NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in aFunctional Complex with Formate Dehydrogenase in Clostridiumautoethanogenum Grown on CO

Shuning Wang,a,b Haiyan Huang,a Jörg Kahnt,a Alexander P. Mueller,c Michael Köpke,c Rudolf K. Thauera

Max Planck Institute for Terrestrial Microbiology, Marburg, Germanya; State Key Laboratory of Microbial Technology, Shangdong University, Jinan, People’s Republic ofChinab; LanzaTech NZ, Ltd., Auckland, New Zealandc

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in thecytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, oneis a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hy-drogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was foundin high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grownon CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different sub-units. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDasubunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenaseproper, 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 and the reversibleformation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyzeCO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formationfrom NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethano-genum on CO (E0= � �520 mV).

Five years ago it was discovered that in butyric acid-formingclostridia, the exergonic reduction of crotonyl coenzyme A

(crotonyl-CoA; E0= � �10 mV) with NADH (E0= � �320 mV) iscoupled with the endergonic reduction of ferredoxin (Fd) (E0= ��400 mV) with NADH (reaction 1) catalyzed by the cytoplasmicbutyryl-CoA dehydrogenase/electron transfer flavoprotein com-plex Bcd/EtfAB (1, 2).

2 NADH � FdOX � crotonyl-CoA → 2 NAD� � Fdred2�

� butyryl-CoA

�Go� � �44 kJ ⁄ mol(1)

The available evidence indicates that electron bifurcation isflavin based: a protein-bound flavin is reduced by NADH to thehydroquinone, which is subsequently reoxidized by crotonyl-CoA to the semiquinone radical that has a redox potential suf-ficiently negative to reduce ferredoxin (3). The proposedmechanism is analogous to the mechanisms of ubiquinone-based electron bifurcation in the bc1 complex of the aerobicrespiratory chain and plastoquinone-based electron bifurca-tion in the b6f-complex in oxygenic photosynthesis (4–6). Themain differences between the electron bifurcation mechanismsare that flavin-based electron bifurcation is associated with cy-toplasmic proteins and operates at more negative redox poten-tials (�300 mV � 200 mV), whereas ubiquinone/plastoqui-none-based electron bifurcation is associated with membranesand operates at more positive redox potentials (�100 mV �200 mV) (3, 7–9).

Since the initial discovery, four other flavin-based electron-bifurcating enzyme complexes have been characterized,

namely, the MvhADG/HdrABC complex from methanogenicarchaea catalyzing reaction 2a (10, 11), the FdhAB/HdrABCcomplex from methanogenic archaea catalyzing reaction 2b(12–14), the NfnAB complex from bacteria and archaea cata-lyzing reaction 3 (15, 16), the HydABC(D) complex from bac-teria catalyzing reaction 4 (17–19), and the caffeyl-CoA reduc-tase/electron transfer flavoprotein complex (CarCDE) fromAcetobacterium woodii catalyzing reaction 5 (20). Each of thesereactions is key to understanding the energy metabolism of theorganism in which it operates.

2 H2 � CoM-S-S-CoB � FdOX → CoM-SH

� CoB-SH � Fdred2� � 2 H�

�Go� � �50 kJ ⁄ mol(2a)

2 HCOO� � CoM-S-S-CoB � FdOX → 2 CO2 � CoM-SH

� CoB-SH � Fdred2�

�Go� � �56 kJ ⁄ mol(2b)

Received 7 June 2013 Accepted 18 July 2013

Published ahead of print 26 July 2013

Address correspondence to Rudolf K. Thauer, thauer@mpi-marburg.mpg.de.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00678-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00678-13

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NADH � Fdred2� � 2 NADP� � H�ª NAD� � FdOX

� 2 NADPH

�Go� � �16 kJ ⁄ mol(3)

NADH � Fdred2� � 3 H�ª NAD� � Fdox � 2 H2

�Go� � �21kJ ⁄ mol(4)

2 NADH � Fdox � caffeyl-CoA → 2 NAD� � Fdred2�

� 2,3-dihydroxyphenyl-propionyl-CoA

�Go� � �40kJ ⁄ mol (estimated)(5)

The free energy changes �G°= associated with the five reactionsunder standard conditions (1 M concentrations of substrates andproducts; partial pressure of gases � 1 bar; pH 7) were calculatedusing an E0= of �400 mV for ferredoxin from Clostridium pasteu-rianum, which harbors two [4Fe4S] clusters with almost the samemidpoint potential (21, 22). It has to be considered, however, thatferredoxins from other anaerobic microorganisms can have muchlower midpoint potentials (23). Ferredoxin II from Moorella ther-moacetica for example, has two [4Fe4S]-clusters, one of which hasa midpoint potential of �487 mV (24). If the free energy changesassociated with reactions 1 to 5 are calculated using an E0= of theFdox/Fdred

2� couple of �500 mV, the free energy changes (�G°=)of reactions 1, 2, and 5 are �20 kJ/mol less exergonic, and those ofreactions 3 and 4 are �20 kJ/mol more exergonic.

When estimating the free energy changes under physiologicalconditions (intracellular concentrations of substrates and prod-ucts) (�G=), it has to be taken into account that in vivo the redoxpotential E= of the NAD�/NADH couple is about �280 mV(E0= � �320 mV), E= of the NADP�/NADPH couple is about�370 mV (E0= � �320 mV) (25) and E= of the Fdox/Fdred

2� cou-ple is considerably more negative than �400 mV, probably near�500 mV (3, 11), although the latter has recently been debated(26). It also has to be considered that under physiological condi-tions the H2 partial pressure (important in reactions 2 and 4) canbe lower than 1 bar � 105 Pa (E0= � �414 mV). For example, inmethanogenic environments the H2 partial pressure is generallynear 10 Pa (E= � �300 mV) (10) and in acetogenic environmentsnear 200 Pa (E= � �350 mV) (27). Reactions 2, 3, and 4 are pHdependent and the intracellular pH can be lower than 7. Taking allof these considerations into account, reactions 1, 2, 3, and 5 aremost likely irreversible in vivo, whereas reaction 4 could proceedin both directions for which there is experimental evidence (19).

Here we report a novel electron-bifurcating [FeFe]-hydroge-nase that is NADP specific rather than NAD specific and thatforms a functional complex with a formate dehydrogenase. Wefound the novel enzyme complex in Clostridium autoethanoge-num (28) growing on CO, which is fermented via the Wood-Ljungdahl pathway (29–31). Via the same pathway the organismcan also grow on H2 and CO2. C. autoethanogenum is a member ofa cluster of acetogenic bacteria including Clostridium ljungdahlii(32) and Clostridium ragsdalei (33), which have a growth pH op-timum near 5.5 and a growth temperature optimum near 37°Cand form acetic acid, ethanol, and 2,3-butanediol during growthon CO (34–36). These organisms belong to a subset of acetogensthat do not contain cytochromes and menaquinone and that arenot dependent on sodium ions for growth.

MATERIALS AND METHODSBiochemicals and enzymes. NAD�, NADP�, NADH, NADPH, flavinadenine dinucleotide (FAD), flavin mononucleotide (FMN), pyruvate,thiamine pyrophosphate, coenzyme A (CoA), acetaldehyde, tetrahy-drofolate (H4F), methyl viologen (MV), benzyl viologen (BV), andphosphotransacetylase from Bacillus stearothermophilus were fromSigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Methyl-tet-rahydrofolate (methyl-H4F) was obtained from Schircks Laboratories(Jona, Switzerland). H2, N2, and CO with highest purity (99.996%)were from Messer (Düsseldorf, Germany). [14C]K2CO3 (1 mCi in 4 mlof H2O � 9.25 MBq/ml) was from Hartmann Analytic (Braunschweig,Germany). All materials for protein purification were obtained fromGE Healthcare (Freiburg, Germany).

Ferredoxin (Fd) (37) and ferredoxin-dependent monomeric [FeFe]-hydrogenase (2) were purified from C. pasteurianum DSM 525. Pyruvate:ferredoxin oxidoreductase was purified from M. thermoacetica DSM 521(38).

Bacterial strains and growth conditions. C. autoethanogenum DSM10061, C. pasteurianum DSM 525, and M. thermoacetica DSM 521 wereobtained from the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Braunschweig, Germany.

C. pasteurianum was cultivated anaerobically at 37°C on a glucose-ammonium medium (39), and M. thermoacetica was grown at 55°C onglucose with 100% CO2 as the gas phase (15). The cells were harvested inthe late-exponential phase.

C. autoethanogenum was grown strictly anaerobically at pH 5 and 37°Cin a 1.2-liter continuous culture at a dilution rate of 1.8 per day using theBioFlo310 system from New Brunswick Scientific; the mineral salts me-dium (0.4 g of MgCl2·6H2O, 0.294 g of CaCl2·6H2O, 0.15 g of KCl, and0.12 g of NaCl per liter) was supplemented per liter with 10 ml of a vitaminsolution [20 mg of biotin, 20 mg of folic acid, 10 mg of pyridoxine hydro-chloride, 50 mg of thiamine, 50 mg of riboflavin, 50 mg of nicotinic acid,50 mg of D-(�)-pantothenate, 50 mg of vitamin B12, 50 mg of p-amino-benzoic acid, and 50 mg of lipoic acid per liter]. In addition to the usualtrace elements (100 �M FeCl3, 50 �M CoCl2, 50 �M NiCl2, 10 �M ZnCl2,10 �M MnCl2, and 10 �M Na2MoO4) the medium contained 10 �Mselenite (Na2SeO3) and 10 �M tungstate (Na2WO3). Ammonium (3.083 gof NH4 acetate) and hydrogen sulfide (0.3 ml of a 0.2 M Na2S solution perh) served as nitrogen and sulfur sources, respectively. The cultures werecontinuously gassed with steel mill waste gas (composition: 42% CO, 36%N2, 20% CO2, and 2% H2; collected from a New Zealand Steel site inGlenbrook, New Zealand) as the sole energy and carbon source (40). ThepH was continuously recorded and maintained at pH 5 by the addition ofNH4OH. Metabolites were measured by high-pressure liquid chromatog-raphy (HPLC) and headspace was analyzed using a micro gas chromato-graph. The steady state cell concentration in the continuous culture of C.autoethanogenum was �3.9 g cells (dry mass) per liter. Before harvest, theculture was adjusted from pH 5 to 6 with K2CO3 and subsequently chilledin an ice water bath. The culture was centrifuged in an anaerobic chamberin 1-liter bottles at 5,000 � g for 10 min. The pelleted cells were resus-pended in 30 ml of 50 mM potassium phosphate pH 7.0 containing 10mM dithiothreitol (DTT), pelleted again by centrifugation, snap-frozenin liquid nitrogen, and stored at �80°C under N2.

Metabolite concentrations were routinely determined using an Agi-lent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA)equipped with a refractive index detector operated at 35°C and an AlltechIOA-2000 organic acid column. The column was kept at 60°C. A 1.25 mMsulfuric acid solution was used as mobile phase with a flow rate of 0.7 mlmin�1. To remove proteins and other cell residues, a 0.4-ml sample vol-ume was mixed with 50 �l of 0.15 M ZnSO4 and 50 �l of 0.15 M Ba(OH)2

and samples were centrifuged at 14,000 � g for 3 min. The supernatant(0.2 ml) was transferred into an HPLC vial, and 5 �l of sample was in-jected into the instrument.

Headspace gas concentrations were measured by gas chromatographyusing a Varian CP-4900 Micro GC with two installed channels. Channel 1

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was a 10-m Mol-Sieve column running at 70°C, with 200 kPa argon and abackflush time of 4.2 s; channel 2 was a 10-m PPQ column running at90°C, with150-kPa helium and no backflush. The injector temperature forboth channels was set at 70°C. Run times were set to 120 s, but all peaks ofinterest usually eluted before 100 s.

Preparation of cell extracts. Frozen cells of C. autoethanogenum (8 g[wet mass]) were suspended in 13 ml of anoxic 50 mM potassium phos-phate (pH 7.0) containing 2 mM DTT, 5 �M FAD, and 5 �M FMN. Afterthe addition of 1.5 mg of DNase I and 0.5 mM MgCl2, the cell suspensionwas passed through a prechilled French pressure cell three times at 120MPa. Before this step, the French pressure cell was first flushed with N2 for15 min and then washed three times with anoxic buffer. Unbroken cellsand cell debris were removed by centrifugation at 20,000 � g and 4°C for30 min. The supernatant was used for enzyme assays. For protein purifi-cation, the supernatant was further centrifuged at 150,000 � g and 4°C for60 min to remove the membrane fraction.

For analysis of the membrane fraction, 3 g of frozen cells of C. auto-ethanogenum was suspended in 6 ml of anoxic 10 mM potassium phos-phate (pH 7.0) containing 2 mM DTT, 5 �M FAD, and 5 �M FMN. Afterthe addition of 5.5 mg of lysozyme, 1.5 mg of DNase I, and 0.5 mM MgCl2,the cell suspension was incubated at 37°C for 40 min under an atmosphereof 100% H2. The membrane fraction was obtained by centrifugation at150,000 � g and 4°C for 60 min.

Enzyme purification. The electron-bifurcating ferredoxin- andNADP-dependent hydrogenase in complex with formate dehydrogenasewas purified under strictly anoxic conditions at room temperature in atype B vinyl anaerobic chamber (Coy, Grass Lake, MI), which was filledwith 95% N2–5% H2 and contained a palladium catalyst for O2 reductionwith H2. Anoxic 50 mM Tris-HCl (pH 7.6) containing 2 mM DTT, 5 �MFAD, and 5 �M FMN (buffer A) was used through the whole process.

The 150,000 � g supernatant (14 ml) containing the cytoplasmic frac-tion with �47 mg of protein ml�1 was fractionated with ammoniumsulfate. The fraction between 40 and 55% ammonium sulfate saturationwas collected by centrifugation at 30,000 � g and 4°C for 30 min. Theprecipitate was dissolved in 7 ml of buffer A containing 0.8 M ammoniumsulfate. After the removal of undissolved proteins by centrifugation, thesupernatant was loaded onto a Phenyl Sepharose high-performance col-umn (2.6 by 12 cm) equilibrated with buffer A containing 0.8 M ammo-nium sulfate. Protein was eluted with a stepwise ammonium sulfate gra-dient (0.80, 0.64, 0.48, 0.32, 0.16, and 0 M; 100 ml of each in buffer A) ata flow rate of 5 ml min�1. The hydrogenase activity was eluted in a peak at0.48 M ammonium sulfate. The pooled fractions were concentrated anddesalted with an Amicon cell with a 50-kDa-cutoff membrane. The con-centrate was then applied onto a Q Sepharose high-performance column(1.6 by 13 cm) equilibrated with buffer A. The column was then washedwith 90 ml of buffer A. Protein was eluted with a 0 to 1 M NaCl lineargradient at a flow rate of 5 ml min�1. The hydrogenase activity was recov-ered in a single peak eluting around 0.4 M NaCl. The fraction was con-centrated, desalted with a 50-kDa-cutoff Amicon filter, and then stored at�20°C in buffer A under an atmosphere of 95% N2–5% H2 until used.

During purification, methyl viologen reduction activities with H2 andwith formate, NADP�-dependent ferredoxin reduction activities with H2

and with formate, and the protein concentration were followed.Specific activity measurements. Enzyme activities were measured un-

der strictly anoxic conditions at 37°C in 1.5-ml anaerobic cuvettes or6.5-ml anaerobic serum bottles (H2 formation and CO2 reduction) sealedwith rubber stoppers and filled with 0.8-ml assay mixtures and N2

(100%), H2 (100%), or CO (100%) at 1.2 � 105 Pa as the gas phase. Afterthe start of the reaction with enzyme, the reduction of NADP� or NAD�

was monitored spectrophotometrically at 340 nm (ε � 6.2 mM�1 cm�1)or at 380 nm (ε � 1.2 mM�1 cm�1), C. pasteurianum ferredoxin reduc-tion was monitored at 430 nm (�εox-red 13.1 mM�1 cm�1), methylviologen reduction was monitored at 578 nm (ε � 9.8 mM�1 cm�1), andbenzyl viologen reduction was monitored at 578 nm (ε � 8.6 mM�1

cm�1). H2 formation was monitored gas chromatographically, and the

reduction of 14CO2 to [14C]formate was monitored by counting 14C in aBeckman LS6500 liquid scintillation counter (Fullerton, CA). One unit (1U) was defined as the transfer of 2 �mol electrons min�1. Protein wasdetermined using the Bio-Rad protein assay (Munich, Germany) withbovine serum albumin as the standard.

CO dehydrogenase. The assay mixtures contained 100 mM Tris-HCl(pH 7.5), 2 mM DTT, and about 30 �M ferredoxin, 1 mM NAD� or 1 mMNADP�. The gas phase was 100% CO.

Reduced ferredoxin:NAD� oxidoreductase. The assay mixtures con-tained 100 mM potassium phosphate (pH 7.0), 2 mM DTT, 15 �M ferre-doxin, and1 mM NAD� or NADP�. The gas phase was 100% CO for thereduction of ferredoxin via the ferredoxin-dependent CO dehydrogenasein the cell extract.

NADH-dependent reduced ferredoxin:NADP� oxidoreductase(Nfn). The assay mixtures contained 100 mM MOPS-KOH (pH 7.0), 10mM 2-mercaptoethanol, 10 �M FAD, 0.5 mM NADP�, 40 mM glucose-6-phosphate, and 2 U glucose-6-phosphate dehydrogenase (NADPH-re-generating system), 10 mM NAD�, and about 25 �M ferredoxin. N2 wasthe gas phase.

Hydrogenase. When the reduction of electron acceptors with H2 wasfollowed, the assay mixtures contained 100 mM Tris-HCl (pH 7.5) (Ta-bles 1 and 2) or 100 mM potassium phosphate (pH as indicated) (Table 3),2 mM DTT, and, where indicated, 25 �M ferredoxin, 1 mM NAD�, 1 mMNADP�, or 10 mM methyl viologen. The gas phase was 100% H2.

When the formation of H2 from reduced ferredoxin and NADPH wasfollowed, the assay mixtures contained 100 mM potassium phosphate(pH as indicated) (Table 3), 2 mM DTT, 1 mM NADPH, and reducedferredoxin-regenerating system (10 mM pyruvate, 0.1 mM thiamine py-rophosphate, 1 mM coenzyme A, 25 �M ferredoxin, 1 U of pyruvate:ferredoxin oxidoreductase from M. thermoacetica, and 5 U of phospho-transacetylase). The gas phase was 100% N2. The serum bottles werecontinuously shaken at 200 rpm to ensure H2 transfer from the liquidphase into the gas phase. Gas samples (0.2 ml) were withdrawn every 1min, and H2 was quantified by gas chromatography (19).

Formate dehydrogenase. The assay mixtures contained 100 mM Tris-HCl (pH 7.5) (Tables 1 and 2) or 100 mM potassium phosphate (pH asindicated) (Table 3), 2 mM DTT, 20 mM sodium formate, and, whereindicated, 25 �M ferredoxin, 1 mM NADP�, 1 mM NAD� or 10 mMmethyl viologen. The gas phase was 100% N2.

Formate dehydrogenase plus hydrogenase. When the formation ofH2 from formate (formate hydrogen lyase activity) was followed, the assaymixtures contained 100 mM Tris-HCl (pH 7.5) (Table 1) or 100 mMpotassium phosphate (pH as indicated) (Table 3), 2 mM DTT, and 20 mMsodium formate. The gas phase was 100% N2. The serum bottles werecontinuously shaken at 200 rpm to ensure H2 transfer from the liquidphase into the gas phase. Gas samples (0.2 ml) were withdrawn every 1min, and H2 was quantified by gas chromatography (19).

When the reduction of CO2 with H2 to formate was measured, theassay mixtures contained 100 mM potassium phosphate (final pH asindicated), 2 mM DTT, and 30 mM [14C]K2CO3 (24,000 dpm/�mol).The gas phase was 100% H2. The serum bottles were continuouslyshaken at 200 rpm to ensure equilibration of the gas phase with theliquid phase. After start of the reaction with enzyme, 100- �l liquidsamples were withdrawn every 1.5 min and added to 1.5-ml Safe-Sealmicrotube containing 100 �l of 150 mM acetic acid to stop the reactionby acidification. The 200-�l mixture was then incubated at 40°C for 10min with shaking at 1,400 rpm in a Thermomixer (type 5436; Eppen-dorf, Germany) to remove all 14CO2 leaving behind the [14C]formateformed. Subsequently, 100 �l of the mixture was added to 5 ml ofQuicksave A scintillation fluid (Zinsser Analytic, Frankfurt, Germany)and analyzed for 14C radioactivity in a Beckman LS6500 liquid scintil-lation counter (Fullerton, CA).

When the reduction of CO2 with reduced ferredoxin and NADPH toformate was monitored, the assay mixtures contained 100 mM potassiumphosphate (final as indicated), 2 mM DTT, 30 mM [14C]K2CO3 (24,000

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dpm/�mol), 1 mM NADPH, and reduced ferredoxin-regenerating sys-tem (10 mM pyruvate, 0.1 mM thiamine pyrophosphate, 1 mM coenzymeA, 25 �M C. pasteurianum ferredoxin, 1 U of pyruvate:ferredoxin oxi-doreductase, and 5 U of phosphotransacetylase). The gas phase was 100%N2. The serum bottles were continuously shaken at 200 rpm to ensureequilibration of the gas phase with the liquid phase. After the start of thereaction with enzyme, 100-�l liquid aliquots were withdrawn every 1.5min and analyzed for formate as described above.

Methylene-H4F dehydrogenase. The assay mixtures contained 100mM morpholinepropanesulfonic acid-KOH (pH 6.5), 50 mM 2-mer-captoethanol, 0.4 mM tetrahydrofolate (H4F), 10 mM formaldehyde,and 0.5 mM NADP� or 0.5 mM NAD�. The gas phase was 100% N2.After start of the reaction with enzyme, the formation of NAD(P)Hand methenyl-H4F were monitored at 350 nm by using an ε of 30.5mM�1 cm�1 [5.6 mM�1 cm�1 for NAD(P)H plus 24.9 mM�1 cm�1

for methenyl-H4F].Methylene-H4F reductase. The assay mixtures contained 100 mM

Tris-HCl (pH 7.5), 20 mM ascorbate, 10 �M FAD, 20 mM benzyl violo-gen, and 1 mM methyl-H4F. Before the start of the reaction with enzyme,benzyl viologen was reduced to an �A578 of 0.3 with sodium dithionite.The gas phase was 100% N2.

Acetaldehyde:ferredoxin oxidoreductase. The assay mixtures con-tained 100 mM Tris-HCl (pH 7.5), 2 mM DTT, 1.1 mM acetaldehyde, andabout 25 �M ferredoxin. The gas phase was 100% N2.

Acetaldehyde dehydrogenase (CoA-acetylating). The assay mixturecontained 100 mM Tris-HCl (pH 7.5), 2 mM DTT, 1.1 mM acetaldehyde,1 mM coenzyme A, and 1 mM NADP� or 1 mM NAD�. The gas phasewas 100% N2.

Alcohol dehydrogenase. The assay mixtures contained 100 mM po-tassium phosphate (pH 6), 2 mM DTT, 1.1 mM acetaldehyde, and 1 mMNADPH or 1 mM NADH. The gas phase was 100% N2.

Pyruvate:ferredoxin oxidoreductase. The assay mixture contained100 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM pyruvate, 1 mM coen-zyme A, 0.1 mM thiamine pyrophosphate, and about 25 �M ferredoxin, 1mM NADP�, or 1 mM NAD�. The gas phase was 100% N2.

2,3-Butanediol dehydrogenase. The assay mixture contained 100mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM acetoin, and 1 mM NADH or1 mM NADPH. The gas phase was 100% N2.

Lactate dehydrogenase. The assay mixture contained 100 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM pyruvate, and 1 mM NADH or 1 mMNADPH. The gas phase was 100% N2.

Glyceraldehyde phosphate dehydrogenase. The assay mixture con-tained 50 mM Tricine-NaOH (pH 8.5), 10 mM 2-mecaptoethanol, 10mM potassium phosphate, 1 mM glyceraldehyde-3-phosphate, and about25 �M ferredoxin, 1 mM NADP�, or 1 mM NAD�. The gas phase was100% N2.

Determination of structural properties. Proteins were separated bySDS-PAGE on 12% Mini-Protean TGX precast gels (Bio-Rad, Munich,Germany) according to the manufacturer’s manual. Where indicated theprotein samples to be analyzed were pretreated with 2-vinylpyridine forthiol function alkylation (41). The gels were stained with Coomassie bril-liant blue G250. The subunit stoichiometry was estimated from the stain-ing using Fluorchem 8800 digital imaging system software from AlphaInnotech, Santa Clara, CA. To obtain subunit sequence information, theprotein bands in the gel were excised and digested with sequencing-grademodified trypsin (Promega, Mannheim, Germany). The resulting peptidemixture was injected onto a PepMap100 C-18 RP nanocolumn (Dionex,Idstein, Germany) and separated on an UltiMate 3000 liquid chromatog-raphy system (Dionex) in a continuous acetonitrile gradient. A Probotmicrofraction collector (Dionex) was used to spot liquid-chromatogra-phy-separated peptides on a MALDI target, mixed with matrix solution(-cyano-4-hydroxycinnamic acid). Matrix-assisted laser desorptionionization–time of flight mass spectrometry (MALDI-TOF/MS) analysiswas carried out on a 4800 Proteomics Analyzer (Applied Biosystems/MDSSciex, Foster City, CA). Tandem MS (MS/MS) data were searched against

an in-house protein sequence database using Mascot (Matrixscience,United Kingdom) embedded into GPS explorer software (19).

The relative molecular mass of the purified hydrogenase-formate de-hydrogenase complex was measured by gel filtration on Superdex 200HR(1.0 by 30 cm) calibrated with standard proteins (Gel Filtration Calibra-tion Kit HMW; GE Healthcare). The column was equilibrated with anoxic50 mM Tris-HCl (pH 7.6) containing 2 mM DTT, 5 �M FAD, 5 �MFMN, and 300 mM NaCl and run at a flow rate of 0.5 ml min�1.

Determination of cofactors. Flavin was identified, and the flavin con-tent of the enzyme was measured as described previously (19). The puri-fied enzyme was first washed by ultrafiltration with a 20-fold volume offlavin-free, anoxic 50 mM Tris-HCl (pH 7.6) containing 2 mM DTT. Thewashed enzyme was then heat denatured in a boiling water bath for 10 minin the dark. After cooling, the denatured protein was removed by centrif-ugation at 13,000 � g and 4°C for 20 min. For identification of the flavinin the enzyme, the supernatant was analyzed by thin-layer chromatogra-phy (TLC) on an RP-18 F254 aluminum sheet (Merck, Darmstadt, Ger-many), with FAD and FMN as standards. The TLC plate was developedwith an aqueous solution of 85% 5 mM ammonium formate and 15%methanol. The presence of flavins was determined by irradiation with UVlight. For quantification of FMN in the enzyme, the UV-Vis spectrum ofthe supernatant of the denatured protein was recorded on a UV-1650PCspectrophotometer (Shimadazu, Japan), and the amount of FMN wascalculated using an ε at 446 nm of 12,200 M�1 cm�1 (42).

The content of iron, molybdenum, tungsten, and selenium in the en-zyme were determined with inductively coupled plasma mass spectrom-etry (ICP-MS; Fachbereich Chemie, Philipps-Universität, Marburg, Ger-many). The enzyme sample was desalted via washing with deionized waterbefore analysis.

Nucleotide sequence accession numbers. The nucleotide sequencesfor hyt-fdh from C. autoethanogenum and C. ragsdalei have been depositedin GenBank under accession numbers KF179065 and KF179066, respec-tively.

RESULTS

Clostridium autoethanogenum was grown on CO (42% CO, 20%CO2, 36% N2, 2% H2) in continuous culture at pH 5, 37°C, and adilution rate of 1.8 day�1 (td � 9.2 h). The culture produced 7 g ofcells (3.5 g protein) liter�1 day�1. They consumed CO at a specificrate of 5 mol liter�1 day�1 (�1 �mol min�1 mg of protein�1) andH2 at a specific rate of 0.1 mol liter�1 day�1 (�0.02 �mol min�1

mg of protein�1) and generated acetic acid at a specific rate of 0.24mol liter�1 day�1 (�0.05 �mol min�1 mg of protein�1), ethanolat a specific rate of 0.49 mol liter�1 day�1 (�0.1 �mol min�1 mgof protein�1), and 2,3-butanediol at a specific rate of 0.06 molliter�1 day�1 (�0.012 �mol min�1 mg of protein�1. For the bio-synthesis of cells (7 g liter�1 day�1 � 3.5 g of C liter�1 day�1), CO2

plus CO were assimilated at a rate of �0.29 mol liter�1 day�1

(�0.06 �mol min�1 mg of protein�1). These data indicated thatin cell extracts of the CO-grown organism, carbon monoxide de-hydrogenase (CO DH) should have a specific activity of at least 1�mol min�1 mg of protein�1 and that other enzymes proposedto be involved in acetyl-CoA formation from CO (Fig. 1)should have specific activities of �0.2 �mol min�1 mg of pro-tein�1. This was found to be the case (Table 1). Only the spe-cific activities of involved oxidoreductases were determined.Their proposed function in acetate, ethanol, and 2,3-butane-diol formation from CO is shown in Fig. 1, which summarizesthe findings of this work.

Specific activities of oxidoreductases in cell extracts. Thesubstrate specificity of individual activities tested indicated thatcell extracts of CO-grown C. autoethanogenum contain a ferredox-in-dependent carbon monoxide dehydrogenase (CO DH; 2.7

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U/mg), a reduced ferredoxin:NAD� oxidoreductase (RnfA-G; 0.6U/mg), an electron-bifurcating NAD�-dependent reduced ferre-doxin:NADP reductase (Nfn; 0.7 U/mg) catalyzing reaction 3, anelectron-bifurcating ferredoxin- and NADP-dependent hydroge-nase (1.1 U/mg) catalyzing reaction 6, a formate-hydrogen lyase(2.4 U/mg) catalyzing reaction 7, an electron-bifurcating ferre-doxin- and NADP-dependent formate dehydrogenase (1.1 U/mg)

catalyzing reaction 8, and an NADP-dependent methylene-tetra-hydrofolate (H4F) dehydrogenase (1.5 U/mg). Methylene-H4F re-ductase activity was also found but only with benzyl viologen asartificial electron donor/acceptor. NADH, NADPH and reducedferredoxin were not oxidized by methylene-H4F. Hydrogen for-mation from formate in the cell extracts was not stimulated byNADP�, NAD�, and/or ferredoxin (Table 1). All of these oxi-

TABLE 1 Specific activities of oxidoreductases involved in acetic acid, ethanol, and2,3-butanediol formation in CO-grown C. autoethanogenuma

a Red highlighting indicates substrates or products whose reduction, oxidation, or formation were monitored;boldfacing indicates relevant specific activities. The activities were determined at 37°C in cell extracts (20,000 �g supernatant) prepared with a French press. For pH and substrate concentrations, see Materials and Methods.Fd, ferredoxin from C. pasteurianum; BV, benzyl viologen; H4F, tetrahydrofolate. One unit (U) equals 2 �molof electrons transferred per min.b In cell extracts prepared with a French press, most of the activity was associated with the soluble fraction.When the cell extracts were obtained by lysis of the cells with lysozyme, the specific activity was only 0.15 U permg, of which almost 100% was membrane associated.c Ferredoxin was reduced by CO via the CO dehydrogenase activity present in the cell extracts.

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TABLE 2 Purification of electron-bifurcating NADP- and ferredoxin- dependent [FeFe]-hydrogenase in complex with formate dehydrogenase fromC. autoethanogenum grown on COa

Purification step

H2 ¡ MVox H2 ¡ NADP� � Fdox Formate ¡ MVox Formate ¡ NADP� � Fdox

Utotal (% yield) U/mg (fold) Utotal (% yield) U/mg (fold) Utotal (% yield) U/mg (fold) Utotal (% yield) U/mg (fold)

Cell extract (150,000 � g) 630,000 (100) 950 (1) 730 (100) 1.1 (1) 13,400 (100) 20.2 (1) 730 (100) 1.1 (1)Phenyl-Sepharose 330,000 (52) 3,400 (3.6) 2,150 (16) 22.3 (1.1)Q Sepharose 270,000 (43) 18,000 (19) 450 (62) 29.2 (27) 2,570 (19) 167 (8) 234 (32) 15.2 (14)a The cell extract (14 ml) with a protein concentration of 47 mg/ml was obtained from 8 g (wet mass) of previously frozen cells by French press treatment and centrifugation. Thepurification yielded 0.9 ml of enzyme solution with a specific activity of 29.2 U/mg and a protein concentration of 17 mg/ml. The activities were measured at 37°C in 100 mMpotassium phosphate (pH 7.5). Fd, ferredoxin from C. pasteurianum; MV, methyl viologen. One unit (U) equals 2 �mol of electrons transferred per min.

TABLE 3 Reactions catalyzed by C. autoethanogenum electron-bifurcating NADP- andferredoxin-dependent [FeFe]-hydrogenase in complex with formate dehydrogenasea

a Red highlighting indicates substrates or products whose reduction, oxidation, or formation were monitored;boldfacing indicates relevant specific activities. The activities were measured at 37°C in 100 mM potassiumphosphate at the indicated pH. Fd, ferredoxin from C. pasteurianum; MV, methyl viologen (10 mM). One unit(U) equals 2 �mol of electrons transferred per min.b Fdred

2�-regenerating system (ferredoxin from C. pasteurianum, pyruvate, thiamine pyrophosphate, coenzymeA, pyruvate:Fd oxidoreductase from M. thermoacetica, and phosphotransacetylase).

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doreductases showed specific activities sufficient to be involved inthe synthesis of acetyl-CoA from CO and CO2 (Fig. 1).

NADPH � Fdred2� � 3 H�ªNADP� � Fdox � 2 H2

�Go� � �21kJ ⁄ mol (6)

H2 � CO2ªHCOO� � H� �Go� � �3kJ ⁄ mol (7)

NADPH � Fdred2� � H� � 2 CO2ª NADP� � Fdox

� 2 HCOO� �Go� � �27kJ ⁄ mol (8)

Under physiological conditions (E= of the Fdox/Fdred2� couple

near �500 mV and of the NADP�/NADPH couple of near �370mV), reactions 6 and 8 most probably proceed only in the direc-tion of H2 and formate formation, respectively, despite the ender-gonicity of these reactions under standard conditions (calculatedwith an E0= of �400 mV for the Fdox/Fdred

2� couple; see the in-troduction).

For the synthesis of ethanol from acetyl-CoA (Fig. 1), acetal-dehyde:ferredoxin oxidoreductase (AFOR), CoA-dependent acet-aldehyde dehydrogenase (ALD), and ethanol dehydrogenase(ADH) were considered (Table 1). The cell extracts of CO-grownC. autoethanogenum catalyzed the reduction of ferredoxin withacetaldehyde (1.9 U/mg), the CoA-dependent reduction ofNADP� (0.08 U/mg) and of NAD� (0.06 U/mg) with acetalde-hyde, and the oxidation of NADPH (0.15 U/mg) and of NADH(0.2 U/mg) with acetaldehyde. The reduction of acetic acid (theacid with a pK � 4.8 has been shown to be the substrate) (43) toacetaldehyde with reduced ferredoxin could not be determined incell extracts because of interfering enzyme activities. However, the

purified acetaldehyde:ferredoxin oxidoreductase from other ace-togen was reported to catalyze the reduction of acetic acid (appar-ent Km � 3 mM) to acetaldehyde at rates comparable to those ofacetaldehyde oxidation to acetic acid (43).

For the synthesis of 2,3-butanediol from acetyl-CoA and CO2

(Fig. 1), pyruvate:ferredoxin oxidoreductase (PFOR) and 2,3-bu-tanediol dehydrogenase (23BDH) are required as oxidoreductases(Table 1). Both enzymes were found in sufficient specific activitiesof 0.2 and 1.3 U/mg, respectively. The reduction of acetoine to2,3-butanediol was observed with both NADPH and NADH. Theapparent Km for NADH was, however, much higher (�1 mM)than that for NADPH (�0.5 mM).

All but one of the enzyme activities were associated with thesoluble cell fraction; the exception was reduced ferredoxin:NAD�

oxidoreductase activity (presumably RnfA-G), which was associ-ated with the membrane fraction, albeit only when the cells werelysed with lysozyme rather than when disrupted via French presstreatment (Table 1). Based on the primary structure of the Rnfsubunits, those interacting with NAD (RnfC) and ferredoxin(RnfB) are peripheral membrane proteins (3, 44, 45), which couldbe only loosely associated with the cytoplasmic membrane.

The genes for all of the enzymes mentioned above have beenfound in the genome of C. autoethanogenum and C. ljungdahlii(36, 46; M. Köpke, unpublished data). Interestingly, the two genesnfnA and nfnB, which normally code for the electron-bifurcatingNAD�-dependent reduced ferredoxin:NADP reductase (Nfn)(15, 16), are fused to one gene in C. autoethanogenum and C.ljungdahlii.

FIG 1 Scheme of the energy metabolism of C. autoethanogenum growing at pH 5 on CO. The proposed role of the electron-bifurcating HytA-E/FdhA complexis shown in green and of the electron-bifurcating Nfn in blue. g, gaseous state; Fd, ferredoxin; H4F, tetrahydrofolate; DH, dehydrogenase; Hyt, electron-bifurcating [FeFe]-hydrogenase dependent on TPN (old name for NADP); Fdh, formate dehydrogenase; Nfn, electron-bifurcating NADH -dependent reducedferredoxin:NADP� oxidoreductase; RnfA-G, reduced ferredoxin:NAD� oxidoreductase. [CO], CO contained in the channel between the active sites of CO DHand acetyl-CoA synthase/decarbonylase that form a multi-enzyme complex (72, 73); ALD, acetaldehyde dehydrogenase; ADH, alcohol dehydrogenase; AFOR,acetaldehyde:Fd oxidoreductase; PFOR, pyruvate:Fd oxidoreductase; 23BDH, 2,3-butanediol dehydrogenase. Of the enzymes involved, only RnfA-G complex ismembrane associated. The reduction of NAD� with reduced ferredoxin is coupled with the build-up of an electrochemical proton potential, which in turn drivesthe phosphorylation of ADP via a membrane-associated FoF1 ATP synthase complex (46, 57).

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Purification of the ferredoxin- and NADP-dependent hydro-genase in complex with formate dehydrogenase. Purification ofthe enzyme complex from 8 g (wet mass) of C. autoethanogenumcells was monitored by measuring the reduction of methyl violo-gen with H2, the NADP-dependent reduction of ferredoxin withH2, the reduction of methyl viologen with formate, the NADP-dependent reduction of ferredoxin with formate, and the proteinconcentration (Table 2). The purification steps involved were am-monium sulfate precipitation, hydrophobic chromatography onPhenyl Sepharose, and anion-exchange chromatography on QSepharose. Via this method, 0.9 ml of enzyme at a concentrationof 17 mg/ml was obtained that exhibited both ferredoxin- andNADP-dependent hydrogenase activity (29.2 U/mg) and ferre-doxin- and NADP-dependent formate dehydrogenase activity(15.2 U/mg) (Table 2) and was composed of seven different pro-teins, as revealed by SDS-PAGE (Fig. 2).

During purification the enzyme was rapidly inactivated in thepresence of only trace amounts of O2, and it lost activity upondilution to much below 1 mg/ml even under strictly anoxic con-ditions. Ferredoxin and NADP� reduction with formate wasmore affected by dilution than ferredoxin and NADP� reductionwith H2. Therefore, activity had to be measured directly after pre-paring a dilution if this became necessary due to the high specificactivity.

In the cell extract the specific activities of NADP- and ferredox-in-dependent hydrogenase and of NADP- and ferredoxin-depen-dent formate dehydrogenase were almost identical (Table 2).Upon purification the specific activity of the NADP- and ferre-doxin-dependent hydrogenase increased 27-fold to 29.2 U/mgwith a 62% yield, whereas the specific activity of the NADP- andferredoxin-dependent formate dehydrogenase increased only 14-fold to 15.2 U/mg with a 32% yield, indicating that during purifi-cation ca. 50% of the formate dehydrogenase activity was lost.When additional purification steps were introduced (e.g., anionchromatography on HiTrap DEAE), the specific hydrogenase ac-tivities no longer increased considerably, whereas the specific for-mate dehydrogenase activities further decreased. Concomitantly,the content of the 78.8-kDa protein (Fig. 2) in the heptamericcomplex decreased, as revealed by SDS-PAGE (data not shown).The decrease probably was due to the partial dissociation and

separation of this protein, which is predicted to harbor theformate dehydrogenase active site (see below), from the hepta-meric complex during purification. Fractions with higherNADP- and ferredoxin-dependent formate dehydrogenase ac-tivity than NADP- and ferredoxin-dependent hydrogenase ac-tivity were never found.

The activity yield of ferredoxin and NADP reduction with H2

was 62%, whereas that of methyl viologen reduction with H2 wasonly 43% (Table 2). We interpreted this finding as indicating thatcell extracts of CO-grown cells of C. autoethanogenum containother hydrogenases that can use methyl viologen as electron ac-ceptor, besides NADP- and ferredoxin-dependent hydrogenase.In the case of formate dehydrogenase, the purification yield was32% for the NADP- and ferredoxin-dependent activity and 19%for the methyl-viologen-reducing activity, which again indicatedthe presence of more than one formate dehydrogenase that canuse methyl viologen as electron acceptor in cell extracts. In thegenome of C. autoethanogenum, there are in fact genes for sixdifferent hydrogenases (five [FeFe]-hydrogenases and one mem-brane-associated [NiFe]-hydrogenase) and for five different for-mate dehydrogenases (two selenium-containing enzymes andthree selenium-free isoenzymes) (M. Köpke, unpublished data).In this respect, it is of interest that the cells of C. autoethanogenum,from which the hydrogenase/formate dehydrogenase complexwas purified, were actually grown on a gas mixture containing H2

(2%), in addition to CO (42%), and that during growth H2 wasoxidized at a specific rate of �0.02 �mol min�1 mg of protein�1

(see above). The hydrogenase involved has not been identified.Subunit composition and encoding genes. The preparation

purified as described in Table 2 contained seven proteins (Fig.2A), which were sequenced via MS to identify the encoding genes.The encoding genes were found in a gene cluster of 10 genes (Fig.3). Reverse transcription-PCR (RT-PCR) analysis (see Table S1and Fig. S1 in the supplemental material) and the presence ofpalindromic termination sequences suggested that the 10 genesare organized in two transcription units. The first putative tran-scription unit consists of the gene (fdhA) for a molybdopterin-and selenocysteine-containing formate dehydrogenase (78.8 kDa)followed at a distance of 84 bp by three genes (moeA, mobB, andfdhD) involved in molybdopterin and formate dehydrogenasebiosynthesis; the proteins encoded by the three genes were notfound in the preparation. The second putative transcription unitat a distance of 295 bp starts with a gene (hytC) for a protein witha [2Fe2S]-cluster (18.1 kDa), followed by a gene (hytB) for aniron-sulfur flavoprotein (65.5 kDa), two genes (hytD and hytE1)for iron sulfur proteins (28.6 and 19.9 kDa), a gene (hytA) for[FeFe]-hydrogenase harboring the H-cluster (51.4 kDa) and agene (hytE2) for another iron-sulfur protein (20.1 kDa) (Fig. 3).The same cluster of 10 genes is found in the genome of both C.ljungdahlii (46) and C. ragsdalei (GenBank accession numberKF179066).

None of the six hyt genes are predicted to code for a proteinwith transmembrane helices. This is noteworthy since one of thesix genes coding for a [FeFe]-hydrogenase in complex with a tung-sten- and selenium-containing formate dehydrogenase in Eubac-terium acidaminophilum has been predicted to code for a mem-brane protein (47). The physiological electron donors/acceptorsfor the enzyme complex from E. acidaminophilum have not beenreported.

The deduced amino acid sequences of the proteins HytA,

FIG 2 SDS-PAGE of purified electron-bifurcating NADP- and ferredoxin-dependent [FeFe]-hydrogenase in complex with formate dehydrogenase(HytA-E/FdhA) from C. autoethanogenum. (A) Sample as such. (B) Samplepretreated with 2-vinylpyridine for thiol function alkylation (41). The encod-ing genes were identified by MALDI-TOF/MS sequence analysis of the sevenprotein bands after their digestion with trypsin. The molecular masses of thesubunits were calculated from amino acid sequences deduced from the encod-ing genes.

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HytB, and HytC from C. autoethanogenum show sequence iden-tity (�35%) to the HydA, HydB, and HydC subunits of the elec-tron-bifurcating NAD- and ferredoxin-dependent [FeFe]-hydro-genases, which are heterotrimeric (Thermotoga maritima and M.thermoacetica) or heterotetrameric enzymes (A. woodii) (17–19).The protein sequence of HytD from A. woodii shows 22% identityto the N terminus of HydA from T. maritima, M. thermoacetica,and A. woodii, which contain the conserved [2Fe2S] and two con-served [4Fe4S] binding motifs. The sequences of HytA, HytB,HytC, and HytD also shows sequence similarity to the subunitsNuoG, NuoF, NuoE, and NuoI of the NADH dehydrogenasecomplex from Escherichia coli. The proteins encoded by geneshytE1 and hytE2 show high sequence similarity to an iron-sulfurcluster containing subunit of dimethyl sulfoxide reductase.

The subunits HytE1 (19.9 kDa) and HytE2 (20.1 kDa), whichare predicted to each contain four [4Fe4S] clusters, formedblurred bands after SDS-polyacrylamide gels (Fig. 2A). The bandssharpened when the preparation was pretreated with 2-vinylpyri-dine to alkylate the cysteines before SDS-PAGE (Fig. 2B).

A scan of the Coomassie brilliant blue stained bands was con-sistent with six of the seven subunits being in equimolar amountsin the complex. Only the formate dehydrogenase subunit (78.8kDa) was in substoichiometric amounts (0.6), consistent with thefinding that during purification formate dehydrogenase activitywas lost (see above). The apparent molecular mass of the enzymecomplex estimated by gel exclusion chromatography on Superdex200HR was nearly 580 kDa, which indicated that the native en-zyme complex is a dimer of the heptamer with a calculated mo-lecular mass of 282.4 kDa.

Cofactor content. From the amino acid sequence of the sevensubunits we deduced that per mol of heteroheptamer, the com-plex should contain 76 iron, 1 molybdenum or tungsten, 1 sele-nium, and 1 FMN or FAD (Fig. 3). The chemical analysis of thepurified complex revealed the presence of about 60 mol of iron,0.3 mol of tungsten, 0.6 mol of selenium, and 0.63 mol of FMN permol of enzyme complex with a protein molecular mass of 282.4kDa. FAD and molybdenum (�0.01 mol per mol) were not found.The high iron-sulfur-cluster content is reflected in a high ε at 430nm of 147.3 mM�1 cm�1 of the enzyme complex in the oxidizedform (see Fig. S2 in the supplemental material).

The cofactor content values given above were based on a pro-tein determination of the samples with Bio-Rad reagent using bo-vine serum albumin as standard. With this method, the proteincontent of iron-sulfur proteins can be overestimated. Assuming a20% lower protein content in the samples, the iron content of theenzyme complex would be 72 rather than 60 mol of iron per mol

and thus very close to the theoretically predicted 76 mol of ironper mol. The contents of tungsten and selenium lower than 1 molper mol is consistent with the finding that the formate dehydro-genase subunit FdhA was present in the enzyme complex only insubstoichiometric amounts.

From the sequence of FdhA, we predicted that the formatedehydrogenase subunit is a selenoprotein as in many other bacte-ria, including Escherichia coli (48). Also, a respective selenocysteininsertion sequence (SECIS element) was found (49). However,sequence analysis left the question open, whether formate dehy-drogenase is a molybdenum protein as, e.g., in E. coli, or a tungstenenzyme as, e.g., in M. thermoacetica (50). The genetic context withclose proximity of fdhA to genes moeA and mob that are putativelyinvolved molybdopterin biosynthesis suggests the formate dehy-drogenase to be molybdenum dependent, but molybdopterin isalso involved in tungsten binding. Our finding that the HytA-E/FdhA complex contained tungsten and not molybdenum, al-though the growth medium was supplemented with bothmolybdate and tungstate, clearly indicated that the formate dehy-drogenase in complex with HytA-E is tungsten specific.

The unusually high iron-sulfur cluster content may be the rea-son why the enzyme complex catalyzes the reduction of methylviologen with H2 at a specific activity more than 100 times higherthan that of ferredoxin and NADP reduction with H2 (Tables 2and 3).

Catalytic properties. The reactions catalyzed by the enzymecomplex are summarized in Table 3. The preparation catalyzedthe reversible coupled reduction of ferredoxin and NADP with H2

(reaction 6), the reversible coupled reduction of ferredoxin andNADP with formate (reaction 8), the reversible reduction of CO2

with H2 to formate (reaction 7), and the reduction of methyl vi-ologen with H2, formate, and NADPH. The controls clearlyshowed that the enzyme complex is NADP rather than NAD spe-cific.

The activities given in Table 2 were routinely determined at pH7.5 and 37°C with the substrate concentrations given in Materialsand Methods. A pH of 7.5 was chosen because all activities of theenzyme complex were highly sensitive toward O2, and at pH 7.5reducing agents such as DTT in the assays removed trace O2 rap-idly enough to allow reproducible activity measurements. Atlower pH, the reproducibility decreased which made it difficult todetermine the pH optima. The highest specific activities weremeasured for NADP and ferredoxin reduction with H2 at pH 6.5,for H2 formation from NADPH and reduced ferredoxin at pH 6,for NADP and ferredoxin reduction with formate at pH 7.5, forCO2 reduction with H2 to formate at pH 7, and for H2 formation

FIG 3 The C. autoethanogenum genomic region around the hyt-fdh gene cluster encoding electron-bifurcating NADP- and ferredoxin-dependent [FeFe]-hydrogenase in complex with formate dehydrogenase. The gene products MoeA, MobB, and FdhD that are not found in the purified enzyme complex areinvolved in molybdopterin cofactor synthesis and formate dehydrogenase maturation. Cofactor binding sites were deduced from the amino acid sequences of theproteins. Sec, selenocysteine; Mo, molybdopterin, to which either molybdate or tungstate can be bound; F, flavin; [H], H-cluster of [6Fe4S], the active site of[FeFe]-hydrogenase.

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from formate at pH 6 (Table 3). In the case of CO2 reduction toformate with H2, it has to be considered that the formate dehy-drogenase uses CO2 rather than bicarbonate as substrate (51) andthat the CO2 concentration at a given amount of bicarbonateadded to the assay increases with decreasing pH. The lowest activ-ity found was for CO2 reduction with reduced ferredoxin andNADPH (Table 3), which is probably because under the assayconditions (100% N2 plus CO2 in the gas phase and continuousequilibration of the gas and liquid phases by shaking) also H2 wasformed at high specific rates (Table 3).

With respect to the substrate concentration dependence of theactivities, we determined only the apparent Km for H2 in ferre-doxin and NADP reduction with H2 because only this Km ap-peared to be of importance for understanding the function of theenzyme complex (see discussion). The apparent Km for H2 wasfound to be 12% H2 in the gas phase (�80 �M) under the exper-imental conditions (see Fig. S3 in the supplemental material).

Stoichiometry of reactions 6 and 8. To determine the stoichi-ometry of NADP and ferredoxin reduction with H2 or with for-mate, we monitored the formation of NADPH at 340 nm and theformation of reduced ferredoxin at 430 nm given that, upon re-duction, C. pasteurianum ferredoxin does not change its absor-bance significantly at 340 nm, and NADH does not absorb at 430nm (19). Per mol of NADP reduced by H2 (see Fig. S4A in thesupplemental material) or formate (see Fig. S4B in the supple-mental material), �0.8 mol of ferredoxin was reduced, a findingin agreement with reactions 6 and 8. In the absence of NADP,ferredoxin was not reduced, and in the absence of ferredoxin,NADP was not reduced.

Energetic coupling. The coupling of the endergonic reductionof ferredoxin with H2 to the exergonic reduction of NADP� withH2 was demonstrated by showing that, at pH 7.0, ferredoxin(E0= � �400 mV) was reduced to �90% by H2 (100% in the gasphase; E0= � �414 mV) in the presence of NADP� (E0= � �320mV) and of the heptameric hydrogenase-formate dehydrogenasecomplex from C. autoethanogenum but that ferredoxin was re-duced only to ca. 55% in the presence of the monomeric [FeFe]-hydrogenase from C. pasteurianum (Fig. 4). The C. pasteurianumhydrogenase, which is not electron bifurcating, catalyzes the re-versible reduction of ferredoxin with H2 in the absence of NAD�

or NADP�. Under the experimental conditions used, equilibriumwas achieved in the presence of this [FeFe]-hydrogenase when55% of the ferredoxin was reduced. Any further reduction musttherefore be energy driven. An almost identical experiment hasrecently been performed to demonstrate that the ferredoxin- andNAD�-dependent electron-bifurcating [FeFe]-hydrogenase fromM. thermoacetica is energy coupling (19).

Inhibition by CO. Carbon monoxide, which is the growth sub-strate of C. autoethanogenum, is known to inhibit most [FeFe]-hydrogenases at relatively low concentrations (52, 53). Inhibitionis competitive with H2 (54, 55). We therefore determined the ap-parent Ki for CO of the reactions catalyzed by the complex. Underthe experimental conditions tested, the following reactions wereinhibited to 50%: the formation of H2 from NADPH and reducedferredoxin at a CO concentration in the gas phase of ca. 0.1% (Fig.5), the reduction of ferredoxin and NADP� with H2 (100%) at aCO concentration of ca. 0.15% (see Fig. S5 in the supplementalmaterial), the formation of H2 from formate at a CO concentra-tion of ca. 0.13% (see Fig. S6 in the supplemental material), thereduction of CO2 with H2 (100%) to formate at a CO concentra-

tion of ca. 0.3% (data not shown), and the reduction of methylviologen (2 mM) with H2 (100%) at a CO concentration of ca.0.05% (data not shown). The different apparent Ki for CO can beexplained by the different experimental conditions (100% H2 or100% N2 in the gas phase) and by the likelihood that the hydro-genase was not always the rate-limiting enzyme in the multi-en-zyme complex catalyzing the four reactions.

Carbon monoxide is not known to inhibit formate dehydroge-nases. Therefore, it was unexpected that reduction of ferredoxinand NADP� with formate was inhibited by CO, with 50% inhibi-tion observed at CO concentrations in the gas phase of ca. 0.06%(Fig. 6), and yet neither the reduction of methyl viologen (2 mM)with formate (20 mM) nor the reduction of methyl viologen (2mM) with NADPH (1 mM) was inhibited by CO (100%) whentested under the conditions described in Table 3. Apparently, in-hibition of the coupled reduction of ferredoxin and NADP withformate was exerted via the hydrogenase in the enzyme complex(HytA-E/FdhA).

DISCUSSION

The finding of an electron-bifurcating ferredoxin- and NADP-dependent hydrogenase in CO-grown C. autoethanogenum wassurprising for two reasons: (i) the electron-bifurcating hydroge-nase in other acetogens, such as A. woodii (sodium ion dependent)and M. thermoacetica (cytochrome-containing), grown on H2 andCO2 is NAD specific (17, 19), and (ii) the NADP-specific enzymeis predicted to catalyze in vivo the formation of H2 rather than theuptake of H2 because of the thermodynamic reasons discussed in

FIG 4 Ferredoxin (Fd) reduction with H2 catalyzed by the electron-bifurcat-ing NADP- and ferredoxin-dependent [FeFe]-hydrogenase in complex withformate dehydrogenase (HytA-E/FdhA) from C. autoethanogenum. As con-trols, Fd reduction with 100% H2 catalyzed by the monomeric Fd-dependent[FeFe]-hydrogenase (Hyd) from C. pasteurianum, as well as the spontaneousreduction of Fd with sodium dithionite (Na2S2O4), were used. The reactionstook place in 1.5-ml anoxic cuvettes closed with rubber stoppers and contain-ing 0.8 ml of 100 mM potassium phosphate (pH 7.0) with 27 �M Fd from C.pasteurianum and, where indicated, C. autoethanogenum HytA-E/FdhA(�0.03 U), C. pasteurianum Hyd (�0.02 U), 1 mM NADP�, or 0.5 mM so-dium dithionite. The gas phase was 100% H2 at 1.2 � 105 Pa. The temperaturewas 37°C. With dithionite 100% of the Fd was reduced, and with H2 plushydrogenase from C. pasteurianum (Hyd) ca. 55% of the Fd was reduced.

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the introduction. During balanced growth on CO, the organismonly occasionally produces H2 Therefore, formation of H2 as anend product of CO fermentation cannot be the main function ofthis electron-bifurcating hydrogenase, which represents in CO-grown cells ca. 6% of the cytoplasmic proteins, as deduced from its27-fold purification to apparent homogeneity at a 62% yield (Ta-ble 2).

An indication to the function of the electron-bifurcating ferre-doxin- and NADP-dependent [FeFe]-hydrogenase (HytA-E) wasthe finding that it forms a functional complex with formate dehy-drogenase (FdhA), as reflected also by the finding that the genesfor the two enzymes cluster together in the genome. The complexcatalyzes both the reversible formation of H2 from reduced ferre-doxin and NADPH in a coupled reaction (reaction 6) and thereversible reduction of CO2 with H2 to formate (reaction 7). Thecomplex also catalyzes the reversible reduction of ferredoxin andNADP with formate (reaction 8). The function of the complex incells growing on CO could therefore be to catalyze the reduction ofCO2 to formate with both reduced ferredoxin and NADPH aselectron donors, which is a step in the Wood-Ljungdahl pathway(Fig. 1). Under physiological conditions the coupled reduction ofCO2 to formate (Eo= � �430 mV) with ferredoxin (E= � �500mV) and NADPH (E= � �370 mV) is slightly exergonic (�2kJ/mol).

In reaction 8, H2 probably is not an intermediate because thecomplex of the electron-bifurcating hydrogenase with formate de-hydrogenase (HytA-E/FdhA) contains only one subunit predicted

to have hydrogenase activity, namely, HytA, and it is difficult toenvisage how only one hydrogenase can simultaneously catalyzeboth the formation and uptake of H2 during reversible ferredoxinand NADP reduction with formate. In keeping with this hypoth-esis, ferredoxin and NADP reduction with formate proceeded atmaximal rates directly after the start of the reaction with formatebefore the H2 concentration had time to build up to the apparentKm concentrations of 80 �M (�12% H2 in the gas phase) (see Fig.S3 in the supplemental material). However, not consistent withthis hypothesis was the finding that ferredoxin and NADP reduc-tion with formate was inhibited by CO at very low concentrations(apparent Ki lower than 0.1% in the gas phase) (Fig. 6). Of thepartial enzyme activities catalyzed by the complex, only hydroge-nase activity was inhibited by CO. Neither the reduction of methylviologen with formate nor the reduction of methyl viologen withNADPH was affected by the hydrogenase inhibitor. One interpre-tation of these results is that the hydrogenase subunit HytA isinvolved in electron transport from formate to ferredoxin andNADP and that not only hydrogenase activity but also this elec-tron transport function of HytA is inhibited by CO.

If CO inhibits the reduction of CO2 to formate with reducedferredoxin and NADPH (reaction 8), then how can the enzymecomplex HytA-E/FdhA be involved in CO2 reduction to formateduring growth on CO? An answer could be that during balancedgrowth on CO the steady-state concentration of CO within thecells is lower than the apparent Ki for CO of 0.6 �M (�0.1% CO inthe gas phase).

The steady-state CO concentration is determined by the solu-bility of CO in water, which is low, by the rate of CO dissolving

FIG 5 Inhibition by CO of H2 formation from NADPH and reduced ferre-doxin catalyzed by the electron-bifurcating NADP- and ferredoxin-dependent[FeFe]-hydrogenase in complex with formate dehydrogenase (HytA-E/FdhA)from C. autoethanogenum. The reaction was performed at 37°C in anoxic6.5-ml serum bottles closed with rubber stoppers and containing 0.8 ml of 100mM potassium phosphate (pH 6) with a 25 �M Fdred

2� regenerating system(ferredoxin from C. pasteurianum, pyruvate, thiamine pyrophosphate, coen-zyme A, pyruvate:Fd oxidoreductase from M. thermoacetica, and phospho-transacetylase) and 1 mM NADPH. The gas phase (5.7 ml) was 100% N2 at1.2 � 105 Pa. CO was injected into the serum bottles with a gas-tight syringe togive the CO concentrations in % indicated. After equilibration of the gas andliquid phases by shaking, the reaction was started by injection of enzyme andmonitored by determining H2 formation gas chromatographically. The insetshows a Dixon plot of the data. At 100% CO in the gas phase at 1 bar pressure,the CO concentration in solution at 37°C is �0.8 mM (74).

FIG 6 Inhibition by CO of ferredoxin (Fd) and NADP reduction with formatecatalyzed by the electron-bifurcating NADP- and ferredoxin-dependent[FeFe]-hydrogenase in complex with formate dehydrogenase (HytA-E/FdhA)from C. autoethanogenum. The reaction took place at 37°C in 1.5-ml anoxiccuvettes closed with rubber stoppers and containing 0.8 ml of 100 mM potas-sium phosphate (pH 7.5) with 25 �M Fd from C. pasteurianum, 1 mM NADP,and 20 mM formate. The gas phase (0.7 ml) was 100% N2 at 1.2 � 105 Pa. COwas injected into the cuvettes with a gas-tight syringe to give CO concentra-tions at the percentages indicated. After equilibration of the gas and liquidphases by shaking, the reaction was started by injection of enzyme and moni-tored by determining the Fd reduction spectrophotometrically at 430 nm. Theinset shows a Dixon plot of the data. At 100% CO in the gas phase at 1 barpressure, the CO concentration in solution at 37°C is �0.8 mM (74).

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from the gas phase into the culture’s water phase, which is slow,and by the rate of CO used up by the bacterium, which can berelatively high. The mass transfer rate is determined by the mixingrate and the CO concentration in the gas phase, and the metabolicrate by the cell concentration and the specific catalytic efficiency(kcat/Km) of CO utilization. The steady-state concentration in-creases when the rate of CO utilization decreases or when the gastransfer rate increases, and vice versa.

The steady-state concentration of CO within the cells of grow-ing C. autoethanogenum cultures is predicted to be considerablylower than the O2 concentrations of �10 �M in cells of growing E.coli cultures (56). This prediction is based on the estimate that forthe synthesis of 1 mol of ATP from ADP and phosphate at least 10times more CO has to be oxidized to CO2 in C. autoethanogenumthan O2 has to be reduced to H2O in E. coli, as deduced from thethermodynamics and ATP yields of the two energy metabolisms.Since the solubility of CO ( � 0.0178 at 35°C) ( is the Bunsensolubility coefficient) is less than that of O2 ( � 0.0246 at 35°C) inwater, this difference in gas consumption rate results in �10-fold-lower steady-state CO concentrations in C. autoethanogenum thanO2 concentrations in E. coli when cultures of the two organismshave the same cell concentration and grow at equal rates.

There is another question that has to be answered. Why wouldan enzyme complex catalyzing in vivo, as proposed, CO2 reductionto formate with reduced ferredoxin and NADPH (reaction 8) beassociated with a hydrogenase? A possible answer is that the en-zyme’s second function is to protect the cells from over-reductionwhen NADP and ferredoxin get too reduced during growth of C.autoethanogenum on CO (E0= � �520 mV). An understanding ofthis function requires a more detailed look at the pathways ofelectrons from CO via ferredoxin, NADP, and NAD into the endproducts acetate, ethanol, and 2,3-butanediol (Fig. 1).

The fermentation starts with a reduction of ferredoxin by COcatalyzed by a ferredoxin-dependent CO dehydrogenase (Table1). The ferredoxin is subsequently reoxidized in four reactions: (i)the reduction of NAD� with reduced ferredoxin catalyzed by themembrane-associated RnfA-G complex, (ii) the reduction ofNADP with reduced ferredoxin and NADH catalyzed by Nfn (re-action 3), (iii) the reduction of acetic acid to acetaldehyde by re-duced ferredoxin catalyzed by acetaldehyde:ferredoxin oxi-doreductase, and (iv) the reduction of acetyl-CoA plus CO2 topyruvate catalyzed by pyruvate:ferredoxin oxidoreductase (Table1). As indicated in the introduction, ferredoxin is considered tooperate in vivo at a redox potential near �500 mV, NADP at aredox potential near �370 mV, and NAD at a redox potential near�280 mV. The redox potential difference between the Fdox/Fdred

2� couple and the NAD�/NADH couple is �200 mV, largeenough to be coupled with electron transport phosphorylationmediated by the membrane-associated RnfA-G complex and anFoF1 ATP synthase. NAD� reduction with ferredoxin is probablythe main coupling site in the energy metabolism of C. autoetha-nogenum growing on CO (44, 45, 57).

In the fermentation (Fig. 1), the rate of ferredoxin reoxidationis most probably limiting, as deduced from the finding that in cellextracts, of the activities tested, the reduction of ferredoxin withCO via CO dehydrogenase had the highest specific activity (Table1). When for whatever reason the rate of ferredoxin reoxidationand thus of CO oxidation slows down, the steady-state concentra-tion of CO in cells will increase, with the result that the reductionof CO2 to formate via the HytA-E/FdhA complex will decrease due

to inhibition by CO, which will further slow down ferredoxinreoxidation. Under these conditions, because of its negative redoxpotential (E0= � �520 mV), CO is likely to reduce ferredoxin,NADP, and NAD to 100% which completely deregulates the me-tabolism.

A way out of this dilemma under these conditions would befor ferredoxin to start being reoxidized by proton reduction toH2. This could be the function of the hydrogenase in the HytA-E/FdhA enzyme complex, namely, to catalyze the formation ofH2 from reduced ferredoxin and NADPH (reaction 6) when thesteady-state concentration of CO gets too high. To be able tocatalyze the formation of H2 in the presence of CO it has to beproposed that the H-cluster of the [FeFe]-hydrogenase be-comes super-reduced when poised in vivo to redox potentials ofthe 100% reduced ferredoxin and NADPH. In the super-re-duced state, the H-cluster no longer binds CO tightly and gen-erates H2 so rapidly that it can outcompete CO even at highconcentrations (58, 59), which is difficult to show experimen-tally but is consistent with present evidence (55, 58–64). Whenvia H2 formation in cultures of C. autoethanogenum growingon CO the steady-state CO concentration in the cells has de-creased again, the H2 formed could be taken up by cells via thereversible formate-hydrogen lyase activity of the HytA-E/FdhAcomplex (reverse of reaction 7).

In the discussion of hydrogenase inhibition by CO in C. auto-ethanogenum, it also has to be considered that the CO-grown cellshave a very high hydrogenase activity potential as determined withmethyl viologen as electron acceptor (950 U per mg of cell extractprotein and 18,000 U per mg of HytA-E/FdhA complex (Table 2).This potential would allow, if realized, H2 formation at significantrates even when 99% of the hydrogenase activity is inhibited byCO at concentrations 100 times higher than the apparent Ki. Tocompensate for CO inhibition, the concentration of the HytA-E/FdhA complex in C. autoethanogenum is probably so high (6% ofthe cytoplasmic proteins).

There are anaerobic bacteria such as Rhodospirillum rubrum(65) and Carboxidothermus hydrogenoformans (66) and anaerobicarchaea such as Thermococcus onnurineus (67) that can grow onCO (100%) forming H2 and CO2 (68). The membrane-associated[NiFe]-hydrogenase involved in these fermentations is very insen-sitive to CO inhibition. When tested in vitro with methyl viologenas electron acceptor, 50% inhibition was observed at a CO con-centration in the gas phase of ca. 50% (69–71). In this regard, it isof interest that in the genome of C. autoethanogenum, as in that ofC. ljungdahlii, there is a gene cluster that encodes for a multisub-unit membrane-associated [NiFe]- hydrogenase.

C. autoethanogenum, like other acetogenic bacteria, can alsogrow on H2 and CO2. Under these growth conditions CO2 has tobe reduced with reduced ferredoxin to CO, which is an interme-diate in the Wood-Ljungdahl pathway (Fig. 1). During growth onH2 and CO2, ferredoxin (E= � �500 mV) must therefore be rer-educed by H2, which is only possible if coupled to a sufficientlyexergonic reaction such as NAD� (E= � �280 mV) reductionrather than NADP� (E= � �370 mV) reduction with H2. Thisexplains why in A. woodii and M. thermoacetica ferredoxin reduc-tion with H2 is catalyzed by an electron-bifurcating ferredoxin-and NAD-dependent [FeFe]-hydrogenase (17, 19). It is thereforelikely that during growth of C. autoethanogenum on H2 and CO2

an NAD-specific electron-bifurcating hydrogenase is synthesized.Moreover, indeed, the genome of C. autoethanogenum harbors

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three gene clusters for three different cytoplasmic heteromeric[FeFe]-hydrogenases, one of which in this communication wasshown to be a ferredoxin- and NADP-dependent electron-bifur-cating [FeFe]-hydrogenase. This leaves one or both of the twoother gene clusters to code for a ferredoxin- and NAD-dependentelectron-bifurcating [FeFe]-hydrogenases when C. autoethanoge-num is growing on H2 and CO2. Cell extracts of CO-grown cellsdid not catalyze a coupled reduction of NAD and ferredoxin withH2 at significant rates (Table 1), indicating that, if present, thegenes for a ferredoxin- and NAD-dependent [FeFe]-hydrogenaseare not expressed during growth on CO.

ACKNOWLEDGMENTS

This study was supported by the Max Planck Society and the Fonds derChemischen Industrie.

We thank Johanna Moll for growing C. pasteurianum and purifying itsferredoxin, Andreas Seubert for the ICP-MS analyses, and WolfgangBuckel for critical reading of the manuscript and helpful suggestions.

REFERENCES1. Herrmann G, Jayamani E, Mai G, Buckel W. 2008. Energy conservation

via electron-transferring flavoprotein in anaerobic bacteria. J. Bacteriol.190:784 –791.

2. Li F, Hinderberger J, Seedorf H, Zhang J, Buckel W, Thauer RK. 2008.Coupled ferredoxin and crotonyl coenzyme a (CoA) reduction withNADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex fromClostridium kluyveri. J. Bacteriol. 190:843– 850.

3. Buckel W, Thauer RK. 2013. Energy conservation via electron bifurcatingferredoxin reduction and proton/Na� translocating ferredoxin oxidation.Biochim. Biophys. Acta 1827:94 –113.

4. Brandt U. 1996. Energy conservation by bifurcated electron-transfer inthe cytochrome bc1 complex. Biochim. Biophys. Acta 1275:41– 46.

5. Brandt U. 1996. Bifurcated ubihydroquinone oxidation in the cyto-chrome bc1 complex by proton-gated charge transfer. FEBS Lett. 387:1– 6.

6. Osyczka A, Moser CC, Daldal F, Dutton PL. 2004. Reversible redoxenergy coupling in electron transfer chains. Nature 427:607– 612.

7. Martin WF. 2012. Hydrogen, metals, bifurcating electrons, and protongradients: the early evolution of biological energy conservation. FEBS Lett.586:485– 493.

8. Nitschke W, Russell MJ. 2012. Redox bifurcations: mechanisms andimportance to life now, and at its origin: a widespread means of energyconversion in biology unfolds. Bioessays 34:106 –109.

9. Zhang F, Zhang Y, Chen M, van Loosdrecht MC, Zeng RJ. 2013. Amodified metabolic model for mixed culture fermentation with energyconserving electron bifurcation reaction and metabolite transport energy.Biotechnol. Bioeng. 110:1884 –1894.

10. Kaster AK, Moll J, Parey K, Thauer RK. 2011. Coupling of ferredoxinand heterodisulfide reduction via electron bifurcation in hydrog-enotrophic methanogenic archaea. Proc. Natl. Acad. Sci. U. S. A. 108:2981–2986.

11. Thauer RK, Kaster AK, Seedorf H, Buckel W, Hedderich R. 2008.Methanogenic archaea: ecologically relevant differences in energy conser-vation. Nat. Rev. Microbiol. 6:579 –591.

12. Costa KC, Lie TJ, Jacobs MA, Leigh JA. 2013. H2-independent growth ofthe hydrogenotrophic methanogen Methanococcus maripaludis. mBio4(2):e00062-13. doi:10.1128/mBio.00062-13.

13. Costa KC, Wong PM, Wang TS, Lie TJ, Dodsworth JA, Swanson I, BurnJA, Hackett M, Leigh JA. 2010. Protein complexing in a methanogensuggests electron bifurcation and electron delivery from formate to het-erodisulfide reductase. Proc. Natl. Acad. Sci. U. S. A. 107:11050 –11055.

14. Lie TJ, Costa KC, Lupa B, Korpole S, Whitman WB, Leigh JA. 2012.Essential anaplerotic role for the energy-converting hydrogenase Eha inhydrogenotrophic methanogenesis. Proc. Natl. Acad. Sci. U. S. A. 109:15473–15478.

15. Huang H, Wang S, Moll J, Thauer RK. 2012. Electron bifurcationinvolved in the energy metabolism of the acetogenic bacterium Moorellathermoacetica growing on glucose or H2 plus CO2. J. Bacteriol. 194:3689 –3699.

16. Wang S, Huang H, Moll J, Thauer RK. 2010. NADP� reduction with

reduced ferredoxin and NADP� reduction with NADH are coupled via anelectron-bifurcating enzyme complex in Clostridium kluyveri. J. Bacteriol.192:5115–5123.

17. Schuchmann K, Müller V. 2012. A bacterial electron-bifurcating hydro-genase. J. Biol. Chem. 287:31165–31171.

18. Schut GJ, Adams MW. 2009. The iron-hydrogenase of Thermotoga ma-ritima utilizes ferredoxin and NADH synergistically: a new perspective onanaerobic hydrogen production. J. Bacteriol. 191:4451– 4457.

19. Wang S, Huang H, Kahnt J, Thauer RK. 2013. A reversible electron-bifurcating ferredoxin- and NAD-dependent [FeFe]-hydrogenase (Hyd-ABC) in Moorella thermoacetica. J. Bacteriol. 195:1267–1275.

20. Bertsch J, Parthasarathy A, Buckel W, Müller V. 2013. An electron-bifurcating caffeyl-CoA reductase. J. Biol. Chem. 288:11304 –11311.

21. Smith ET, Bennett DW, Feinberg BA. 1991. Redox properties of 2[4Fe-4S] ferredoxins. Anal. Chim. Acta 251:27–33.

22. Smith ET, Feinberg BA. 1990. Redox properties of several bacterial ferre-doxins using square wave voltammetry. J. Biol. Chem. 265:14371–14376.

23. Boll M, Fuchs G, Tilley G, Armstrong FA, Lowe DJ. 2000. Unusualspectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxinof Thauera aromatica. Biochemistry 39:4929 – 4938.

24. Bender G, Ragsdale SW. 2011. Evidence that ferredoxin interfaces withan internal redox shuttle in acetyl-CoA synthase during reductive activa-tion and catalysis. Biochemistry 50:276 –286.

25. Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinow-itz JD. 2009. Absolute metabolite concentrations and implied enzymeactive site occupancy in Escherichia coli. Nat. Chem. Biol. 5:593–599.

26. Bar-Even A. 2013. Does acetogenesis really require especially low reduc-tion potential? Biochim. Biophys. Acta 1827:395– 400.

27. Poehlein A, Schmidt S, Kaster AK, Goenrich M, Vollmers J, Thürmer A,Bertsch J, Schuchmann K, Voigt B, Hecker M, Daniel R, Thauer RK,Gottschalk G, Müller V. 2012. An ancient pathway combining carbondioxide fixation with the generation and utilization of a sodium ion gra-dient for ATP synthesis. PLoS One 7:e33439. doi:10.1371/journal.pone.0033439.

28. Abrini J, Naveau H, Nyns EJ. 1994. Clostridium autoethanogenum, sp.nov., an anaerobic bacterium that produces ethanol from carbon monox-ide. Arch. Microbiol. 161:345–351.

29. Drake HL, Daniel SL. 2004. Physiology of the thermophilic acetogenMoorella thermoacetica. Res. Microbiol. 155:869 – 883.

30. Lindahl PA. 2012. Metal-metal bonds in biology. J. Inorg. Biochem. 106:172–178.

31. Ragsdale SW, Pierce E. 2008. Acetogenesis and the Wood-Ljungdahlpathway of CO2 fixation. Biochim. Biophys. Acta 1784:1873–1898.

32. Tanner RS, Miller LM, Yang D. 1993. Clostridium ljungdahlii sp. nov., anacetogenic species in clostridial rRNA homology group I. Int. J. Syst. Bac-teriol. 43:232–236.

33. Huhnke RL, Lewis RS, Tanner RS. 2010. Isolation and characterizationof novel clostridial species. U.S. patent 7,704,723.

34. Daniell J, Köpke M, Simpson SD. 2012. Commercial biomass syngasfermentation. Energies 5:5372–5417.

35. Köpke M, Mihalcea C, Bromley JC, Simpson SD. 2011. Fermentativeproduction of ethanol from carbon monoxide. Curr. Opin. Biotechnol.22:320 –325.

36. Köpke M, Mihalcea C, Liew F, Tizard JH, Ali MS, Conolly JJ, Al-SinawiB, Simpson SD. 2011. 2,3-Butanediol production by acetogenic bacteria,an alternative route to chemical synthesis, using industrial waste gas. Appl.Environ. Microbiol. 77:5467–5475.

37. Schönheit P, Wäscher C, Thauer RK. 1978. Rapid procedure for purifi-cation of ferredoxin from clostridia using polyethyleneimine. FEBS Lett.89:219 –222.

38. Wahl RC, Orme-Johnson WH. 1987. Clostridial pyruvate oxidoreduc-tase and the pyruvate-oxidizing enzyme specific to nitrogen fixation inKlebsiella pneumoniae are similar enzymes. J. Biol. Chem. 262:10489 –10496.

39. Jungermann K, Thauer RK, Leimenstoll G, Decker K. 1973. Function ofreduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolyticclostridia. Biochim. Biophys. Acta 305:268 –280.

40. Heijstra BD, Kern E, Köpke M, Segovia S, Liew F. 2012. Novel bacteriaand methods of use thereof. WO patent 2012015317.

41. Sechi S, Chait BT. 1998. Modification of cysteine residues by alkylation.A tool in peptide mapping and protein identification. Anal. Chem. 70:5150 –5158.

42. Aliverti A, Curti B, Vanoni MA. 1999. Identifying and quantitating FAD

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase

October 2013 Volume 195 Number 19 jb.asm.org 4385

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

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Dec

embe

r 20

21 b

y 12

2.35

.44.

41.

and FMN in simple and in iron-sulfur-containing flavoproteins. MethodsMol. Biol. 131:9 –23.

43. Huber C, Skopan H, Feicht R, White H, Simon H. 1995. Pterin cofactor,substrate specificity, and observations on the kinetics of the reversibletungsten-containing aldehyde oxidoreductase from Clostridium ther-moaceticum: preparative reductions of a series of carboxylates to alcohols.Arch. Microbiol. 164:110 –118.

44. Biegel E, Müller V. 2010. Bacterial Na�-translocating ferredoxin:NAD�

oxidoreductase. Proc. Natl. Acad. Sci. U. S. A. 107:18138 –18142.45. Biegel E, Schmidt S, Gonzalez JM, Müller V. 2011. Biochemistry, evo-

lution and physiological function of the Rnf complex, a novel ion-motiveelectron transport complex in prokaryotes. Cell. Mol. Life Sci. 68:613–634.

46. Köpke M, Held C, Hujer S, Liesegang H, Wiezer A, Wollherr A,Ehrenreich A, Liebl W, Gottschalk G, Dürre P. 2010. Clostridium ljung-dahlii represents a microbial production platform based on syngas. Proc.Natl. Acad. Sci. U. S. A. 107:13087–13092.

47. Graentzdoerffer A, Rauh D, Pich A, Andreesen JR. 2003. Molecular andbiochemical characterization of two tungsten- and selenium-containingformate dehydrogenases from Eubacterium acidaminophilum that are as-sociated with components of an iron-only hydrogenase. Arch. Microbiol.179:116 –130.

48. Böck A, Forchhammer K, Heider J, Baron C. 1991. Selenoproteinsynthesis: an expansion of the genetic code. Trends Biochem. Sci. 16:463–467.

49. Engelberg-Kulka H, Liu Z, Li C, Reches M. 2001. An extended Esche-richia coli “selenocysteine insertion sequence” (SECIS) as a multifunc-tional RNA structure. Biofactors 14:61– 68.

50. Andreesen JR, Makdessi K. 2008. Tungsten, the surprisingly positivelyacting heavy metal element for prokaryotes. Ann. N. Y. Acad. Sci. 1125:215–229.

51. Vorholt JA, Thauer RK. 1997. The active species of ’CO2= utilized byformylmethanofuran dehydrogenase from methanogenic Archaea. Eur. J.Biochem. 248:919 –924.

52. Adams MW. 1990. The structure and mechanism of iron-hydrogenases.Biochim. Biophys. Acta 1020:115–145.

53. Shima S, Thauer RK. 2007. A third type of hydrogenase catalyzing H2

activation. Chem. Rec. 7:37– 46.54. Thauer RK, Kaufer B, Zahringer M, Jungermann K. 1974. The reaction

of the iron-sulfur protein hydrogenase with carbon monoxide. Eur. J.Biochem. 42:447– 452.

55. Goldet G, Brandmayr C, Stripp ST, Happe T, Cavazza C, Fontecilla-Camps JC, Armstrong FA. 2009. Electrochemical kinetic investigations ofthe reactions of [FeFe]-hydrogenases with carbon monoxide and oxygen:comparing the importance of gas tunnels and active-site electronic/redoxeffects. J. Am. Chem. Soc. 131:14979 –14989.

56. Potzkei J, Kunze M, Drepper T, Gensch T, Jaeger KE, Buchs J. 2012.Real-time determination of intracellular oxygen in bacteria using a genet-ically encoded FRET-based biosensor. BMC Biol. 10:28. doi:10.1186/1741-7007-10-28.

57. Tremblay PL, Zhang T, Dar SA, Leang C, Lovley DR. 2013. The Rnfcomplex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD� oxidoreductase essential for autotrophic growth. mBio 4(1):e00406-12. doi:10.1128/mBio.00406-12.

58. Foster CE, Kramer T, Wait AF, Parkin A, Jennings DP, Happe T,

McGrady JE, Armstrong FA. 2012. Inhibition of [FeFe]-hydrogenases byformaldehyde and wider mechanistic implications for biohydrogen acti-vation. J. Am. Chem. Soc. 134:7553–7557.

59. Adamska A, Silakov A, Lambertz C, Rudiger O, Happe T, Reijerse E,Lubitz W. 2012. Identification and characterization of the “super-reduced” state of the H-cluster in [FeFe] hydrogenase: a new buildingblock for the catalytic cycle? Angew. Chem. Int. Ed. Engl. 51:11458 –11462.

60. Bennett B, Lemon BJ, Peters JW. 2000. Reversible carbon monoxidebinding and inhibition at the active site of the Fe-only hydrogenase. Bio-chemistry 39:7455–7460.

61. Lemon BJ, Peters JW. 1999. Binding of exogenously added carbon mon-oxide at the active site of the iron-only hydrogenase (CpI) from Clostrid-ium pasteurianum. Biochemistry 38:12969 –12973.

62. Lemon BJ, Peters JW. 2000. Photochemistry at the active site of thecarbon monoxide inhibited form of the iron-only hydrogenase (CpI). J.Am. Chem. Soc. 122:3793–3794.

63. Roseboom W, De Lacey AL, Fernandez VM, Hatchikian EC, AlbrachtSP. 2006. The active site of the [FeFe]-hydrogenase from Desulfovibriodesulfuricans. II. Redox properties, light sensitivity and CO-ligand ex-change as observed by infrared spectroscopy. J. Biol. Inorg. Chem. 11:102–118.

64. Silakov A, Kamp C, Reijerse E, Happe T, Lubitz W. 2009. Spectroelec-trochemical characterization of the active site of the [FeFe] hydrogenaseHydA1 from Chlamydomonas reinhardtii. Biochemistry 48:7780 –7786.

65. Kerby RL, Ludden PW, Roberts GP. 1995. Carbon monoxide-dependentgrowth of Rhodospirillum rubrum. J. Bacteriol. 177:2241–2244.

66. Svetlichny VA, Sokolova TG, Gerhardt M, Ringpfeil M, Kostrikina NA,Zavarzin GA. 1991. Carboxydothermus hydrogenoformans gen. nov., sp.nov., a CO-utilizing thermophilic anaerobic bacterium from hydrother-mal environments of Kunashir island. Syst. Appl. Microbiol. 14:254 –260.

67. Kim MS, Bae SS, Kim YJ, Kim TW, Lim JK, Lee SH, Choi AR, Jeon JH,Lee JH, Lee HS, Kang SG. 2013. CO-dependent H2 production by genet-ically engineered Thermococcus onnurineus NA1. Appl. Environ. Micro-biol. 79:2048 –2053.

68. Hedderich R, Forzi L. 2005. Energy-converting [NiFe] hydrogenases:more than just H2 activation. J. Mol. Microbiol. Biotechnol. 10:92–104.

69. Bonam D, Lehman L, Roberts GP, Ludden PW. 1989. Regulation ofcarbon monoxide dehydrogenase and hydrogenase in Rhodospirillumrubrum: effects of CO and oxygen on synthesis and activity. J. Bacteriol.171:3102–3107.

70. Fox JD, Kerby RL, Roberts GP, Ludden PW. 1996. Characterization ofthe CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrumand the gene encoding the large subunit of the enzyme. J. Bacteriol. 178:1515–1524.

71. Soboh B, Linder D, Hedderich R. 2002. Purification and catalytic prop-erties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydo-thermus hydrogenoformans. Eur. J. Biochem. 269:5712–5721.

72. Drennan CL, Doukov TI, Ragsdale SW. 2004. The metalloclusters ofcarbon monoxide dehydrogenase/acetyl-CoA synthase: a story in pictures.J. Biol. Inorg. Chem. 9:511–515.

73. Seravalli J, Ragsdale SW. 2000. Channeling of carbon monoxide duringanaerobic carbon dioxide fixation. Biochemistry 39:1274 –1277.

74. Air Liquide. 1976. Gas encyclopeadia. Elsevier Science, Amsterdam,Netherlands.

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