2).4 At Ks = 1, [SQ]2 = [Q] × [HQ], both lines intersect or cross each other. At Ks > 1, the SQ is stable and the potentials are normal, whereas at Ks < 1, it leads to “crossed-over potentials” as required for electron bifurcation.4 Therefore, a major goal is to measure Ks, which is experimentally difficult to verify because the concentration of the semiquinone is expected to be very low. If log Ks = −14 and [Q] = [HQ] = 10 mM, then [SQ] = 10 μM. Here, it should be mentioned that the reduction potentials of R

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

(−89) = −254 mV. Hence, the first oxidation of UQH− by ISP has become an endergonic reaction driven by the exergonic reduction of cytochrome bL, just the apparent reversal of the overall process. Thus, both reductions are strongly coupled.20 This behavior is also consistent with the observation of the first oxidation as a rate-limiting step of the bifurcation.23,26

free flavin are crossed-over: EmQ/SQ = −314 ± 8 mV and EmSQ/HQ = −124 mV resulting in Ks = 10−3.2 ≪ 1 (eq 25).115 In contrast, the reduction potentials of α-FAD of EtfAB-Bcd (section 3.2.1) as well as those of flavodoxin are not crossedover: Em Q/SQ = −60 mV and Em SQ/HQ = −420 mV18 leading to Ks = 10+6.1. This high value indicates that, in a 1:1 mixture of quinone and hydroquinone of flavodoxin, the semiquinone is the predominant species, which agrees well with the observed high stability of the blue neutral semiquinone.

4.2. NfnAB Complex

For the reduction potentials of the Q/SQ and SQ/HQ transitions of b-FAD in NfnI to be determined, photon excited transient absorption spectroscopy (TAS) was applied.109 NfnI was incubated with substoichiometric amounts of NADPH, which transferred both electrons to b-FAD. The consecutive bifurcation reduced all three iron−sulfur clusters and a-FAD, whereby b-FADH− became reoxidized to b-FAD. A short laser beam of “sufficient energy” (amount not indicated by the authors) excited b-FAD to the highly oxidizing species b-FAD*, which abstracted one electron from the closest available donor, the proximal [4Fe-4S]+ cluster, to form the mechanistically relevant anionic semiquinone (ASQ). This unstable species relaxed with a half-life of 10 ps (10−11 s) as measured by TAS via its absorption at 366 nm. With oxidized [4Fe-4S]2+ clusters, no ASQ was observed. Using this half-life of 10 ps, the driving force, and the distance dependence of the electron transfer118 between the ASQ of b-FAD and the proximal [4Fe-4S]2+ cluster (5.4 Å), the one-electron low-potential of b-FAD was calculated as EQ/ASQ = −911 mV (pH 8), which is ΔE = −911 − (−276) = −635 mV more negative than the two-electron reduction potential of −276 mV. Hence, the high-potential of b-FAD amounts to EASQ/HQ = −276 − (−635) = +359 mV, which separates the two one-electron reduction potentials by ΔE = 2 × 0.635 = 1.27 V, leading to log Ks = −ΔE × F (2.3 RT)−1 = −21.5 (eq 24). Surprisingly, log Ks for b-FAD appears to be 7 orders of magnitude lower than that for quinones (−14 to −15). Whether this is valid for all electron-bifurcating flavins remains to be established. In their subsequent paper, the authors, who conducted the TAS experiment, point out that the identification of such “a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process”.119 Of course, the mere detection of such an ASQ is not sufficient because as shown above the crossed reduction potentials and distances of the lowand high-potential acceptors are equally important (see section 4.4 and Table 1). Electron bifurcation in Nfn has been proposed to start by hydride transfer from the substrate NADPH to b-FAD yielding b-FADH−, which is oxidized to b-FAD•− thermodynamically uphill from E1 = +359 mV (SQ/HQ) by the high-potential [2Fe-2S]2+ cluster (Em = +80 mV), ΔE = +279 mV (Figure 17). The extremely unstable anionic semiquinone (Em = −911 mV) formed instantaneously reduces the proximal [4Fe-4S]2+ cluster (Em = −718 mV; ΔE = −193 mV), which transfers the electron further via the distal [4Fe-4S]2+ cluster (Em = −513 mV) to ferredoxin (E ≈ −500 to −400 mV). Finally, the reduced [2Fe2S]+ cluster reduces a-FAD (Em = −276 mV) to a-FAD•− or aFADH•, again an endergonic reaction, ΔE = +356 mV. Repetition of the whole process leads to two reduced ferredoxins as products and a-FADH−, which reduces the second substrate NAD to the product NADH.109 The reviewers doubt whether the two consecutive endergonic reactions in the high-potential branch of the bifurcation really take place. Perhaps the separation of the two one-electron reduction

4.1. Cytochrome bc1 Complex

The low concentration of the SQ of ubiquinone excluded a direct measurement of Ks by redox titrations of the wild-type complex III (cytochrome bc1).20 Using mutants and inhibitors of cytochrome bc 1 of the photosystem from Rhodobacter capsulatus,116 a log Ks = −14 to −15 for ubiquinone bound to the Qo-site has been estimated.23,117 Conversion of this number by eq 25 yields a span of the reduction potentials between the first and second oxidation of UQH− as ΔE = 827−886 mV (average 857 mV). Because the two-electron reduction potential of UQ amounts to +90 mV, the reduction potentials are E1 = 90 + 0.5 × 857 = +518 mV and E2 = 90−0.5 × 857 = −343 mV for the first and second step, respectively (Figure 16). However, the

Figure 16. Redox midpoint potentials involved in electron bifurcation by the cytochrome bc1 complex. Red arrows, electron flow; green solid arrows, proton flow (down, outward; up, from the inside); dashed green arrows, movement of the ubiquinones in the membrane; see also Figure 3. The one-electron midpoint potentials (Q/SQ and SQ/HQ) of the bifurcating UQH2 at the Qo site are based on direct EPR measurements with photosynthetic membranes from Rhodobacter capsulatus containing a knockout cytochrome bH mutant in the presence and absence of various inhibitors. Stimulation of the reaction center by light provided UQH2 as well as oxidized Rieske [2Fe-2S] and cytochrome c1, which initiated electron bifurcation resulting in a longer lived UQo semiquinone at a higher concentration.117 The figure has been constructed using data from Bergdoll et al.20

reduction potential of the Rieske iron−sulfur protein (ISP) in R. capsulatus is Em = +274 mV and that for cytochrome bL is Em = −89 mV. Therefore, the first electron of the bifurcation will reside mainly on the quinol, but as soon as it overcomes the barrier of ΔE = 518−274 mV = +244 mV to ISP, the resulting semiquinone immediately reduces cytochrome bL, ΔE = −343 − S

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Table 1. Properties of the Three Well-Characterized Electron-Bifurcating Complexes complex overall balanced reaction direction in vivo bifurcating cofactor; EmQ/HQ log Ks bifurcating cofactor; EmSQ/HQ − EmQ/SQ = ΔE 1st hipa accb, Em; Rc barrier for the 1st electron 2nd hip acc, Em; R 1st lowpd acc, Em; R 2nd lowp acc, Em; R conformational change of 1st hip acc

cytochrome bc1e

NfnAB

EtfAB-Bcd

2UQoH2 + 2cyt c + UQi ⇋ 2UQo + 2cyt c− + UQiH2 + 2ΔμH+ forward, reversible UQH2; + 90 mV −14 − (−15) +518 − (−343) = 857 mV

2NADPH + NAD+ + 2Fd ⇋ 2NADP+ + NADH + H+ + 2 Fd− reverse, reversible b-FADH−; − 276 mV −21.5 +359 − (−911) = 1270 mV

2NADH + crotonyl-CoA + 2Fd → 2NAD+ + butyryl-CoA + 2Fd− forward, irreversible β-FADH−; −279 mV −15.2 (?) +171 − (−729) = 900 mV

Rieske ISP, +285 mV; 14.8 Åf 518−285 = 233 mV cyt c1, ∼ +300 mV; 7.8 Å cyt bL, −60 mV; ∼6 Å cyt bH, +82 mV; 7.9 Å >20 Å

[2Fe-2S], +80 mV; 13 − 15 Å 359 − 80 = 279 mV a-FAD, −276 mV; 9.6 Å [4Fe-4S]p, −711 mV; 5.4 Å [4Fe-4S]d, −513 mV; 9.6 Å 1−2Å

α-FAD•−, −136 mV; 14 Å 171 − (−136) = 305 mV δ-FAD, ∼0 mV; 8 Å ferredoxin, −420 mV; ∼6 Å 23 Å

a

Hip, high-potential. bAcc, acceptor. cR, distance between cofactors. dLowp, low-potential. eData from R. capsulatus. fDistance between ISP and the inhibitor stigmatellin at the place of UQ.24

Figure 17. Redox midpoint potentials reported to be involved in electron bifurcation by the NADH-dependent ferredoxin: NADP reductase complex, NfnAB. The one-electron midpoint potential (Q/SQ) of the bifurcating b-FAD is based on photon-excited transient absorption spectroscopy and calculations with partially assumed parameters.109 Therefore, the derived value certainly does not represent the Q/SQ potential of the ground state, which should be at least 300 mV more positive. As a consequence, the SQ/HQ potential should be at least 300 mV more negative. For comparison, the reduction potentials of free flavin115 and flavodoxin18 are indicated on the left side (see above). The figure has been constructed using data from Lubner et al.109

Bifurcation starts with a two-electron reduction of β-FAD to β-FADH− by NADH (Figure 18). As soon as the first electron of β-FADH− surmounts the energetic barrier to the semiquinone of α-FAD (ΔE = +171 − (−136) = +307 mV), the extremely low-potential semiquinone of β-FAD (β-FADH• or βFAD•−; Em = −729 mV) reduces ferredoxin, making the EtfAB partial reaction slightly exergonic as indicated by the green dashed line in Figure 18, which represents the average of the reduction potentials of ferredoxin (Fd/Fd−) and α-FAD (SQ/ HQ): 1/2(−405 − 136) = −270.5 mV, 8.5 mV more positive than −279 mV, the two-electron reduction potential of β-FAD. This endergonic one-electron reduction of α-FAD•− ensures a tight coupling with the exergonic reduction of ferredoxin, as observed experimentally.3 The formed α-FADH− bound to domain II of EtfAB swings over from β-FAD (B-state) to δ-FAD

potentials of b-FAD by 1.27 V is much too large (see legend to Figure 17). 4.3. EtfAB-Bcd Complex

For the EtfAB-Bcd complex, no Ks is available yet. Therefore, log Ks = −21.5 of Nfn has been attempted. For β-FAD of EtfAB, however, such a large separation of the one-electron reduction potentials appears not necessary because the low-potential acceptor ferredoxin (Em = −420 mV) has a 300 mV higher potential than the proximal [4Fe-4S] cluster (Em = −711 mV), the corresponding acceptor in Nfn. If one assumes a log Ks = −15.2, close to that of ubiquinone, the one-electron reduction potentials become separated by ΔE = 900 mV leading for βFAD to SQ/HQ, E1 = −279 + 450 = +171 mV and to Q/SQ, E2 = −279 − 450 = −729 mV.47 T

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

Figure 18. Redox midpoint potential proposed to be involved in electron bifurcation by the EtfAB-Bcd complex. The span of the one-electron midpoint potentials (Em Q/SQ − Em SQ/HQ) of the bifurcating β-FAD were taken from cytochrome bc1. For comparison, the reduction potentials of free flavin and flavodoxin are indicated on the left side (see above). Adapted with permission from ref 47. Copyright 2017, Nature Publishing Group.

(D-state), which enables a smooth one-electron transfer to δFAD, ΔE = −10 − (−136) = +126 mV. Binding of a new molecule of NADH close to β-FAD most likely triggers the return of α-FAD•− to the B-state, where it accepts another electron and swings over to Bcd to generate δ-FADH−, which reduces crotonyl-CoA to butyryl-CoA (Em = −10 mV).47

process itself remains to be established. The distances for the low-potential electrons from the bifurcating cofactors to the acceptors with ∼6 Å are also equal but much shorter. Thus, the bifurcating low-potential electron must have a much faster electron transfer rate than the high-potential electron. Calculations for the Nfn system revealed for the bifurcating low-potential electron a transfer rate of 1011 s−1, whereas the high-potential electron moves ∼106-times slower.109 This is consistent with the known fact that, in the bc1 system, the highpotential electron transfer comprises the rate-limiting step.23,26 Furthermore, these rates dismiss the idea that a conformational change is necessary for bifurcation to prevent the high-potential electron from flowing back to the donor. When the highpotential electron reaches its acceptor, there is no time for slow conformational changes because the ∼106-times faster reduction of the low-potential acceptor blocks the return of the electron. In summary, the electron bifurcating systems are a nice example for convergent evolution. The three evolutionary unrelated enzyme complexes, for which the redox profiles are shown in Figures 16−18, appear to have independently adjusted their reduction potentials and distances to optimally conduct this electron bifurcation process. A paper by the BETCy group recently appeared in which several bifurcation scenarios of NfnI without crossed-over potentials were analyzed.121 In principle, such bifurcation mechanisms appear to be possible. However, in all uncrossed cases, the anionic semiquinone had a much longer lifetime than in those with crossed-over potentials. Therefore, the authors came to the conclusion “The dual advantage of crossed potentials in minimizing the presence of a highly reactive intermediate and of avoiding back ET (electron transfer) of the first electron may be physiologically compelling.”

4.4. Comparison of the Three Well-Characterized Electron-Bifurcating Complexes

The structures of several QBEB enzyme complexes and of two FBEB have been determined. The main properties of these systems are listed in Table 1. Whereas cytochrome bc1 with UQ as cofactor works with the exception of cytochrome bL only with positive potentials, those of the flavin based electron bifurcations are shifted by almost 400 mV to the negative side, as required in the anaerobic world. In the quinone system, the one-electron reduction potentials of the bifurcating cofactor are separated by almost 900 mV, those of the flavin system Nfn are even ∼1300 mV apart. Although the potentials could not be measured for Etf-Bcd, 900 mV fitted best to this system (Figure 18). Interestingly, in all three systems the first electron from the bifurcating cofactor has to overcome the high barrier of 270 ± 35 mV to reach the high-potential acceptor. Furthermore, the distances from the bifurcating cofactor to the high-potential acceptor of 13−14 Å are identical in the three systems, just the maximal distance for an efficient electron transfer.120 After bifurcation, the high-potential acceptors undergo conformational changes, which brings the reduced cofactor closer to the next acceptor. In cytochrome bc1 and Etf-Bcd, the changes are large (∼25 Å), whereas in Nfn, only a short movement of 2 Å has been observed. The large conformational changes in cytochrome bc1 and Etf-Bcd are probably due to the location of the final high-potential accepting cofactors, cytochrome c1 and δ-FAD of Bcd, on separate enzymes, whereas in Nfn, both acceptors are present in the same a-subunit. Whether an even smaller movement is a necessary requirement for the bifurcation U

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

AUTHOR INFORMATION

REFERENCES

Corresponding Author

(1) Mitchell, P. The Protonmotive Q Cycle: a General Formulation. FEBS Lett. 1975, 59, 137−139. (2) Herrmann, G.; Jayamani, E.; Mai, G.; Buckel, W. Energy Conservation via Electron-Transferring Flavoprotein in Anaerobic Bacteria. J. Bacteriol. 2008, 190, 784−791. (3) Li, F.; Hinderberger, J.; Seedorf, H.; Zhang, J.; Buckel, W.; Thauer, R. K. Coupled Ferredoxin and Crotonyl-Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri. J. Bacteriol. 2008, 190, 843− 850. (4) Nitschke, W.; Russell, M. J. Redox Bifurcations: Mechanisms and Importance to Life Now, and at its Origin: a Widespread Means of Energy Conversion in Biology Unfolds. BioEssays 2012, 34, 106−109. (5) Buckel, W.; Thauer, R. K. Energy Conservation via Electron Bifurcating Ferredoxin Reduction and Proton/Na+ Translocating Ferredoxin Oxidation. Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 94−113. (6) Metcalf, W. W. Classic Spotlight: Electron Bifurcation, a Unifying Concept for Energy Conservation in Anaerobes. J. Bacteriol. 2016, 198, 1358. (7) Peters, J. W.; Lubner, C. Electron Bifurcation Makes the Puzzle Pieces Fall Energetically into Place in Methanogenic Energy Conservation. ChemBioChem 2017, 18, 2295−2297. (8) Peters, J. W.; Miller, A. F.; Jones, A. K.; King, P. W.; Adams, M. W. Electron Bifurcation. Curr. Opin. Chem. Biol. 2016, 31, 146−152. (9) Boiangiu, C. D.; Jayamani, E.; Brügel, D.; Herrmann, G.; Kim, J.; Forzi, L.; Hedderich, R.; Vgenopoulou, I.; Pierik, A. J.; Steuber, J.; Buckel, W. Sodium Ion Pumps and Hydrogen Production in Glutamate Fermenting Anaerobic Bacteria. J. Mol. Microbiol. Biotechnol. 2006, 10, 105−119. (10) Hess, V.; Schuchmann, K.; Müller, V. The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and is Reversibly Coupled to the Membrane Potential. J. Biol. Chem. 2013, 288, 31496−31502. (11) Biegel, E.; Müller, V. Bacterial Na+-Translocating Ferredoxin:NAD+ Oxidoreductase. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 18138−18142. (12) Meuer, J.; Kuettner, H. C.; Zhang, J. K.; Hedderich, R.; Metcalf, W. W. Genetic Analysis of the Archaeon Methanosarcina barkeri Fusaro Reveals a Central Role for Ech Hydrogenase and Ferredoxin in Methanogenesis and Carbon Fixation. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5632−5637. (13) Buckel, W.; Kung, J. W.; Boll, M. The Benzoyl-Coenzyme A Reductase and 2-Hydroxyacyl-Coenzyme A, a Dehydratase Radical Enzyme Family. ChemBioChem 2014, 15, 2188−2194. (14) Thauer, R. K.; Jungermann, K.; Decker, K. Energy Conservation in Chemotrophic Anaerobic Bacteria. Bacteriol. Rev. 1977, 41, 100− 180. (15) Sato, K.; Nishina, Y.; Setoyama, C.; Miura, R.; Shiga, K. Unusually High Standard Redox Potential of Acrylyl-CoA/PropionylCoA Couple Among Enoyl-CoA/Acyl-CoA Couples: A Reason for the Distinct Metabolic Pathway of Propionyl-CoA from Longer Acyl-CoAs. J. Biochem. 1999, 126, 668−675. (16) Walsh, C. Naturally-Occurring 5-Deazaflavin Coenzymes Biological Redox Roles. Acc. Chem. Res. 1986, 19, 216−221. (17) Tietze, M.; Beuchle, A.; Lamla, I.; Orth, N.; Dehler, M.; Greiner, G.; Beifuss, U. Redox Potentials of Methanophenazine and CoB-S-SCoM, Factors Involved in Electron Transport in Methanogenic Archaea. ChemBioChem 2003, 4, 333−335. (18) Hans, M.; Bill, E.; Cirpus, I.; Pierik, A. J.; Hetzel, M.; Alber, D.; Buckel, W. Adenosine Triphosphate-Induced Electron Transfer in 2Hydroxyglutaryl-CoA Dehydratase from Acidaminococcus fermentans. Biochemistry 2002, 41, 5873−5882. (19) Li, B.; Elliott, S. J. The Catalytic Bias of 2-Oxoacid: Ferredoxin Oxidoreductase in CO2 Evolution and Reduction Through a Ferredoxin-Mediated Electrocatalytic Assay. Electrochim. Acta 2016, 199, 349−356.

*E-mail: buckel@staff.uni-marburg.de. ORCID

Wolfgang Buckel: 0000-0002-8280-1260 Notes

The authors declare no competing financial interest. Biographies Wolfgang Buckel, born in 1940, received his diploma in chemistry (1965) and his Dr. rer. nat. in biochemistry (1968) at the Ludwigs Maximilian Universität (LMU) in Munich, Germany. He was a postdoctoral fellow at LMU in the Institute of Feodor Lynen working with Hermann Eggerer (1968−1970) and at the University of California at Berkeley in the group of Horace A. Barker (1970− 1971). Then, he worked and taught at the Universität Regensburg, Germany, as Research Associate in biochemistry. In 1987, he became Professor of Microbiology at the Philipps-Universität Marburg, Germany. After his retirement from the university in 2008, he became a Fellow of the Max-Planck Society at the Max-Planck Institute of Terrestrial Microbiology in Marburg until 2017. His research interests are mainly mechanisms of enzyme action. Starting with citrate synthesis and cleavage, he soon turned to the unusual dehydrations of 2- and 4hydroxyacyl-CoA to 2-enoyl-CoA, which he found to proceed via radical intermediates. A second field of interest are the bioenergetics of anaerobic bacteria, where he has codiscovered the sodium pumping decarboxylases and ferredoxin-NAD reductase (Rnf) as well as proposed flavin-based electron bifurcation. Rudolf K. Thauer, born in 1939, received his Ph.D. in biochemistry (1968) at the University of Freiburg, Germany. He was a postdoctoral fellow in the groups of Karl Decker at the University of Freiburg and of Harland G. Wood at Case Western University in Cleveland, USA. In 1972, he was appointed as Associated Professor for biochemistry at the Ruhr University in Bochum and in 1976 Full Professor for Microbiology at the Philipps-University Marburg. In 1991, he additionally became Director of the newly founded Max Planck Institute for Terrestrial Microbiology in Marburg. After his retirement at the end of 2007, he continued to have an active lab until end of 2014, when he turned 75. Since then, he has remained active in committees of the German National Academy of Sciences Leopoldina and in Scientific Advisory Boards of several companies interested in biofuel production and mitigation of microbial methane formation. The bioenergetics of anaerobic microorganisms and their biochemistry has always been his main scientific interest. He was involved in the experiments that in 2008 led Wolfgang Buckel to propose the new coupling mechanism of flavinbased electron bifurcation.

ACKNOWLEDGMENTS The authors thank Dr. Wolfgang Nitschke (Bioénergétique et Ingénierie des Protéines, CNRS/AMU, Marseille, France) for his helpful introduction into the theory of electron bifurcation. We further acknowledge Dr. Ulrich Ermler (Max-Planck-Institut fü r Biophysik, Frankfurt, Germany), Dr. Nilanjan Pal Chowdhury (University of Washington, Seattle, USA; present address Mikrobiologie, Universität Frankfurt, Germany), Dr. Dirk Tischler (Bergakademie Freiberg, Germany), and Dr. Inês ́ Cardoso Pereira (Instituto de Tecnologia Quimica e Biológica (ITQB)−Universidade Nova de Lisboa, Portugal) for their help and discussion. Both authors gratefully acknowledge funding by the Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany. V

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

(40) Koelzer, S. Aufrenigung und Charakterisierung des Butyryl-CoADehydrogenase/ETf Komplexes aus Clostridium tetanomorphum. Diploma Thesis, Philipps-Universität, Marburg, Germany, 2008. (41) Chowdhury, N. P.; Mowafy, A. M.; Demmer, J. K.; Upadhyay, V.; Koelzer, S.; Jayamani, E.; Kahnt, J.; Hornung, M.; Demmer, U.; Ermler, U.; Buckel, W. Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and ButyrylCoA Dehydrogenase (Bcd) of Acidaminococcus fermentans. J. Biol. Chem. 2014, 289, 5145−5157. (42) Chowdhury, N. P. On the Mechanism of Electron Bifurcation by Electron Transferring Flavoprotein and Butyryl-CoA Dehydrogenase. Doctoral Thesis, Philipps-Universität, Marburg, Germany, 2014. (43) Roberts, D. L.; Frerman, F. E.; Kim, J. J. Three-Dimensional Structure of Human Electron Transfer Flavoprotein to 2.1-Å Resolution. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 14355−14360. (44) Roberts, D. L.; Salazar, D.; Fulmer, J. P.; Frerman, F. E.; Kim, J. J. Crystal Structure of Paracoccus denitrif icans Electron Transfer Flavoprotein: Structural and Electrostatic Analysis of a Conserved Flavin Binding Domain. Biochemistry 1999, 38, 1977−1989. (45) Sato, K.; Nishina, Y.; Shiga, K. Interaction Between NADH and Electron-Transferring Flavoprotein from Megasphaera elsdenii. J. Biochem. 2013, 153, 565−572. (46) Campbell, C.; Adeolu, M.; Gupta, R. S. Genome-Based Taxonomic Framework for the Class Negativicutes: Division of the Class Negativicutes into the Orders Selenomonadales Emend., Acidaminococcales Ord. Nov. and Veillonellales Ord. Nov. Int. J. Syst. Evol. Microbiol. 2015, 65, 3203−3215. (47) Demmer, J. K.; Chowdhury, N. P.; Selmer, T.; Ermler, U.; Buckel, W. The Semiquinone Swing in the Bifurcating Electron Transferring Flavoprotein/Butyryl-CoA Dehydrogenase Complex from Clostridium dif f icile. Nat. Commun. 2017, 8, 1577. (48) Thamer, W.; Cirpus, I.; Hans, M.; Pierik, A. J.; Selmer, T.; Bill, E.; Linder, D.; Buckel, W. A Two [4Fe-4S]-Cluster-Containing Ferredoxin as an Alternative Electron Donor for 2-Hydroxyglutaryl-CoA Dehydratase from Acidaminococcus fermentans. Arch. Microbiol. 2003, 179, 197−204. (49) Chowdhury, N. P.; Klomann, K.; Seubert, A.; Buckel, W. Reduction of Flavodoxin by Electron Bifurcation and Sodium Iondependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf). J. Biol. Chem. 2016, 291, 11993−12002. (50) Baldwin, R. L.; Milligan, L. P. Electron Transport in Peptostreptococcus elsdenii. Biochim. Biophys. Acta, Spec. Sect. Enzymol. Subj. 1964, 92, 421−432. (51) Chowdhury, N. P.; Kahnt, J.; Buckel, W. Reduction of Ferredoxin or Oxygen by Flavin-Based Electron Bifurcation in Megasphaera elsdenii. FEBS J. 2015, 282, 3149−3160. (52) Wood, P. M. The Two Redox Potentials for Oxygen Reduction to Superoxide. Trends Biochem. Sci. 1987, 12, 250−251. (53) Hansen, B.; Bokranz, M.; Schönheit, P.; Kröger, A. ATP Formation Coupled to Caffeate Reduction by H2 in Acetobacterium woodii NZval6. Arch. Microbiol. 1988, 150, 447−451. (54) Hess, V.; Gonzalez, J. M.; Parthasarathy, A.; Buckel, W.; Müller, V. Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii: a Coenzyme A Loop Saves Energy for Caffeate Activation. Appl. Environ. Microbiol. 2013, 79, 1942−1947. (55) Bertsch, J.; Parthasarathy, A.; Buckel, W.; Müller, V. An ElectronBifurcating Caffeyl-CoA Reductase. J. Biol. Chem. 2013, 288, 11304− 11311. (56) Schuchmann, K.; Müller, V. A Bacterial Electron-Bifurcating Hydrogenase. J. Biol. Chem. 2012, 287, 31165−31171. (57) Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; Van Dien, S. J.; Rabinowitz, J. D. Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia coli. Nat. Chem. Biol. 2009, 5, 593−599. (58) Futai, M. Membrane D-Lactate Dehydrogenase from Escherichia coli. Purification and Properties. Biochemistry 1973, 12, 2468−2474. (59) Hashimoto, T.; Hussien, R.; Brooks, G. A. Colocalization of MCT1, CD147, and LDH in Mitochondrial Inner Membrane of

(20) Bergdoll, L.; Ten Brink, F.; Nitschke, W.; Picot, D.; Baymann, F. From Low- to High-Potential Bioenergetic Chains: Thermodynamic Constraints of Q-Cycle Function. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 1569−1579. (21) Chance, B. Spectra and Reaction Kinetics of Respiratory Pigments of Homogenized and Intact Cells. Nature 1952, 169, 215− 221. (22) Crofts, A. R. The Cytochrome bc1 Complex: Function in the Context of Structure. Annu. Rev. Physiol. 2004, 66, 689−733. (23) Crofts, A. R.; Hong, S.; Wilson, C.; Burton, R.; Victoria, D.; Harrison, C.; Schulten, K. The Mechanism of Ubihydroquinone Oxidation at the Qo-Site of the Cytochrome bc1 Complex. Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 1362−1377. (24) Hunte, C.; Koepke, J.; Lange, C.; Rossmanith, T.; Michel, H. Structure at 2.3 A Resolution of the Cytochrome bc1 Complex from the Yeast Saccharomyces cerevisiae Co-Crystallized with an Antibody Fv Fragment. Structure 2000, 8, 669−684. (25) Iwata, S.; Lee, J. W.; Okada, K.; Lee, J. K.; Iwata, M.; Rasmussen, B.; Link, T. A.; Ramaswamy, S.; Jap, B. K. Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome bc1 Complex. Science 1998, 281, 64−71. (26) Kramer, D. M.; Schoepp, B.; Liebl, U.; Nitschke, W. Cyclic Electron Transfer in Heliobacillus mobilis Involving a MenaquinolOxidizing Cytochrome bc Complex and an RCI-Type Reaction Center. Biochemistry 1997, 36, 4203−4211. (27) Xia, D.; Yu, C. A.; Kim, H.; Xia, J. Z.; Kachurin, A. M.; Zhang, L.; Yu, L.; Deisenhofer, J. Crystal Structure of the Cytochrome bc1 Complex from Bovine Heart Mitochondria. Science 1997, 277, 60−66. (28) Zhang, Z.; Huang, L.; Shulmeister, V. M.; Chi, Y. I.; Kim, K. K.; Hung, L. W.; Crofts, A. R.; Berry, E. A.; Kim, S. H. Electron Transfer by Domain Movement in Cytochrome bc1. Nature 1998, 392, 677−684. (29) Zhu, J.; Egawa, T.; Yeh, S. R.; Yu, L.; Yu, C. A. Simultaneous Reduction of Iron-Sulfur Protein and Cytochrome bL During Ubiquinol Oxidation in Cytochrome bc1 Complex. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 4864−4869. (30) Tikhonov, A. N. The Cytochrome b6 f Complex at the Crossroad of Photosynthetic Electron Transport Pathways. Plant Physiol. Biochem. 2014, 81, 163−183. (31) Myer, Y. P.; Saturno, A. F.; Verma, B. C.; Pande, A. Horse Heart Cytochrome c. The Oxidation-Reduction Potential and Protein Structures. J. Biol. Chem. 1979, 254, 11202−11207. (32) Refojo, P. N.; Teixeira, M.; Pereira, M. M. The Alternative Complex III: Properties and Possible Mechanisms for Electron Transfer and Energy Conservation. Biochim. Biophys. Acta, Bioenerg. 2012, 1817, 1852−1859. (33) Barker, H. A.; Kamen, M. D.; Bornstein, B. T. The Synthesis of Butyric and Caproic Acids from Ethanol and Acetic Acid by Clostridium kluyveri. Proc. Natl. Acad. Sci. U. S. A. 1945, 31, 373−381. (34) Angenent, L. T.; Richter, H.; Buckel, W.; Spirito, C. M.; Steinbusch, K. J.; Plugge, C. M.; Strik, D. P.; Grootscholten, T. I.; Buisman, C. J.; Hamelers, H. V. Chain Elongation with Reactor Microbiomes: Open-Culture Biotechnology To Produce Biochemicals. Environ. Sci. Technol. 2016, 50, 2796−2810. (35) Thauer, R. K.; Jungermann, K.; Henninger, H.; Wenning, J.; Decker, K. The Energy Metabolism of Clostridium kluyveri. Eur. J. Biochem. 1968, 4, 173−180. (36) Schoberth, S.; Gottschalk, G. Considerations on the Energy Metabolism of Clostridium kluyveri. Arch. Microbiol. 1969, 65, 318−328. (37) Lehman, T. C.; Thorpe, C. Alternate Electron Acceptors for Medium-Chain Acyl-CoA Dehydrogenase: Use of Ferricenium Salts. Biochemistry 1990, 29, 10594−10602. (38) Nakos, G.; Mortenson, L. Purification and Properties of Hydrogenase, an Iron Sulfur Protein, from Clostridium pasteurianum W5. Biochim. Biophys. Acta 1971, 227, 576−583. (39) Thauer, R. K.; Buckel, W. Flavin-based electron bifurcation, ferredoxin, flavodoxin, and anaerobic respiration with protons (Ech) or NAD+ (Rnf) as electron acceptors: A historical review. Frontiers in Microbiology 2018, 9, 401. W

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

L6Muscle Cells: Evidence of a Mitochondrial Lactate Oxidation Complex. Am. J. Physiol. Endocrinol. Metab. 2006, 290, E1237−1244. (60) Jacobs, R. A.; Meinild, A. K.; Nordsborg, N. B.; Lundby, C. Lactate Oxidation in Human Skeletal Muscle Mitochondria. Am. J. Physiol. Endocrinol. Metab. 2013, 304, E686−694. (61) Weghoff, M. C.; Bertsch, J.; Müller, V. A novel Mode of Lactate Metabolism in Strictly Anaerobic Bacteria. Environ. Microbiol. 2015, 17, 670−677. (62) Brockman, H. L.; Wood, W. A. D-Lactate Dehydrogenase of Peptostreptococcus elsdenii. J. Bacteriol. 1975, 124, 1454−1461. (63) Brockman, H. L.; Wood, W. A. Electron-Transferring Flavoprotein of Peptostreptococcus elsdenii that Functions in the Reduction of Acrylyl-Coenzyme A. J. Bacteriol. 1975, 124, 1447−1453. (64) Edgren, T.; Nordlund, S. Two Pathways of Electron Transport to Nitrogenase in Rhodospirillum rubrum: The Major Pathway is Dependent on the Fix Gene Products. FEMS Microbiol. Lett. 2006, 260, 30−35. (65) Lonjers, Z. T.; Dickson, E. L.; Chu, T. P.; Kreutz, J. E.; Neacsu, F. A.; Anders, K. R.; Shepherd, J. N. Identification of a New Gene Required for the Biosynthesis of Rhodoquinone in Rhodospirillum rubrum. J. Bacteriol. 2012, 194, 965−971. (66) Ledbetter, R. N.; Garcia Costas, A. M.; Lubner, C. E.; Mulder, D. W.; Tokmina-Lukaszewska, M.; Artz, J. H.; Patterson, A.; Magnuson, T. S.; Jay, Z. J.; Duan, H. D.; et al. The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of LowPotential Reducing Equivalents for Nitrogenase Catalysis. Biochemistry 2017, 56, 4177−4190. (67) Hetzel, M.; Brock, M.; Selmer, T.; Pierik, A. J.; Golding, B. T.; Buckel, W. Acryloyl-CoA Reductase from Clostridium propionicum. An Enzyme Complex of Propionyl-CoA Dehydrogenase and ElectronTransferring Flavoprotein. Eur. J. Biochem. 2003, 270, 902−910. (68) Poehlein, A.; Schlien, K.; Chowdhury, N. P.; Gottschalk, G.; Buckel, W.; Daniel, R. Complete Genome Sequence of the Amino Acid-Fermenting Clostridium propionicum X2 (DSM 1682). Genome Announc. 2016, 4, e00294-16. (69) Cardon, B. P.; Barker, H. A. Two New Amino-Acid-Fermenting Bacteria, Clostridium propionicum and Diplococcus glycinophilus. J. Bacteriol. 1946, 52, 629−634. (70) Cardon, B. P.; Barker, H. A. Amino Acid Fermentations by Clostridium propionicum and Diplococcus glycinophilus. Arch. Biochem. 1947, 12, 165−180. (71) Dörner, C.; Schink, B. Clostridium homopropionicum sp. nov., a New Strict Anaerobe Growing with 2-, 3-, or 4-Hydroxybutyrate. Arch. Microbiol. 1990, 154, 342−348. (72) Garvie, E. I. Bacterial Lactate Dehydrogenases. Microbiol. Rev. 1980, 44, 106−139. (73) Kröger, A.; Biel, S.; Simon, J.; Gross, R.; Unden, G.; Lancaster, C. R. Fumarate Respiration of Wolinella succinogenes: Enzymology, Energetics and Coupling Mechanism. Biochim. Biophys. Acta, Bioenerg. 2002, 1553, 23−38. (74) Lancaster, C. R.; Gorss, R.; Haas, A.; Ritter, M.; Mantele, W.; Simon, J.; Kröger, A. Essential Role of Glu-C66 for Menaquinol Oxidation Indicates Transmembrane Electrochemical Potential Generation by Wolinella succinogenes Fumarate Reductase. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13051−13056. (75) Seeliger, S.; Janssen, P. H.; Schink, B. Energetics and Kinetics of Lactate Fermentation to Acetate and Propionate via MethylmalonylCoA or Acrylyl-CoA. FEMS Microbiol. Lett. 2002, 211, 65−70. (76) Aboulnaga, E.-H.; Pinkenburg, O.; Schiffels, J.; El-Refai, A.; Buckel, W.; Selmer, T. Butyrate Production in Escherichia coli: Exploitation of an Oxygen Tolerant Bifurcating Butyryl-CoA Dehydrogenase/Electron Transferring Flavoprotein Complex from Clostridium dif f icile. J. Bacteriol. 2013, 195, 3704−3713. (77) Edgren, T.; Nordlund, S. The f ixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase. J. Bacteriol. 2004, 186, 2052−2060. (78) Byron, C. M.; Stankovich, M. T.; Husain, M.; Davidson, V. L. Unusual Redox Properties of Electron-Transfer Flavoprotein from Methylophilus methylotrophus. Biochemistry 1989, 28, 8582−8587.

(79) Munk, A. C.; Copeland, A.; Lucas, S.; Lapidus, A.; Del Rio, T. G.; Barry, K.; Detter, J. C.; Hammon, N.; Israni, S.; Pitluck, S.; et al. Complete Genome Sequence of Rhodospirillum rubrum Type Strain (S1). Stand. Genomic Sci. 2011, 4, 293−302. (80) Garcia Costas, A. M.; Poudel, S.; Miller, A. F.; Schut, G. J.; Ledbetter, R. N.; Fixen, K. R.; Seefeldt, L. C.; Adams, M. W. W.; Harwood, C. S.; Boyd, E. S.; Peters, J. W. Defining Electron Bifurcation in the Electron Transferring Flavoprotein Family. J. Bacteriol. 2017, 199, e00440-17. (81) Schut, G. J.; Adams, M. W. The Iron-Hydrogenase of Thermotoga maritima Utilizes Ferredoxin and NADH Synergistically: A New Perspective on Anaerobic Hydrogen Production. J. Bacteriol. 2009, 191, 4451−4457. (82) Verhagen, M. F.; O’Rourke, T.; Adams, M. W. The Hyperthermophilic Bacterium, Thermotoga maritima, Contains an Unusually Complex Iron-Hydrogenase: Amino Acid Sequence Analyses Versus Biochemical Characterization. Biochim. Biophys. Acta, Bioenerg. 1999, 1412, 212−229. (83) Sazanov, L. A.; Hinchliffe, P. Structure of the Hydrophilic Domain of Respiratory Complex I from Thermus thermophilus. Science 2006, 311, 1430−1436. (84) Berrisford, J. M.; Baradaran, R.; Sazanov, L. A. Structure of Bacterial Respiratory Complex I. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 892−901. (85) Gnandt, E.; Dörner, K.; Strampraad, M. F. J.; de Vries, S.; Friedrich, T. The Multitude of Iron-Sulfur Clusters in Respiratory Complex I. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 1068−1072. (86) Gnandt, E.; Schimpf, J.; Harter, C.; Hoeser, J.; Friedrich, T. Reduction of the Off-Pathway Iron-Sulphur Cluster N1a of Escherichia coli Respiratory Complex I Restrains NAD+ Dissociation. Sci. Rep. 2017, 7, 8754. (87) Dörner, K.; Vranas, M.; Schimpf, J.; Straub, I. R.; Hoeser, J.; Friedrich, T. Significance of [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I. Biochemistry 2017, 56, 2770−2778. (88) Bar-Even, A. Does Acetogenesis Really Require Especially Low Reduction Potential? Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 395−400. (89) Wang, S.; Huang, H.; Kahnt, J.; Thauer, R. K. A Reversible Electron-Bifurcating Ferredoxin- and NAD-Dependent [FeFe]-Hydrogenase (HydABC) in Moorella thermoacetica. J. Bacteriol. 2013, 195, 1267−1275. (90) Wang, S.; Huang, H.; Kahnt, J.; Mueller, A. P.; Köpke, M.; Thauer, R. K. NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO. J. Bacteriol. 2013, 195, 4373−4386. (91) Mock, J.; Zheng, Y.; Mueller, A. P.; Ly, S.; Tran, L.; Segovia, S.; Nagaraju, S.; Köpke, M.; Dürre, P.; Thauer, R. K. Energy Conservation Associated with Ethanol Formation from H2 and CO2 in Clostridium autoethanogenum Involving Electron Bifurcation. J. Bacteriol. 2015, 197, 2965−2980. (92) Wang, S.; Huang, H.; Kahnt, J.; Thauer, R. K. Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase. Appl. Environ. Microbiol. 2013, 79, 6176−6179. (93) Hoke, K. R.; Cobb, N.; Armstrong, F. A.; Hille, R. Electrochemical Studies of Arsenite Oxidase: An Unusual Example of a Highly Cooperative Two-Electron Molybdenum Center. Biochemistry 2004, 43, 1667−1674. (94) Kaster, A. K.; Moll, J.; Parey, K.; Thauer, R. K. Coupling of Ferredoxin and Heterodisulfide Reduction via Electron Bifurcation in Hydrogenotrophic Methanogenic Archaea. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2981−2986. (95) Thauer, R. K.; Kaster, A. K.; Seedorf, H.; Buckel, W.; Hedderich, R. Methanogenic Archaea: Ecologically Relevant Differences in Energy Conservation. Nat. Rev. Microbiol. 2008, 6, 579−591. (96) Wagner, T.; Koch, J.; Ermler, U.; Shima, S. Methanogenic Heterodisulfide Reductase (HdrABC-MvhAGD) Uses Two Noncubane [4Fe-4S] Clusters for Reduction. Science 2017, 357, 699−703. X

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews

Review

(97) Costa, K. C.; Lie, T. J.; Xia, Q.; Leigh, J. A. VhuD Facilitates Electron Flow from H2 or Formate to Heterodisulfide Reductase in Methanococcus maripaludis. J. Bacteriol. 2013, 195, 5160−5165. (98) Costa, K. C.; Wong, P. M.; Wang, T.; Lie, T. J.; Dodsworth, J. A.; Swanson, I.; Burn, J. A.; Hackett, M.; Leigh, J. A. Protein Complexing in a Methanogen Suggests Electron Bifurcation and Electron Delivery from Formate to Heterodisulfide Reductase. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11050−11055. (99) Sowers, K. R.; Baron, S. F.; Ferry, J. G. Methanosarcina acetivorans sp. nov., an Acetotrophic Methane-Producing Bacterium Isolated from Marine Sediments. Appl. Environm. Microbiol. 1984, 47, 971−978. (100) Yan, Z.; Wang, M.; Ferry, J. G. A Ferredoxin- and F420H2Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea. mBio 2017, 8, e02285-16. (101) Ramos, A. R.; Grein, F.; Oliveira, G. P.; Venceslau, S. S.; Keller, K. L.; Wall, J. D.; Pereira, I. A. The FlxABCD-HdrABC Proteins Correspond to a Novel NADH Dehydrogenase/Heterodisulfide Reductase Widespread in Anaerobic Bacteria and Involved in Ethanol Metabolism in Desulfovibrio vulgaris Hildenborough. Environ. Microbiol. 2015, 17, 2288−2305. (102) Ramos, A. R.; Keller, K. L.; Wall, J. D.; Pereira, I. A. The Membrane QmoABC Complex Interacts Directly with the Dissimilatory Adenosine 5′-Phosphosulfate Reductase in Sulfate Reducing Bacteria. Front. Microbiol. 2012, 3, 137. (103) Boll, M.; Einsle, O.; Ermler, U.; Kroneck, P. M.; Ullmann, G. M. Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase. J. Mol. Microbiol. Biotechnol. 2016, 26, 119−137. (104) Mock, J.; Wang, S.; Huang, H.; Kahnt, J.; Thauer, R. K. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica. J. Bacteriol. 2014, 196, 3303−3314. (105) de Graef, M. R.; Alexeeva, S.; Snoep, J. L.; Teixeira de Mattos, M. J. The Steady-State Internal Redox State (NADH/NAD) Reflects the External Redox State and is Correlated with Catabolic Adaptation in Escherichia coli. J. Bacteriol. 1999, 181, 2351−2357. (106) Wang, S.; Huang, H.; Moll, J.; Thauer, R. K. NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri. J. Bacteriol. 2010, 192, 5115−5123. (107) Huang, H.; Wang, S.; Moll, J.; Thauer, R. K. Electron Bifurcation Involved in the Energy Metabolism of the Acetogenic Bacterium Moorella thermoacetica Growing on Glucose or H2 plus CO2. J. Bacteriol. 2012, 194, 3689−3699. (108) Demmer, J. K.; Huang, H.; Wang, S.; Demmer, U.; Thauer, R. K.; Ermler, U. Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure. J. Biol. Chem. 2015, 290, 21985−21995. (109) Lubner, C. E.; Jennings, D. P.; Mulder, D. W.; Schut, G. J.; Zadvornyy, O. A.; Hoben, J. P.; Tokmina-Lukaszewska, M.; Berry, L.; Nguyen, D. M.; Lipscomb, G. L.; et al. Mechanistic Insights into Energy Conservation by Flavin-Based Electron Bifurcation. Nat. Chem. Biol. 2017, 13, 655−659. (110) Nguyen, D. M. N.; Schut, G. J.; Zadvornyy, O. A.; TokminaLukaszewska, M.; Poudel, S.; Lipscomb, G. L.; Adams, L. A.; Dinsmore, J. T.; Nixon, W. J.; Boyd, E. S.; et al. Two Functionally Distinct NADP+Dependent Ferredoxin Oxidoreductases Maintain the Primary Redox Balance of Pyrococcus f uriosus. J. Biol. Chem. 2017, 292, 14603−14616. (111) Demmer, J. K.; Rupprecht, F. A.; Eisinger, M. L.; Ermler, U.; Langer, J. D. Ligand Binding and Conformational Dynamics in a FlavinBased Electron-Bifurcating Enzyme Complex Revealed by HydrogenDeuterium Exchange Mass Spectrometry. FEBS Lett. 2016, 590, 4472− 4479. (112) Berry, L.; Poudel, S.; Tokmina-Lukaszewska, M.; Colman, D. R.; Nguyen, D. M. N.; Schut, G. J.; Adams, M. W. W.; Peters, J. W.; Boyd, E. S.; Bothner, B. H/D Exchange Mass Spectrometry and Statistical Coupling Analysis Reveal a Role for Allostery in a Ferredoxin-Dependent Bifurcating Transhydrogenase Catalytic Cycle. Biochim. Biophys. Acta, Gen. Subj. 2018, 1862, 9.

(113) Ma, K.; Adams, M. W. Sulfide Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus f uriosus: a New Multifunctional Enzyme Involved in the Reduction of Elemental Sulfur. J. Bacteriol. 1994, 176, 6509−6517. (114) Hagen, W. R.; Silva, P. J.; Amorim, M. A.; Hagedoorn, P. L.; Wassink, H.; Haaker, H.; Robb, F. T. Novel Structure and Redox Chemistry of the Prosthetic Groups of the Iron-Sulfur Flavoprotein Sulfide Dehydrogenase from Pyrococcus f uriosus; Evidence for a [2Fe2S] Cluster with Asp(Cys)3 Ligands. JBIC, J. Biol. Inorg. Chem. 2000, 5, 527−534. (115) Anderson, R. F. Energetics of the One-Electron Reduction Steps of Riboflavin, FMN and FAD to their Fully Reduced Forms. Biochim. Biophys. Acta, Bioenerg. 1983, 722, 158−162. (116) Berry, E. A.; Huang, L. S.; Saechao, L. K.; Pon, N. G.; ValkovaValchanova, M.; Daldal, F. X-Ray Structure of Rhodobacter Capsulatus Cytochrome bc1: Comparison with its Mitochondrial and Chloroplast Counterparts. Photosynth. Res. 2004, 81, 251−275. (117) Zhang, H.; Osyczka, A.; Dutton, P. L.; Moser, C. C. Exposing the Complex III Qo Semiquinone Radical. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 883−887. (118) Page, C. C.; Moser, C. C.; Chen, X.; Dutton, P. L. Natural Engineering Principles of Electron Tunnelling in Biological OxidationReduction. Nature 1999, 402, 47−52. (119) Hoben, J. P.; Lubner, C. E.; Ratzloff, M. W.; Schut, G. J.; Nguyen, D. M. N.; Hempel, K. W.; Adams, M. W. W.; King, P. W.; Miller, A. F. Equilibrium and Ultrafast Kinetic Studies Manipulating Electron Transfer: A Short-Lived Flavin Semiquinone is not Sufficient for Electron Bifurcation. J. Biol. Chem. 2017, 292, 14039−14049. (120) Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L. Nature of Biological Electron Transfer. Nature 1992, 355, 796−802. (121) Zhang, P.; Yuly, J. L.; Lubner, C. E.; Mulder, D. W.; King, P. W.; Peters, J. W.; Beratan, D. N. Electron Bifurcation: Thermodynamics and Kinetics of Two-Electron Brokering in Biological Redox Chemistry. Acc. Chem. Res. 2017, 50, 2410−2417.

Y

DOI: 10.1021/acs.chemrev.7b00707 Chem. Rev. XXXX, XXX, XXX−XXX