Reduction of flavodoxin by electron bifurcation and sodium Ion-dependent reoxidation by NAD+ catalyzed by ferredoxin-NAD+ reductase (Rnf)?

Article abstract of DOI:10.1074/jbc.M116.726299

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

Full text of DOI:10.1074/jbc.M116.726299

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 23, pp. 11993–12002, June 3, 2016
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Reduction of Flavodoxin by Electron Bifurcation and Sodium
Ion-dependent Reoxidation by NAD؉ Catalyzed by
Ferredoxin-NAD؉ Reductase (Rnf)*
Received for publication, March 8, 2016, and in revised form, April 1, 2016 Published, JBC Papers in Press, April 5, 2016, DOI 10.1074/jbc.M116.726299
Nilanjan Pal Chowdhury‡§, Katharina Klomann‡, Andreas Seubert¶, and Wolfgang Buckel‡§1
From the ‡Laboratorium fu¨r Mikrobiologie, Fachbereich Biologie and Synmikro and the ¶Fachbereich Chemie, Philipps-Universita¨t,
35032 Marburg, and the §Max-Plank-Institut fu¨r terrestrische Mikrobiologie, 35043 Marburg, Germany
Electron-transferring flavoprotein (Etf) and butyryl-CoA de-
hydrogenase (Bcd) from Acidaminococcus fermentans catalyze
the endergonic reduction of ferredoxin by NADH, which is also
driven by the concomitant reduction of crotonyl-CoA by
NADH, a process called electron bifurcation. Here we show that
recombinant flavodoxin from A. fermentans produced in Esch-
erichia coli can replace ferredoxin with almost equal efficiency.
After complete reduction of the yellow quinone to the blue
semiquinone, a second 1.4 times faster electron transfer affords
the colorless hydroquinone. Mediated by a hydrogenase, pro-
tons reoxidize the fully reduced flavodoxin or ferredoxin to the
semi-reduced species. In this hydrogen-generating system, both
electron carriers act catalytically with apparent Km ؍ 0.26 ␮M
ferredoxin or 0.42 ␮M flavodoxin. Membrane preparations of
A. fermentans contain a highly active ferredoxin/flavodoxin-
In 2008, however, it was established that the butyrate-forming
pathway contributes to hydrogen formation and energy conser-
vation, because the exergonic reduction of crotonyl-CoA to
butyryl-CoA by NADH is also tightly coupled to the endergonic
Ϫ 2
reduction of ferredoxin (Fd ) by NADH. This process has
been called electron bifurcation, because the two electrons sup-
plied by NADH (E°Ј ϭ Ϫ320 mV) move to different potentials:
one electron goes to the high potential crotonyl-CoA (E°Ј ϭ
Ϫ10 mV), and the other goes to the low potential ferredoxin
(E°Ј ϭ Ϫ405 mV; EЈ Յ Ϫ500 mV). Repetition of this process
affords butyryl-CoA and two reduced ferredoxins (Fd2Ϫ) (5–7).
This “energy-rich” electron carrier gives rise to molecular
hydrogen and/or recycles NADH mediated by a ferredoxin-
ϩ
NAD reductase also called ferredoxin/flavodoxin-NAD
reductase (Rnf) (8, 9). The energy difference between reduced
؉
ϩ
NAD reductase (Rnf) that catalyzes the irreversible reduction
ferredoxin and NAD of about 200 mV is proposed to be used
of flavodoxin by NADH to the blue semiquinone. Using flavo-
doxin hydroquinone or reduced ferredoxin obtained by electron
bifurcation, Rnf can be measured in the forward direction,
whereby one NADH is recycled, resulting in the simple equa-
by this integral membrane enzyme to generate an electrochem-
ϩ
ϩ
ical H or Na gradient. In Acidaminococcus fermentans (10)
and Megasphaera elsdenii (11), both of which belong to the
Negativicutes of the Clostridia (12), the electron-bifurcating
reduction of crotonyl-CoA is catalyzed by two enzymes. One is
a heterodimeric electron-transferring flavoprotein (EtfAB),
which contains one FAD on each subunit (␣ and ␤-FAD on
subunit A and B, respectively), and the other is a homotetra-
meric butyryl-CoA dehydrogenase (Bcd) with one FAD
(␦-FAD) on each subunit. Based on protein crystallography and
enzymatic assays, we propose that NADH reduces ␤-FAD to
؉
؉
tion: crotonyl-CoA ؉ NADH ؉ H ؍ butyryl-CoA ؉ NAD
with Km ؍ 1.4 ␮M ferredoxin or 2.0 ␮M flavodoxin. This reaction
؉
؉
requires Na (Km ؍ 0.12 mM) or Li (Km ؍ 0.25 mM) for activity,
؉
indicating that Rnf acts as a Na pump. The redox potential of
the quinone/semiquinone couple of flavodoxin (Fld) is much
higher than that of the semiquinone/hydroquinone couple.
With free riboflavin, the opposite is the case. Based on this
behavior, we refine our previous mechanism of electron
bifurcation.
Ϫ
the hydroquinone (␤-FADH ), which delivers one electron fur-
ther to the high potential ␣-FAD that is 14 Å apart to become a
Ϫ
⅐
stable anionic semiquinone (␣-FAD ). The remaining low
⅐
potential and highly reactive ␤-FADH donates its electron fur-
Ϫ
The fermentation of sugars and amino acids to butyrate is a
characteristic feature of anaerobic bacteria of the Firmicutes
phylum. In the human gut, butyrate is derived from the micro-
bial degradation of fibers, i.e. oligosaccharides or peptides that
are non-digestible by human enzymes (1, 2). Butyrate nourishes
the epithelia cells of the gut and thus is essential for human
health (3, 4). Until recently, the production of butyrate by bac-
teria was regarded as a mere disposal for reducing equivalents.
⅐
ther to ferredoxin. Then ␣-FAD , located on a flexible domain,
swings to the FAD of Bcd (␦-FAD) and transfers its electron to
Ϫ
⅐
yield ␦-FADH . Finally, the second round generates ␦-FADH ,
which reduces crotonyl-CoA to butyryl-CoA (10, 11).
It has been known for a long time that under iron-limiting
conditions, flavodoxin (Fld) replaces ferredoxin (13). One well
studied organism in this respect is A. fermentans, which grows
at equal rates either at 7 ␮M or at 45 ␮M Fe2ϩ in the medium. At
7 ␮M Fe2ϩ, the cells contain 3.5 ␮mol flavodoxin gϪ1 of protein
(1.4 mM) but only 0.02 ␮mol ferredoxin gϪ1 of protein, whereas
* This work was supported in part by a grant from the German Research
Foundation (DFG). The authors declare that they have no conflicts of inter-
est with the contents of this article.
1 To whom correspondence should be addressed: Laboratorium fu¨r 2 The abbreviations used are: Fd, ferredoxin; Etf, electron-transferring flavo-
Mikrobiologie, Fachbereich Biologie, Karl-von-Frisch-Str. 8, 35032
Marburg. Tel.: 49-6421-28-22088; Fax: 49-6421-28-28979; E-mail:
buckel@staff.uni-marburg.de.
protein; Bcd, butyryl-CoA dehydrogenase; Fld, flavodoxin; Rnf, ferredoxin-
NAD reductase; Rf, riboflavin; ICP-MS, inductively coupled plasma mass
spectrometry; MPa, megapascals.
JUNE 3, 2016•VOLUME 291•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 11993

Role of Flavodoxin in Electron Bifurcation and Rnf
at 45 ␮M Fe2ϩ, the flavodoxin content decreases to 0.2 ␮mol CoA and butyryl-CoA was performed on a C18 Kinetex column
g
Ϫ1 and that of ferredoxin increases to 1.0 Ϯ 0.1 ␮mol gϪ1 of (5-␮m particle size, 100-Å pore size, 250 ϫ 4.6 mm, Phenome-
protein (0.4 mM) (14). Flavodoxin from A. fermentans is a pro- nex, Aschaffenburg, Germany) at a flow rate of 1 ml/min in 50
tein (molecular mass ϭ 14.5 kDa) with FMN as tightly bound mM KH2PO4, pH 5.3, and 5% acetonitrile. During 20 min, a
cofactor. Ferredoxin from the same organism is a smaller pro- linear gradient up to 60% acetonitrile was applied. For detection
tein (molecular mass ϭ 5.6 kDa) that contains two [4Fe-4S] of respective CoA esters from the assay mixtures, samples were
clusters. The redox potentials of both electron carriers from acidified with concentrated HCl to pH 2.0, centrifuged, and
A. fermentans have been measured. In the case of ferredoxin, filtered to remove the denatured protein. Samples of 10 ␮l were
the first electron encounters a potential of E1°Ј ϭ Ϫ340 mV loaded on the HPLC column.
Ϫ
(Fd/Fd ), and the second electron encounters a potential of
Preparation of Ti(III) Citrate—Ti(III) citrate was prepared as
E2°Ј ϭ Ϫ405 mV (Fd /Fd2Ϫ) (14); with flavodoxin, the corre- described (18). TiCl3 in HCl (12%, 0.5 ml) was added to 5 ml of
sponding potentials are E1°Ј ϭ Ϫ60 mV and E2°Ј ϭ Ϫ430 mV 0.2 M sodium citrate. After 10 min, the mixture was neutralized
(15). Hence, from a thermodynamic point of view, flavodoxin with a saturated sodium carbonate solution to pH 7.0. The pre-
can easily replace ferredoxin, if only the half/fully reduced car- pared Ti(III) citrate was stored under anaerobic conditions in
riers with the low redox potentials (E2°Ј) are involved. In a pre- air-tight bottles.
Ϫ
vious study, evidence was obtained that ferredoxin cycles
Recombinant DNA Methods—Genomic DNA was isolated
between half/fully reduced states (Fd /Fd2Ϫ) during electron from A. fermentans cells using a genomic DNA preparation kit
transfer from Etf to hydrogenase and back (10). This study will (Fermentas, Thermo Fisher) according to the manufacturer’s
show that flavodoxin behaves in a similar manner; it cycles instructions. The flavodoxin protein from A. fermentans was
Ϫ
⅐
between the neutral blue semiquinone (FldH ) and the colorless identified earlier by N-terminal sequencing (15). The gene cor-
Ϫ
hydroquinone (FldH ) states, which are much easier to detect responding to the protein, Acfer_0269 (19), was amplified by
than the brownish/colorless Fd/Fd /Fd2Ϫ states. Furthermore, PCR using genomic DNA as the template with the forward
Ϫ
the generation of two-electron reduced ferredoxin and flavo- primer 5Ј-AAGCTCTTCAATGAGCAAAATCGCAGTGGT-
doxin by electron bifurcation without the use of artificial elec- GTTCT and the reverse primer 5Ј-AAGCTCTTCACCCTGC-
ϩ
tron donors allowed the confirmation of the postulated Na
CAGCGCCTCTCCCAG. The PCR product was cloned into
the p-ENTRY vector and introduced in E. coli DH5␣ by chem-
ical transformation. The cloned gene sequence was confirmed
and then transferred to the expression vector pASG IBA 33 and
dependence of the Rnf from A. fermentans.
Materials and Methods
Growth of Microorganisms—A. fermentans strain VR4 (DSM finally transferred to E. coli BL21-DE3. The colonies harboring
20731) was grown anaerobically on the glutamate/yeast the plasmid were used for overproduction of the protein.
extract/biotin medium as described earlier (16). Clostridium
Purification of Flavodoxin—E. coli cells overproducing the
tetanomorphum DSM 526 was grown on the same glutamate recombinant flavodoxin (10 g of wet mass) were suspended in
medium but without added biotin. Clostridium pasteurianum 50 mM potassium phosphate/150 mM NaCl, pH 7.0 (buffer A)
DSM 525 was grown on a medium containing 100 mM glucose, and disrupted by three passages through a French press at 140
70 mM NaHCO3, and yeast extract (2 g/liter). E. coli strains MPa. Cell debris and membranes were separated by centrifu-
DH5␣ and BL21-DE3 were grown aerobically at 37 °C in Stand- gation at 150,000 ϫ g for 45 min at 4 °C to obtain a membrane-
ard I nutrient broth (Merck, Darmstadt, Germany) using an free extract. The extract was passed through a 45-␮m filter and
orbital shaker. For growth of E. coli with inserted plasmids, the loaded on a 5-ml Ni-Sepharose column (HiTrap, GE Health-
medium was supplemented with carbenicillin (50 ␮g/ml). For care, Mu¨nchen, Germany), which was pre-equilibrated with
overproduction of flavodoxin, 2 liters of Standard I broth, sup- buffer A. All purification steps were performed at 4 °C, unless
plemented with carbenicillin, was inoculated with 25 ml of an otherwise mentioned. After loading the membrane-free
overnight culture of BL21-DE3 that has been transformed with extract, the column was washed with 10 column volumes of
the pASG-IBA33 expression vector (IBA, Go¨ttingen, Germany) buffer A to remove contaminating proteins. The protein was
containing the flavodoxin gene of A. fermentans. The culture eluted with 250 mM imidazole in buffer A. The purified protein
was grown until it reached OD600 ϭ 0.6, and then anhydrotet- was desalted and concentrated; FMN in molar excess was
racycline (200 ␮g/liter, IBA) was added to induce the expres- added and incubated overnight in the dark at 4 °C. Chromatog-
sion of the recombinant gene during the further growth for the raphy on a Superdex 75 pg (GE Healthcare) yielded homoge-
next 12 h at room temperature. The harvested cell paste was nous protein and removed excess FMN. The protein was stored
stored at Ϫ80 °C until use.
at Ϫ80 °C until use.
Synthesis of CoA Esters—Crotonyl-CoA and butyryl-CoA
Purification of Ferredoxin—The purification was performed
under strict anoxic conditions under an atmosphere of 95% N2
were synthesized by acylation of CoASH in aqueous 1
M
KHCO3 using 1 M crotonic anhydride or butyric anhydride in and 5% H2 (Coy Anaerobic Chamber) (14). Frozen C. tetano-
acetonitrile with a slight molar excess. After acidification, the morphum cells (10 g) were suspended in 50 mM potassium
CoA thioesters were purified over C18 columns and stored as phosphate, pH 6.8 (buffer B), and passed three times through a
lyophilized powders at Ϫ80 °C (17). The concentration of cro- French press at 140 MPa. The supernatant obtained by centrif-
ϩ
tonyl-CoA was calibrated by the NAD -dependent ␤-oxida- ugation at 150,000 ϫ g for 1 h at 4 °C was loaded on a DEAE
tion to acetyl-CoA and acetyl phosphate as described for the column, which was pre-equilibrated by buffer B. The column
assay of glutaconyl-CoA decarboxylase (16). HPLC of crotonyl- was washed with 2 column volumes of buffer B, and the protein
11994 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 23•JUNE 3, 2016

Role of Flavodoxin in Electron Bifurcation and Rnf
was eluted with a gradient of 0–100% 1 M NaCl achieved over 10 with 100 ␮M crotonyl-CoA. The decrease in NADH concentra-
column volumes (600 ml). Active fractions of ferredoxin, iden- tion was monitored at 340 nm, ⑀ ϭ 6.3 mMϪ1 cmϪ1 (24). Stoi-
tified by the bifurcation assay (see below), were pooled and chiometric amounts of flavodoxin (50 ␮M) were used in place of
concentrated by ultrafiltration with a 3-kDa membrane (Cen- ferredoxin/hydrogenase and monitored at 340 nm and also at
tricon, Merck Millipore, Darmstadt, Germany). The concen- 450 nm (oxidized flavin) and 578 (blue flavin semiquinone).
trated protein sample was chromatographed on a Superdex 75 Usually all measurements were repeated three times and the
column pre-equilibrated with 150 mM NaCl in buffer B. The error bars (average with S.D.) were inserted into the figures
fractions containing ferredoxin were identified by their dark when appropriate.
brown color, concentrated, and stored at Ϫ80 °C under anaer-
Determination of Na and Other Elements—After running the
obic conditions. The ferredoxin concentration was measured bifurcation/Rnf reaction, the whole assay was analyzed by
by its iron content (14), and a molecular mass of 6 kDa was used. inductively coupled plasma mass spectrometry (ICP-MS). The
The concentrations of flavodoxin and ferredoxin in A. fermen- protein samples and buffer blanks were diluted 100-fold with
tans cells listed as ␮mol/g of protein (14) were converted to mM ultrapure water and spiked with 10 ␮g yttrium (89Y)/kg as inter-
by assuming a cell volume of 2.5 ml/g of protein (20).
nal standard. The calibration of the ICP-MS was performed in
Preparation of Hydrogenase—Frozen C. pasteurianum cells the concentration range 0.1–100 ␮g/kg using dilutions of a
(5 g) were suspended in 10 ml of buffer B and broken by three Merck ICP multi-element standard solution IV (Merck product
passages through a French press at 140 MPa (20,000 p.s.i.) number 111355). The elemental contents of Li, Na, Mg, K, Mn,
under strict anoxic conditions. The broken cells were centri- Fe, Co, Ni, Cu, and Zn in the protein sample were determined
fuged at 7,000 ϫ g for 20 min. The supernatant was heated to by ICP-MS using an Agilent 7900 ICP-MS with HEN nebulizer
55–60 °C for 10–15 min under a hydrogen atmosphere. After- and cooled Scott spray chamber under standard operating con-
ward the extract was cooled in ice for 30 min. The precipitated ditions. The instrument was auto-tuned to robust plasma con-
7
proteins were removed by centrifugation at 20,000 ϫ g for 30 ditions to avoid matrix effects. The isotopes Li, 23Na, 24Mg,
min. The supernatant containing hydrogenase was stored at 26Mg, 39K, 55Mn, 56Fe, 57Fe, 59Co, 60Ni, 62Ni, 63Cu, 65Cu, 64Zn,
Ϫ80 °C until use (21).
66Zn and 67Zn were measured under NoGas, He collision, and
Electron-transferring Flavoprotein and Butyryl-CoA Dehy- H2 reaction mode conditions. Some isotopes were strongly
drogenase—Recombinant Etf from A. fermentans with a C-ter- interfered from the K-containing matrix (mainly 55Mn) in the
minal His tag at the ␣-subunit produced in E. coli and native NoGas mode and therefore rejected. The assay used to deter-
Bcd from A. fermentans were prepared as reported earlier (10). mine the dependence of Na contained 43 Ϯ 1 ␮M Na, which was
Both proteins were incubated with excess FAD overnight in mainly introduced by the 50 mM potassium phosphate buffer,
dark at 4 °C. Excess FAD was removed by using a desalting PD pH 6.8. In addition, about 8 ␮M Mg, 2.5 ␮M Fe, 0.8 ␮M Ni, and 2
10 column (GE Healthcare). The FAD content of the proteins ␮M Zn were detected.
Ϫ1
was calculated using ⑀450 ϭ 11.3 mMϪ1 cm
.
Results
Characterization of Recombinant Flavodoxin from A.
Preparation of Membrane Extracts—Wet-packed cells of
A. fermentans (20 g) were suspended in buffer B and broken by
four passages through a French press at 140 MPa under strict fermentans—The flavodoxin with a C-terminal His tag was
anoxic conditions. Cell debris was removed by centrifugation affinity-purified from a cell lysate of E. coli BL21 (DE3) harbor-
20,000 ϫ g for 20 min at 4 °C. The crude extract was centrifuged ing the pASG IBA33 vector with the flavodoxin gene as
at 150,000 ϫ g for 60 min. The supernatant was stored for fur- described under “Materials and Methods.” To assess its homo-
ther purification of soluble proteins, and the membrane pellet geneity, the protein was subjected to gel filtration at pH 7.0
was homogenized and washed twice with buffer B by centrifug- whereby several peaks were observed. Each of the two most
ing at 150,000 ϫ g for 30 min. The obtained inverted vesicles prominent peaks, when analyzed by SDS-PAGE, revealed a sin-
ϩ
ϩ
were used for Na /Li dependence assays. For other kinetic gle band around the 15-kDa protein marker. Both proteins were
measurements, the vesicles were solubilized with 2% n-dode- identified to be flavodoxin by peptide mapping with MALDI-
cyl-␤-D-maltoside in buffer B supplemented with 0.5 M NaCl TOF mass spectrometry, showing that the flavodoxin tends to
and then well homogenized. The homogenate was kept on ice make a mixture of oligomeric and monomeric forms. The
for 30 min and centrifuged at 150,000 ϫ g for 30 min. The molecular mass of the monomeric form (15.37 kDa) fits to
supernatant was used for the experiments (16).
the calculated value based on the amino acid sequence from the
Analytical Methods—Protein concentrations were estimated protein (14.54 kDa) and the His tag (0.84 kDa). The UV-visible
with the Bradford assay (22) (Bio-Rad-Microassay reagent, Bio- spectrum of the purified flavodoxin (Fig. 1) exhibited the two
Rad Laboratories, Munich, Germany). Bovine serum albumin characteristic peaks at 375 and 448 nm with a shoulder at 464
(Sigma, Darmstadt, Germany) was used as standard. SDS- nm. The flavodoxin was heated at 80 °C for 5 min, and the
PAGE was performed as described (23). All enzyme assays were yellow compound in the supernatant of the precipitated protein
performed under an atmosphere of 95% N2 and 5% H2. The was identified as FMN by TLC and reverse phase HPLC. The
bifurcation assay, i.e. the measurement of the reduction of cro- FMN content of the protein was quantified by using the known
Ϫ1
tonyl-CoA to butyryl-CoA, was done in a quartz cuvette (total absorbance coefficients at ␭ ϭ 450 nm (⑀ ϭ 11.3 mMϪ1 cm
)
volume 500 ␮l, d ϭ 1 cm) using, unless otherwise indicated, 0.5 and ␭ ϭ 375 nm (⑀ ϭ 10.7 mM cmϪ1) (25). Flavodoxin as
␮M EtfAf, 1 ␮M BcdAf, 1 ␮M ferredoxin, 30 ␮g of crude hydro- isolated contained only 0.2 mol of FMN per monomer, which
genase, and 250 ␮M NADH in buffer B. The reaction was started was raised to 0.5–0.6 upon overnight incubation with a molar
Ϫ1
JUNE 3, 2016•VOLUME 291•NUMBER 23
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Role of Flavodoxin in Electron Bifurcation and Rnf
FIGURE 2. Titration of Etf and flavodoxin with NADH. Black spectrum (A450 ϭ
0.17), 10 ␮M Etf containing 13 ␮M FAD. Red spectrum (A450 ϭ 0.30), after the
addition of 20 ␮M flavodoxin containing 10 ␮M FMN. This mixture was
reduced stepwise with NADH: purple trace (A450 ϭ 0.24), 5 ␮M NADH; brown
trace (A450 ϭ 0.18), 10 ␮M; pink trace (A450 ϭ 0.14), 12.5 ␮M; blue trace (A450 ϭ
0.12), 15 ␮M; gray trace (A450 ϭ 0.08), 20 ␮M.
FIGURE1. ReductionofflavodoxinbyTi(III)citrate. Blacktrace,oxidizedFld;
⅐
blue trace, semiquinone FldH reduced by 0.1 mM Ti(III)citrate; red trace, com-
pletely reduced FldHϪ by 5 mM Ti(III)citrate.
excess of FMN in the dark at 4 °C followed by gel filtration to
remove the unbound FMN. In contrast, the FMN content of
the flavodoxin purified from A. fermentans was reported to achieved. To study the reduction of flavodoxin during the elec-
be 1.0 mol/mol of protein (15), which suggests that upon tron bifurcation process, catalytic amounts of electron-bifur-
overproduction in E. coli, about half of the recombinant fla- cating flavoprotein from A. fermentans (EtfAf; 0.5 ␮M) and
vodoxin folded into a structure unable to bind FMN, which butyryl-CoA dehydrogenase (BcdAf; 1.0 ␮M subunit), 125 ␮M
could be due to the presence of the His tag. The production NADH, and 75 ␮M flavodoxin (43 ␮M FMN) were mixed under
of flavodoxin from Anabaena without a tag in E. coli yielded strict anaerobic conditions, and the UV-visible spectrum of the
pure holo-flavodoxin with a ratio of A465/A274 ϭ 0.17 (26), oxidized Fld was obtained. Upon the addition of 30 ␮M croto-
whereas the ratio of A465/A274 obtained with the recombi- nyl-CoA, the peak at 450 nm decreased and that of the blue
⅐
nant protein in this study was 0.146. The addition of 0.1 mM semiquinone (FldH ) at 578 nm, which remained stable for
Ti(III)citrate to 35 ␮M flavodoxin (22 ␮M FMN) at pH 6.8 more than 3 min, became visible. Upon further addition of 50
reduced the absorbance at 375 and 446 nm, and two new ␮M crotonyl-CoA, the semiquinone was reduced to the hydro-
Ϫ
stable peaks at 500 and 578 nm appeared (Fig. 1), which are quinone (FldH ). The complete reduction of flavodoxin indi-
⅐
characteristic for the blue neutral semiquinone (FldH ) (27). cated that an effective redox potential of E2Ј Յ Ϫ500 mV was
Upon increasing the concentration of Ti(III)citrate to 5 mM, reached. The same behavior was observed in the bifurcation
both peaks disappeared and the spectrum changed to that of process with ferredoxin (6, 29). To get more insight into the
Ϫ
completely reduced flavodoxin (FldH ).
kinetics of this reduction, we simultaneously measured the
Etf Exhibits Low Diaphorase Activity—Upon incubation of absorbance changes with time at three different wavelengths,
flavodoxin with NADH, the absorbance at 450 nm remained NADH at 340 nm, quinone at 450 nm, and semiquinone at 578
unchanged. In the presence of Etf, however, flavodoxin was nm (Fig. 3). After starting the incubation of Etf, Bcd, flavodoxin,
slowly reduced by NADH to the blue semiquinone. As shown in and NADH with crotonyl-CoA, we observed NADH oxidation,
Fig. 2, stepwise addition of 2.5 ␮M NADH to a mixture of 10 ␮M quinone reduction, and semiquinone formation. Only after the
Etf (13 ␮M FAD) and 20 ␮M flavodoxin (10 ␮M FMN) reduced quinone was quantitatively reduced to the semiquinone did an
the 450 nm peak. The lower peak at 375 nm shifted to 370 nm ϳ1.4 times faster reduction to the hydroquinone take place. At
and decreased more slowly until the height of both peaks this point, the apparent rate of NADH consumption increased
became equal (about 7.5–10 ␮M NADH), indicating the known accordingly. This result showed that the thermodynamically
formation of the red anionic semiquinone of the ␣-FAD of Etf more facile reduction of the quinone to the semiquinone
(28, 29). At 12.5 ␮M NADH, the blue neutral semiquinone of (E1°Ј ϭ Ϫ60 mV) occurred first and was followed by the more
flavodoxin began to appear and was fully present at 15 ␮M difficult reduction to the hydroquinone (E2°Ј ϭ Ϫ430 mV) (15).
NADH: theoretical at 13 ϩ 0.5 ϫ 10 ϭ 18 ␮M NADH. At Ն17.5 In contrast, the rates of the two reductions did not follow the
␮M NADH, a peak at 340 nm became visible due to excess of thermodynamics, indicating that at the Etf, the semiquinone is
NADH. Hence Etf catalyzes the slow reduction of flavodoxin to a better electron acceptor than the quinone.
the semiquinone by NADH, which is called diaphorase activity:
Hydrogenase Catalyzes the Reoxidation of Flavodoxin Hy-
Ϫ1
specific activity 0.04 units (mg of Etf)
.
droquinone—In our previous bifurcation assays, the hydrogen-
Electron Bifurcation Reduces Flavodoxin to the Hydro- ase from C. pasteurianum recycled oxidized ferredoxin con-
quinone—Due to the low redox potential of the semiquinone/ comitant with hydrogen formation (6, 29). With 100 ␮M
hydroquinone couple (E°Ј ϭ Ϫ430 mV), NADH can reduce NADH, 50 ␮M crotonyl-CoA, 0.5 ␮M Etf, and 1.0 ␮M Bcd (opti-
flavodoxin only to the semiquinone (E°Ј ϭ Ϫ60 mV). With elec- mal Etf/Bcd ratio) from A. fermentans, specific activities up to
tron bifurcation, however, the low potential can easily be
V
max ϭ 3.2 units (mg of Etf)Ϫ1 were achieved. The apparent Km
VOLUME 291•NUMBER 23•JUNE 3, 2016
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Role of Flavodoxin in Electron Bifurcation and Rnf
FIGURE 5. Reduction of flavodoxin by electron bifurcation and reoxida-
tion by NAD؉ mediated by Rnf. Black trace, spectrum of 50 ␮M flavodoxin.
The addition of 0.5 ␮M Etf, 1.0 ␮M Bcd, 250 ␮M NADH, and 100 ␮M crotonyl-
CoA reduced Fld to FldHϪ (Reaction 1) (red trace). Subsequent addition of Rnf
Ϫ
caused the oxidation FldH to FldH by the formed NADϩ (Reaction 5). The
⅐
reaction cycled until the excess NADH had consumed all crotonyl-CoA, yield-
⅐
ing the spectrum of the pure FldH (blue trace).
FIGURE 3. Kinetics of the reduction of flavodoxin. To an incubation with
0.5 ␮M Etf and 1 ␮M Bcd was added 250 ␮M NADH at t ϭ 50 s and 50 ␮M Fld
(29 ␮M FMN) at t ϭ 150 s. The reaction was started with 75 ␮M crotonyl-
CoA at t ϭ 250 s and measured simultaneously at three wavelengths: 340
nm, black trace; 450 nm, purple trace; 578 nm, blue trace.
crotonyl-CoA) was observed earlier with ferredoxin (29). At the
end of the reaction when crotonyl-CoA and/or NADH are
consumed, the semiquinone cannot be reduced anymore
(Reaction 3).
Etf ϩ Bcd: Crotonyl-CoA ϩ 2 NADH ϩ Fld ϩ 2 Hϩ
3 Butyryl-CoA ϩ 2 NADϩ ϩ FldHϪ
Reaction 1
⅐
Hydrogenase: 2 FldHϪ ϩ 2 Hϩ ϭ 2 FldH ϩ H2
Reaction 2
⅐
Etf ϩ Bcd: Crotonyl-CoA ϩ 2 NADH ϩ 2 FldH
3 Butyryl-CoA ϩ 2 NADϩ ϩ 2 FldHϪ
Reaction 3
Sum (2 ϩ 3): Crotonyl-CoA ϩ 2 NADH ϩ 2 Hϩ
FIGURE4. RateofNADHoxidation(units(mgofEtf)Ϫ1)asafunctionofFld
concentration. The cuvette contained 0.5 ␮M Etf, 1.0 ␮M Bcd, 100 ␮M NADH,
and hydrogenase. Error bars indicate Ϯ S.D.
ϩ
3 Butyryl-CoA ϩ 2 NAD ϩ H2
Reaction 4
for ferredoxin was determined as 0.26 Ϯ 0.03 ␮M (10). This
system worked equally well with Fld, exhibiting a similar Vmax ϭ
Rnf Mediates the Reoxidation of Either Flavodoxin Hydroqui-
1.2 units (mg of Etf)Ϫ1 and an apparent Km ϭ 0.42 Ϯ 0.05 ␮M, none or Reduced Ferredoxin—Membranes of A. fermentans sol-
based on a content of 0.5 FMN/Fld (Fig. 4). Initially, Fld is ubilized with dodecylmaltoside contain a very active Rnf that
Ϫ
reduced to the hydroquinone (FldH , Reaction 1). In the next catalyzes the exergonic oxidation of NADH with ferricyanide,
Ϫ
step, the hydrogenase catalyzes the oxidation of FldH to the specific activity ϭ 20 units (mg of protein)Ϫ1 (8, 30, 31). Now
⅐
semiquinone (FldH , Reaction 2), because further oxidation to electron bifurcation allowed the complete reduction of
the quinone (Fld) is not possible due to the much more positive ferredoxin or flavodoxin, with which the reaction could be
redox potential of E°Ј ϭ Ϫ60 mV than that of hydrogen, E°Ј ϭ measured in the physiologic direction with the natural elec-
Ϫ
⅐
Ϫ414 mV. FldH is reduced again to FldH by electron bifur- tron donor (Reaction 5 with flavodoxin) (Fig. 5). Together
cation (Reaction 3). In summary, crotonyl-CoA and 2 protons with Reaction 3, the simple Reaction 6 for the reduction of
are reduced with 2 NADH to butyryl-CoA and hydrogen (Reac- crotonyl-CoA to butyryl-CoA by NADH was obtained. This
tion 4), whereby flavodoxin cycles between the semiquinone stoichiometry could be verified experimentally (1.09
and hydroquinone states. The same stoichiometry (2 NADH/ NADH/crotonyl-CoA, Fig. 6).
JUNE 3, 2016•VOLUME 291•NUMBER 23
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Role of Flavodoxin in Electron Bifurcation and Rnf
FIGURE 6. Stoichiometry of consumed NADH as a function of the added
crotonyl-CoA according to Reaction 6. The assays contained 250 ␮M NADH,
0.5 ␮M Etf, 1.0 ␮M Bcd, 5 ␮M Fld, and 35 ␮g of solubilized membrane protein
from A. fermentans as source of Rnf; S.D. was Ϯ2%.
⅐
Rnf: 2 FldHϪ ϩ NADϩ ϩ Hϩ 3 2 FldH ϩ NADH
Reaction 5
Sum (3 ϩ 5): Crotonyl-CoA ϩ NADH ϩ Hϩ
3 butyryl-CoA ϩ NADϩ
Reaction 6
In this system, the apparent Km ϭ 2.0 Ϯ 0.4 ␮M flavodoxin
and Vmax ϭ 1.1 units (mg of Etf)Ϫ1 as well as Km ϭ 1.4 Ϯ 0.1 ␮M
ferredoxin and Vmax ϭ 1.3 units (mg of Etf)Ϫ1 were measured.
At the end of the reaction, when either NADH or crotonyl-CoA
was consumed, flavodoxin was quantitatively converted to the
semiquinone as shown in Fig. 5, blue trace. This figure exhibits the
UV-visible spectrum of the blue semiquinone of flavodoxin
between 400 and 700 nm with two peaks around 510 and 580 nm.
Sodium Dependence of Rnf—It was postulated that Rnfs from
A. fermentans and many clostridia use the energy difference of
FIGURE7. RequirementofNa؉ orLi؉ forRnfactivity. Shownaretheratesof
NADH oxidation (⌬A340/⌬t) according to Reaction 6 in the presence of 250 ␮M
NADH, 100 ␮M crotonyl-CoA, 0.5 ␮M Etf, 1.0 ␮M Bcd, 5 ␮M Fld, and 50 ␮g of
membrane vesicles from A. fermentans as the source of Rnf. The data were
fitted by the modified Michaelis-Menten equation: v ϭ Vmax ϫ (X ϩ X0) ϫ
(Km ϩ X ϩ X0)Ϫ1, where X signifiestheconcentrationsof Naϩ or Liϩ addedand
X0 signifies the concentration of Naϩ ϭ 0.043 mM already present in the assay
mixes. Error bars indicate Ϯ S.D.
Discussion
about 200 mV between reduced ferredoxin or flavodoxin and
Our results clearly demonstrate that flavodoxin can replace
ϩ
NADH to generate an electrochemical Na gradient for ATP ferredoxin as acceptor in the Etf ϩ Bcd bifurcating system of
synthesis (7, 8) as shown recently for Rnf from Acetobacterium A. fermentans, as donor of the [FeFe]hydrogenase of C. pasteu-
woodii (9, 32). Using inverted membrane vesicles from C. tet- rianum, and as donor/acceptor of the Rnf of A. fermentans.
anomorphum and A. fermentans in two assays, with either Thus flavodoxin or its semiquinone is reduced by NADH to the
NADH or Ti(III) citrate ϩ ferredoxin as donor and ferricyanide hydroquinone via electron bifurcation and reoxidized back to
ϩ
ϩ
or NAD as acceptor, respectively, a scalar Na dependence the semiquinone catalyzed either by the hydrogenase or by
could be not be detected. Furthermore, when the pH was raised RnfABCDGE (Acfer_0108-0113) (19). The advantage of study-
ϩ
above 7.0, Ti(III) citrate reduced NAD directly in the absence ing these reactions with flavodoxin is the facile identification of
of ferredoxin and Rnf (30). In the reaction with inverted mem- the blue neutral semiquinone, whereas with ferredoxin, the Fd,
brane vesicles from A. fermentans, Etf, Bcd, flavodoxin, NADH, Fd , and Fd2Ϫ states are not easily differentiated. Thus it could
Ϫ
and crotonyl-CoA (Reaction 6), a sodium or lithium ion depen- be shown that the quinone was almost quantitatively reduced to
dence could indeed be observed (Fig. 7). For the determination the semiquinone before it was further reduced to the hydroqui-
ϩ
ϩ
of the apparent Km ϭ 120 Ϯ 20 ␮M Na or 275 Ϯ 50 ␮M Li , the none as expected from thermodynamics (Fig. 3). In contrast,
ϩ
Na concentrations of the assays before the addition of defined the reduction of the flavodoxin semiquinone to the hydroqui-
amounts of NaCl or LiCl were measured with ICP-MS. The none proceeded ϳ1.4 times faster than the reduction of the
ϩ
lowest Na concentration in the assay mixture was 43 ␮M, quinone to the semiquinone. Hence a one-electron transfer to a
ϩ
whereas that of Li was close to zero.
compound with an unpaired electron most likely proceeds over
11998 JOURNAL OF BIOLOGICAL CHEMISTRY
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Role of Flavodoxin in Electron Bifurcation and Rnf
FIGURE 8. Proposed sodium ion gradient phosphorylation driven by the reduction of crotonyl-CoA in A. fermentans. The whole picture is a modification
of that taken from Ref. 9. The presence of an F1Fo ATP synthase was deduced from the genome of A. fermentans. The model of Rnf was constructed from data
obtained from Rnf of C. tetanomorphum (30) and NADH-quinone oxidoreductase (Nqr) of V. cholerae (33). For the structure of the Etf-Bcd complex see Ref. 10.
ϩ
The now confirmed Na dependence of Rnf fits well to the
a lower barrier than the addition of one electron to a closed
ϩ
shell. Alternatively, the easier one-electron reduction of the Na bioenergetics of A. fermentans (Fig. 8). This organism fer-
semiquinone could be an adaption to the physiologic condi- ments glutamate via the radical dehydration of (R)-2-hydroxy-
ϩ
tions under which flavodoxin cycles between semiquinone and glutaryl-CoA (37) followed by the Na -dependent decarboxy-
hydroquinone, whereas the quinone hardly exists.
lation of the product glutaconyl-CoA to crotonyl-CoA (35, 38).
In this work, we showed for the first time that Rnf from A. fer- Disproportionation of crotonyl-CoA yields acetate, butyrate, and
ϩ
mentans indeed required Na for activity. The failure of earlier hydrogen; the latter stems from reduced flavodoxin or ferredoxin
attempts to verify this effect was certainly due to the application generated by electron bifurcation with crotonyl-CoA and NADH.
ϩ
of artificial electron acceptors and donors. The heterohexam- The Na -pumping enzymes of this organism are glutaconyl-CoA
eric Rnf from C. tetanomorphum contains riboflavin, non-co- decarboxylase and Rnf, both of which most likely translocate two
ϩ
valently bound riboflavin-5Ј-phosphate, two riboflavin-5Ј- Na per turnover. According to the proposed pathway of gluta-
ϩ
phosphates covalently bound to RnfG and RnfD, about five mate fermentation, the decarboxylase contributes 2 Na /gluta-
ϩ
ϩ
iron-sulfur clusters (30), and probably one iron atom coordi- mate and Rnf adds 2 Na /5 glutamate (7). Because 1 Na /
nated by RnfA and RnfE as in the homologous NADH-quinone glutamate is most likely required for the import, from 2 ϩ 0.4 Ϫ
ϩ
ϩ
oxidoreductase (Nqr) of Vibrio cholerae (33) (Fig. 8). Thus one 1 ϭ 1.4 Na /glutamate are left for the Na -dependent ATP syn-
of this plethora of cofactors could transfer an electron directly thase,leadingto1.4/4ϭ0.35ATP/glutamateviaiongradientphos-
to ferricyanide or accept one electron from Ti(III)citrate with- phorylation. The contribution of substrate level phosphorylation
ϩ
out passing the Na -translocating site. Here we used electron amounts to 3 ATP/5 glutamate, which sums up to 0.95 ATP/glu-
bifurcation for the generation of flavodoxin hydroquinone tamate, the maximum theoretical yield (7).
(Fld2Ϫ) in the absence of Ti(III)citrate and were able to demon-
The electron-bifurcating Etf ϩ Bcd system has been shown
ϩ
ϩ
strate the Na and the Li dependences of Rnf from A. fermen- to provide a stable steady state concentration of almost com-
ϩ
tans. The apparent Km value of 120 Ϯ 20 ␮M Na at pH 6.8 is pletely reduced ferredoxin or flavodoxin (EЈ Յ Ϫ500 mV). Thus
similar to those of Rnf from A. woodii with Km values of 201 Ϯ in addition to hydrogenase and Rnf, this system also allows us to
ϩ
30 ␮M Na at pH 6 and 155 Ϯ 39 ␮M at pH 7.7 (34). The Km study other enzymatic reactions in which these reduced elec-
ϩ
values for Li also lie in the same range for both Rnfs. Surpris- tron carriers are substrates. The reaction rates are easily fol-
ingly, these values are much lower than those of the biotin- lowed via the oxidation of NADH. Examples are the synthases
ϩ
containing Na pumps glutaconyl-CoA decarboxylase from that form 2-oxo acids from acyl-CoA and CO2, such as pyruvate
A. fermentans (35) or oxaloacetate decarboxylase from Kleb- synthase, or the tungsten-containing aldehyde dehydroge-
siella pneumoniae (36), which exhibit apparent Km values of nases, which are also proposed to catalyze the reverse reactions,
ϩ
ϩ
1.0–1.5 mM Na and 25–100 mM Li .
the reduction of free carboxylic acids to aldehydes. Further-
JUNE 3, 2016•VOLUME 291•NUMBER 23
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Role of Flavodoxin in Electron Bifurcation and Rnf
FIGURE9.Proposedmechanismofelectronbifurcationasdeducedfromtheredoxpotentialsofriboflavinandflavodoxin.Theboldlinesandlettersindicatethe
postulated one-electron flows, endergonic from NADH to ferredoxin or flavodoxin and exergonic to crotonyl-CoA. See “Discussion” for further details.
more, the Etf ϩ Bcd system enables us to monitor even very trum. This spectrum is also obtained when the ␣-FAD is removed
difficult conversions such as nitrogen fixation or benzoyl-CoA by treatment of the Etf with KBr (28, 29).
reduction just by NADH oxidation at 340 nm.
␤-FAD is tightly bound to Etf between domain III, the ␤-sub-
Stimulated by the inverted redox potentials of flavodoxin as unit, and domain I of the ␣-subunit. It exhibits a more closed
compared with those of free FAD or riboflavin (39), we propose conformation, different from that of ␣-FAD and without the
a mechanism of electron bifurcation with Etf ϩ Bcd (Fig. 9). internal hydrogen bond. Therefore we conclude that the redox
Crystallographic studies showed that ␣-FAD of Etf from A. fer- potentials of ␤-FAD are not reversed and similar to those of free
mentans is bound to domain II of the ␣-subunit with a flavo- riboflavin. Unfortunately, only the two-electron redox poten-
doxin-like fold (10). The similarity ␣-FAD with flavodoxin is tial of the related Etf from M. elsdenii could be measured as
Ϫ
manifested by the ability of both proteins to stabilize a semiqui- E°Ј ϭ Ϫ279 mV for ␤-FAD/FADH (28). Together with the
none, a red anionic semiquinone in the case of ␣-FAD and the one-electron redox potentials of riboflavin (Rf), E°ЈRf1 ϭ Ϫ314
Ϫ
⅐
⅐
protonated blue form in the case of flavodoxin. The formation mV for Rf/RfH and E°ЈRf2 ϭ Ϫ124 mV for RfH /RfH (39), the
of stable semiquinones is due to the inverse redox potentials of real redox potentials of ␤-FAD could be E°Ј␤ ϭ Ϫ279 Ϯ
1
flavodoxin (see above) and ␣-FAD. The redox potentials of
⁄ (E°ЈRf1 Ϫ E°ЈRf2) ϭ Ϫ374 mV and Ϫ184 mV. Due to interac-
2
␣-FAD in the Etf from M. elsdenii (EtfMe), a bacterium closely tion with the protein, the separation between E°Ј␤1 and E°Ј␤2
related to A. fermentans, have been reported as E°Ј1 ϭ ϩ 81 mV could be even larger, which is assumed in Fig. 9.
Ϫ
Ϫ
⅐
⅐
for ␣-FAD/FAD and E°Ј2 ϭ Ϫ136 mV for ␣-FAD /FADH
Using these estimated redox potentials for ␣- and ␤-FAD,
(28). Although both potentials are much higher than the corre- one can construct a possible mechanism for electron bifurca-
sponding potentials of flavodoxin (Ϫ60 and Ϫ430 mV, respec- tion. As shown by crystallographic studies, NADH binds close
Ϫ
tively), there are indications that they decrease considerably to ␤-FAD and reduces it to ␤-FADH (29). Due to the ability of
when Etf forms a complex with Bcd as assumed in Fig. 9 (10, 40). ␣-FAD to stabilize a semiquinone, only one electron is shifted
Ϫ
⅐
Otherwise the reduction of crotonyl-CoA to butyryl-CoA (E°Ј ϭ from the low ␤-FADH /FADH level (about Ϫ90 mV, Fig. 9) to
Ϫ
⅐
Ϫ
⅐
Ϫ10 mV) by ␣-FAD would be difficult. Upon titration of Etf ␣-FAD/FAD (about Ϫ60 mV), which is 14 Å apart. According
1
1
4
with ⁄ NADH( ⁄ NADH/FAD), thestableredsemiquinoneanion to the law of energy conservation, the other electron at the
2
⅐
(␭max ϭ370 nm), whose concentration is significantly increased in ␤-FAD/FADH level becomes “red hot” (about Ϫ520 mV) (41)
the presence of Bcd (10), is observed (see Fig. 2 and Ref. 28). A and reduces the much more closely located ferredoxin that is
special feature of ␣-FAD is an internal hydrogen bond between the only 6 Å apart (29). Because the isoalloxazine ring of FMN sits
⅐
4Ј-OH of its ribityl side chain to either N1 or to the C2ϭO group of on the surface of flavodoxin, an electron from ␤-FADH to this
the isoalloxazine ring. This bond, as well as several other interac- acceptor probably has to pass the same distance. Now the flex-
Ϫ
⅐
tions with the surrounding amino acid residues, perturbs the UV- ible domain II carrying ␣-FAD must undergo a large confor-
visible spectrum of ␣-FAD in such a way that an extra absorption mational change to interact with ␦-FAD of Bcd, which, in the
band around 400 nm between the two prominent peaks of the complex of human Etf and medium chain acyl-CoA dehydro-
FAD quinone at 375 and 446 nm is observed. This band at 400 nm genase, has been shown to be 30 Å apart (42). After reduction of
⅐
is quenched by reduction of Etf with NADH in a 1:1 ratio, whereby ␦-FAD to the semiquinone (␦-FADH ), a second round of bifur-
Ϫ
the remaining still oxidized ␤-FAD exhibits a normal flavin spec- cation generates ␦-FADH that transfers a hydride to crotonyl-
12000 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 23•JUNE 3, 2016

Role of Flavodoxin in Electron Bifurcation and Rnf
⅐
proach to investigate the intestinal butyrate-producing bacterial commu-
nity. Microbiome 1, 8
CoA, yielding butyryl-CoA. Alternatively, ␦-FADH reduces
crotonyl-CoA to an allylic ketyl radical (43, 44), and the second
5. Herrmann, G., Jayamani, E., Mai, G., and Buckel, W. (2008) Energy con-
servation via electron-transferring flavoprotein in anaerobic bacteria. J.
Bacteriol. 190, 784–791
⅐
␦-FADH completes the reduction to butyryl-CoA.
This mechanism is actually similar to that of the “redox see-
saw” originally proposed by Peter Mitchell (41, 55), although
the redox potentials of the bifurcating ␤-FAD are normal and
not inverted. The inverted redox potentials of ␣-FAD are nec-
essary to generate the high potential one-electron acceptor able
to form a stable semiquinone, which has to be transported over
30 Å to ␦-FAD of Bcd. Other bifurcating systems use iron sulfur
clusters as high potential electron acceptors; established cases
are the Rieske [2Fe-2S] cluster in the Q-cycle of complex III of
the respiratory chain (45) and the [4Fe-4S] cluster N3 close to
FMN of NADH-quinone oxidoreductase (Nuo, complex I) (46).
The Rieske [2Fe-2S] cluster has a function very similar to that of
␣-FAD. It accepts the high potential electron from ubihydro-
quinone (UQH2) and swings over to cytochrome c1, which
becomes reduced. It has been proposed that in complex I, the
6. Li, F., Hinderberger, J., Seedorf, H., Zhang, J., Buckel, W., and Thauer, R. K.
(2008) Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction
with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex
from Clostridium kluyveri. J. Bacteriol. 190, 843–850
7. Buckel, W., and Thauer, R. K. (2013) Energy conservation via electron
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doxin oxidation. Biochim. Biophys. Acta 1827, 94–113
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Hedderich, R., Vgenopoulou, I., Pierik, A. J., Steuber, J., and Buckel, W.
(2005) Sodium ion pumps and hydrogen production in glutamate fer-
menting anaerobic bacteria. J. Mol. Microbiol. Biotechnol. 10, 105–119
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10. Chowdhury, N. P., Mowafy, A. M., Demmer, J. K., Upadhyay, V., Koelzer,
S., Jayamani, E., Kahnt, J., Hornung, M., Demmer, U., Ermler, U., and
Buckel, W. (2014) Studies on the mechanism of electron bifurcation cat-
alyzed by electron transferring flavoprotein (Etf) and Butyryl-CoA dehy-
drogenase (Bcd) of Acidaminococcus fermentans. J. Biol. Chem. 289,
5145–5157
Ϫ
two electrons of FMNH bifurcate; one goes to the high poten-
tial [4Fe-4S] cluster N3, and the other goes to the lower poten-
tial [2Fe-2S] cluster N1a. Although the electron on N3 moves
forward via six additional clusters to the ubiquinone, the other
electron is stored on N1a until the way is free to follow the first
electron. Thus the formation of a reactive oxygen species-form-
11. Chowdhury, N. P., Kahnt, J., and Buckel, W. (2015) Reduction of ferre-
doxin or oxygen by flavin-based electron bifurcation in Megasphaera els-
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12. Campbell, C., Adeolu, M., and Gupta, R. S. (2015) Genome-based taxo-
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ord. nov., and Veillonellales ord. nov. Int. J. Syst. Evol. Microbiol. 65,
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⅐
ing FMNH is avoided (46). With the exception of Etf ϩ Bcd, all
other bifurcating systems also contain iron-sulfur clusters,
which possibly serve as high potential electron acceptors (47–
54). Probably these clusters can be more easily tuned to the
required redox potentials than flavins. Hence the Etf ϩ Bcd
system appears to be a special case among the bifurcating fla-
voprotein complexes, in which the flavodoxin-like ␣-FAD
adopts the function of an iron-sulfur cluster.
13. Mayhew, S. G., and Tollin, G. (1992) General properties of flavodoxins. in
Chemistry and Biochemistry of Flavoenzymes (Mu¨ller, F., ed), pp.
389–426, CRC Press, Inc., Boca Raton
14. Thamer, W., Cirpus, I., Hans, M., Pierik, A. J., Selmer, T., Bill, E., Linder,
D., and Buckel, W. (2003) A two [4Fe-4S]-cluster-containing ferredoxin as
an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase
from Acidaminococcus fermentans. Arch. Microbiol. 179, 197–204
15. Hans, M., Bill, E., Cirpus, I., Pierik, A. J., Hetzel, M., Alber, D., and Buckel,
W. (2002) Adenosine triphosphate-induced electron transfer in 2-hy-
droxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Bio-
chemistry 41, 5873–5882
Author Contributions—N. P. C. and W. B. designed the study and
wrote the manuscript. K. K. performed initial experiments. N. P. C.
produced and purified recombinant flavodoxin and studied its reac-
tions with Etf ϩ Bcd and Rnf. A. S. performed the metal analysis. All
authors analyzed the results and approved the final version of the
manuscript.
16. Buckel, W. (1986) Biotin-dependent decarboxylases as bacterial sodium
pumps: purification and reconstitution of glutaconyl-CoA decarboxylase
from Acidaminococcus fermentans. Methods Enzymol. 125, 547–558
17. Parthasarathy, A., Pierik, A. J., Kahnt, J., Zelder, O., and Buckel, W. (2011)
Substrate specificity of 2-hydroxyglutaryl-CoA dehydratase from Clos-
tridium symbiosum: toward a bio-based production of adipic acid. Bio-
chemistry 50, 3540–3550
Acknowledgments—We thank Professor Rudolf K. Thauer, Max-
Planck-Institut fu¨r terrestrische Mikrobiologie, Marburg, for very
helpful discussions and advice, and Professor Johann Heider,
Philipps-Universita¨t Marburg, for help with the modified Michaelis-
Menten equation. We are indebted to Gabriela Mielke, Institut fu¨r
Pharmazeutische Chemie, Philipps-Universita¨t Marburg, for initial
18. Zehnder, A. J. B., and Wuhrmann, K. (1976) Titanium(III) citrate as a
nontoxic oxidation-reduction buffering system for the culture of obligate
anaerobes. Science 194, 1165–1166
ϩ
Na -determinations by flame photometry.
19. Chang, Y. J., Pukall, R., Saunders, E., Lapidus, A., Copeland, A., Nolan, M.,
Glavina Del Rio, T., Lucas, S., Chen, F., Tice, H., Cheng, J. F., Han, C.,
Detter, J. C., Bruce, D., Goodwin, L., et al. (2010) Complete genome se-
quence of Acidaminococcus fermentans type strain (VR4). Stand. Genomic
Sci. 3, 1–14
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12002 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 23•JUNE 3, 2016

Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent
Reoxidation by NAD + Catalyzed by Ferredoxin-NAD+ Reductase (Rnf)
Nilanjan Pal Chowdhury, Katharina Klomann, Andreas Seubert and Wolfgang Buckel
J. Biol. Chem. 2016, 291:11993-12002.
doi: 10.1074/jbc.M116.726299 originally published online April 5, 2016
Access the most updated version of this article at doi: 10.1074/jbc.M116.726299
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