Tài liệu Báo cáo khoa học: Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes docx

Reconstitution of coupled fumarate respiration in liposomes
by incorporating the electron transport enzymes isolated
from
Wolinella succinogenes
Simone Biel
1
,Jo¨ rg Simon
1
, Roland Gross
1
, Teresa Ruiz
2
, Maarten Ruitenberg
3
and Achim Kro¨ ger
1
1
Institut fu
¨
r Mikrobiologie, Johann Wolfgang Goethe-Universita
¨
t, Frankfurt am Main, Germany;
2
Max-Planck-Institut fu
¨
r Biophysik,
Abteilung Strukturbiologie, Frankfurt am Main, Germany;
3
Max-Planck-Institut fu
¨
r Biophysik, Abteilung Biophysikalische Chemie,

Frankfurt am Main, Germany
Hydrogenase and fumarate reductase isolated from Woli-
nella succinogenes were incorporated into liposomes con-
taining menaquinone. The two e nzymes were found to be
oriented solely to the outside of the resulting proteolipo-
somes. The proteoliposomes catalyzed fumarate reduction
by H
2
which generated an electrical p roton potential (Dw ¼
0.19 V, negative inside) i n the same direction as that gen-
erated by fumarate respiration in cells of W. succinogenes.
The H
+
/e ratio brought abo ut by f umarate reduction with
H
2
in proteoliposomes in the presence of v alinomycin and
external K
+
was a pproximately 1 . T he same Dw and H
+
/e
ratio was associated with the r eduction of 2,3-dimethyl-1,4-
naphthoquinone (DMN) by H
2
in proteoliposomes con-
taining menaquinone and h ydrogenase with or without
fumarate reductase. Proteoliposomes containing menaqui-
none and fumarate reductase with o r without hydrogenase
catalyzed fumarate reduction by DMNH

2
which did not
generate a Dw. Incorporation of formate dehydrogenase
together with fumarate reductase and menaquinone
resulted in proteoliposomes catalyzing the reduction of
fumarate or DMN by formate. Both reactions generated a
Dw of 0.13 V ( negative inside). The H
+
/e ratio of formate
oxidation by menaquinone or DMN was close to 1. The
results d emonstrate f or the first time that coupled fumarate
respiration can be restored in liposomes using the well
characterized electron transport enzymes isolated from
W. succinogenes. The results support the view that Dw
generation is coupled to menaquinone reduction by H
2
or
formate, but not to menaquinol oxid ation by fumarate. Dw
generation is probably caused by proton uptake from the
cytoplasmic side of the membrane during menaquinone
reduction, and by the coupled release of protons from H
2
or
formate o xidation on the p eriplasmic side. This mechanism
is supported b y t he properties o f t wo hydrogenase m utants
of W. succinogenes which indicate that the s ite of quinone
reduction is close to the cytoplasmic surface of the
membra ne.
Keywords: fumarate respiration; Wolinella succinogenes;
proteoliposomes; H

+
/ e ratio; hydrogenase.
The electron transport chain catalyzing fumarate respiration
with H
2
(reaction a) or formate (reaction b) in Wolinella
succinogenes consists of fumarate reductase, menaquinone
(MK), and either hydrogenase or formate dehydrogenase
(Fig. 1 ).
H
2
þ Fumarate ! Succinate ðaÞ
HCO
À
2
þ Fumarate þ H
2
O ! HCO
À
3
þ Succinate ðbÞ
The enzymes were isolated and the corresponding genes
were sequenced [2,3]. Each of the t hree enzymes consists of
two hydrophilic subunits and a di-heme cytochrome b
which is integrated in the membrane [4–7]. The i ron–sulfur
subunits (HydA, FdhB, FrdB) mediate electron transfer
from the catalytic subunits to the cytochromes b or vice
versa [8]. The di-heme cytochromes b of hydrogenase and of
formate dehydrogenase carry the sites of MK reduction,
and are similar in their sequences [6,9,10]. Menaquinol

(MKH
2
) is oxidized a t the di-heme cytochrome b of
fumarate reductase [4,5].
The dehydrogenases (hydrogenase and formate dehy-
drogenase) catalyze the reduction of the water soluble
MK analogue 2,3-dimethyl-1,4-naphthoquinone (DMN)
by their respective substrates (reaction c and d). The site
of DMN reduction is located on HydC [6]. Fumarate
reductase catalyzes DMNH
2
oxidation by fumarate
(reaction e). The site of DMNH
2
oxidation is located
on FrdC [4].
H
2
þ DMN ! DMNH
2
ðcÞ
Correspondence to A. Kro
¨
ger, Institut fu
¨
r Mikrobiologie, Johann
Wolfgang Goethe-Universita
¨
t, Marie- Curie-Str. 9, D-60439 Frankfurt
am Main, Germany.

Fax: + 49 69 79829527, Tel.: + 4 9 6 9 79829507,
E-mail:
Abbreviations: DMN, 2,3-dimethyl-1,4-naphthoquinone; DMNH
2
,
hydroquinone of DMN; FCCP, carbonyl cyanide p-tri-
fluoromethoxyphenylhydrazone; FdhA/B/C, formate dehydrogenase;
FrdA/B/C, fumarate reductase; HQNO, 2-(n-heptyl)-4-hydroxyquin-
oline-N-oxide; HydA/B/C, hydrogenase A/B/C of W. succinogenes;
MK, menaquinone; MKH
2
, hydroquinone of MK; methyl-MK, 5- or
8-methyl-MK; TAME, N-a-tosyl-
L
-arginyl-O-methylester; TPP
+
,
tetraphenylphosphonium; TPB
–
, tetraphenylboranate; Dp, electro-
chemical proton potential (proton motive force) across a membr ane
(in volts); Dw, e lectrical proton potential across a membrane (in volts).
(Received 6 December 2001, r evised 12 February 2002, accepted 21
February 20 02)
Eur. J. Biochem. 269, 1974–1983 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02842.x
HCO
À
2
þ DMN þ H
þ

! CO
2
þ DMNH
2
ðdÞ
DMNH
2
þ Fumarate ! DMN þ Succinate ðeÞ
The substrate sites of hydrogenase and of formate
dehydrogenase are exposed to the bacterial periplasm,
whereas that of fumarate reductase faces the cytoplasm
(Fig. 1) [1,11]. From the crystal structure of fumarate
reductase it is obvious that the protons consumed by
fumarate reduction at the catalytic site of FrdA are taken
up from the cytoplasmic side of the membrane [8]. The
protons liberated by the oxidation of H
2
or formate a t the
catalytic sites of the enzymes on HydB or FdhA are
probably released on the periplasmic s ide of t he membrane.
This is suggested by the crystal structures of related
enzymes. The periplasmic Nickel hydrogenases isolated
from sulfate reducing bacteria consist of two subunits
which are similar to HydA and HydB of W. succinogenes
hydrogenase. As suggested by the structures of those
enzymes, H
2
is split into protons and electrons at the a ctive
site [12,13]. The protons are released at the surface of the
catalytic subunit. The electrons are passed by three

consecutive iron–sulfur centers to a cytochrome c which
binds to the surface of the iron–sulfur subunit. The
corresponding electron acceptor in the case of W. succin-
ogenes hydrogenase is the di-heme cytochrome b HydC
which is a subunit of the enzyme.
Escherichia coli formate dehydrogenase-N consists of
three different subunits whose sequences resemble those o f
W. s uccinogenes formate dehydrogenase [14]. A s seen from
its crystal structure, the subunits of the E. coli enzyme are
arranged as depict ed in Fig. 1 [ 14]. A cavity in the catalytic
subunit of E. coli formate dehydrogenase-N extends from
the surface to the molybdenum ion where formate is
oxidized. The electrons derived from formate are likely to be
passed to the iron–sulfur center close to the molybdenum.
The products, CO
2
and proton s, are probably released
through t he cavity. A similar mechanism is likely to apply
for W. succinogenes formate dehydrogenase. The C-termi-
nus of the iron–sulfur subunit (FdnH) of E. coli formate
dehydrogenase-N forms a membrane-spanning helix [14].
This applies also t o HydA of W. succinogenes [1]. The helix
is predicted to be absent in FdhB [7].
Cells of W. succinogenes ca talyzing fumarate respiration
with H
2
(reaction a) or formate (re action b) were found to
develop a Dw of 0.14 or 0.16 V (negative inside) [15,16]. The
corresponding DpH across the membrane was found to be
negligible. A similar Dw was generated in cells by H

2
or
formate oxidation with DMN (reaction c or d) [16]. In
contrast, DMNH
2
oxidation by fumarate (reaction e) was
not coupled to Dw generation. Inverted vesicles of the
W. su ccinogenes membrane catalyzed fumarate respiration
with H
2
, which generated a Dw ¼ 0.18 V (positive inside)
[15]. The corresponding H
+
/e ratio was close to 1. The
reduction of DMN by H
2
catalyzed by these vesicles
generated a much lower Dw,andtheH
+
/e ratio was below
0.5.
Proteoliposomes containing fumarate reductase, vita-
min K
1
, and either formate dehydrogenase or hydro-
genase were found to catalyze fumarate reduction by
formate or H
2
at the expected specific activities [17–19].
The two reactions were not coupled to Dpgenerationor

the Dp generated was very low [19]. In this paper, we
address the following questions: (a) can coupled fumarate
respiration be restored by incorporating the isolated
enzymes into liposomes containing menaquinone; (b) is
the Dp generated by menaquinone reduction with H
2
or
formate, by menaquinol oxidation with fumarate, or by
both reactions; and (c) what is the mechanism of Dp
generation.
EXPERIMENTAL PROCEDURES
Preparation of proteoliposomes
Phosphatidylcholine w as prepared from egg yolk a ccording
to Singleton et al. leaving out the chromatographic steps
[20]. Di-palmitoyl phosphatidate was purchased from
Fluka. MK w as extracted f rom the membrane fraction of
W. su ccinogenes and separated from methyl-MK by H PLC
[21]. MK and methyl-MK o f W. succinogenes carry a side
chain with six isoprene units. Phosphatidylcholine (50 mg)
and phosphatidate (5 mg) were dissolved in a mixture of
CHCl
3
and methanol ( 2 : 1, v/v). A fter the a ddition of MK
(10 lmolÆg phospholipid
)1
), the solvents were evaporated,
and the residue was sonicated at 0 °Cin50m
M
Hepes
(adjusted t o pH 7.5 with KOH) until minimum t urbidity of

the s uspension. The r esulting suspension of sonic liposomes
contained 10 g phospholipidÆL
)1
.
Proteoliposomes containing hydrogenase and fumarate
reductase were prepared according to a procedure previously
described [22]. Dodecyl- b-
D
-maltoside (0.8 gÆg phospho-
lipid
)1
) was added to a suspension of sonic liposomes
containing MK (1 g phospholipidÆL
)1
in 50 m
M
Hepes at
pH 7.5), and the mixture was stirred for at least 3 h at
room temperature. After the addition of hydrogenase
(20 mgÆg phospholipid
)1
) prepared a ccording to [ 6] and/or
fumarate reductase [18] (0.18 g Æg phospholipid
)1
), stirring
was continued for 1 h. For removal of detergent, B io-Beads
SM-2 ( Bio-Rad) (0.24 gÆmL
)1
) w ere added a nd stirring was
continued for 1 h.

Fig. 1. Composition and orientation of the enzymes involved in fumarate
respiration o f W. succinogenes. Fuma rate reductase (FrdA, B, C) and
formate d ehydrogenase (FdhA, B , C) are i ntegrated in t he membrane
by th eir di-heme cytochrome b subunits (FrdC and F dhC). Hydro-
genase (HydA, B, C) is integrated in the membrane by its di-heme
cytochrome b sub unit (HydC) and the C -terminal hydrop hobic stretch
of HydA [1]. HydC and FdhC carry the sites of MK reduction. MKH
2
is oxidized at FrdC. Ni, catalytic site of h ydrogenase; M o, molybde-
num ion coordinated by molybd opterin guanine dinu cleotide; Fe/S,
iron–sulfur centers; Cyt. b,di-hemecytochromeb.
Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1975
Proteoliposomes containing formate dehydrogenase and
fumarate reductase were prepared using sonic liposomes
with MK (10 g phospholipidÆL
)1
) in a buffer (adjusted to
pH 7.3 with KOH) containing 95 m
M
Hepes, 2 m
M
malonate, and 1 m
M
azide. After the addition of formate
dehydrogenase [18] (40 mgÆg p hospholipid
)1
)andfuma-
rate reductase (0.16 g Æg phospholipid
)1
), the mixture was

frozen in liquid N
2
and then thawed a t room temperature.
Freeze-thawing was repeated twice. The detergent intro-
duced with the enzyme preparations was removed by
stirring the m ixture for 1 h with Bio-Beads SM-2
(0.5 g ÆmL
)1
). The suspension was sonicated (Branson
sonifier equipped with a microtip) for 20 s at 0 °Cbefore
use.
Enzymic activities of proteoliposomes and protein
The reduction of fumarate by H
2
or formate was
recorded as the absorbance difference at 270 minus
290 nm (De ¼ 0.4 5 m
M
)1
Æcm
)1
) in a buffer (50 m
M
Hepes, pH 7.5, 37 °C) containing 2 m
M
fumarate and
flushed with H
2
[or N
2

when formate (10 m
M
)wasused
as electron donor]. DMN (0.2 m
M
) reduction by H
2
or
formate (10 m
M
)aswellasDMNH
2
(0.2 m
M
) oxidation
by fumarate (1 m
M
) was recorded in the same buffer
using the same wavelength pair (De ¼ 15.2 m
M
)1
Æcm
)1
).
Methyl viologen reduction by H
2
was recorded at
578 nm (e ¼ 9.8 m
M
)1

Æcm
)1
)inaH
2
-saturated buffer
(0.15
M
glycine, pH 9.5, 37 °C). The unit of activity (U)
corresponds to the transfer of 2 lmol electronsÆmin
)1
.
Protein was determined using the Biuret method with
KCN [23].
Determination of Dw
The TPP
+
electrode was constructed according to [24].
Proteoliposomes were suspended (0.4 g phospholipidÆL
)1
)
in 50 m
M
Hepes buffer (pH 7.5, 25 °C) which was flushed
with H
2
(or N
2
when formate w as used). The T PP
+
electrode

was calibrated by adding known a mounts of TPP
+
before
the electron transport was started by the addition of the
substrates . Dw wa s calculated from t he TPP
+
concentrations
within the proteoliposomes ( T
i
) and in the medium (T
e
)using
the Nernst equation. T
i
was calculated from the maximum
amount of TPP
+
(T
s
,inmolÆg phospholipid
)1
)takenup
from the medium i n the steady state o f electron t ransport
according t o E qn (1) [ 16,25].
ðT
i
Þ
n þ 1
¼ T
e

þ
T
s
À V
i
ðT
i
Þ
n
K
ln
ðT
i
Þ
n
T
e
ð1Þ
V
i
(3.5 mL Æg phospholipid
)1
) represents the average inter-
nal volume of the proteoliposomes which was obtained
from the amount of phosphate retained by proteoliposomes
prepared in the presence of 50 m
M
phosphate, after gel
filtration using a Sephacryl S-1000 SF (Pharmacia) c olumn.
The binding constant K (53 mLÆg phospholipid

)1
)was
calculated from the amount of TPP
+
absorbed by the
proteoliposomal membrane at various concentrations of
TPP
+
.TheinternalTPP
+
concentration (T
i
)
n+1
was
calculated from an assumed value of (T
i
)
n
(Eqn 1). Using
the value so obtained, calculation was repeated until (T
i
)
n+1
was consistent with (T
i
)
n
.
Measurement of H

+
/e ratios
Proteoliposomes containing hydrogenase, MK, and fuma-
rate reductase were prepared as described a bove, however,
the preparation buffer contained 95 m
M
Hepes (adjusted to
pH 7.3 by KOH). The suspension was dialyzed for 14 h
against buffer C (50 l
M
Hepes, 45 m
M
KCl and 50 m
M
sucrose, pH 7.3, 0 °C), flushed with H
2
. After valinomycin
(0.5 lmolÆg phospholipid
)1
) and phenol red (60 l
M
)had
been added, the suspension (1 g phospholipidÆL
)1
)was
mixed with fumarate (5 m
M
) or DMN (50–100 l
M
)in

buffer C at 25 °C. A stop-flow spectrophotometer was used
for mixing [26]. Ten volumes o f the suspension were mixed
with one volume of sub strate. The amount of protons
released was calculated from the absorbance change of
phenol red at 550 nm.
For buffer exchange, proteoliposomes containing for-
mate dehydrogenase and fumarate reductase were subjected
to gel fi ltration using a Sephadex G -25 column (Pharmacia)
equilibrated with N
2
-flushed buffer C at room temperature.
After the addition of valinomycin and phenol red (see
above), the suspension was mixed with buffer C containing
either formate (100 m
M
) and DMN (50–100 l
M
)orformate
(100 m
M
)at25°C.
Phenol red absorbance at 550 nm was calibrated using
tryptic hydrolysis of N-a-tosyl-
L
-arginyl-O-methylester
(TAME) according to [27]. In this reaction, one proton
is released per mol of substrate. The proteoliposomal
suspension containing 5 l
M
trypsin was mixed with

TAME (9.1 or 18.2 l
M
final concentration) in buffer C
at 25 °C.
Construction of
W. succinogenes hydC
mutants
The hydC mutants of W. succinogenes were constructed
by transforming the deletion mutant DhydABC with
derivatives of pHydcat [1]. Plasmid pHydcat contains the
entire hydABC operon and integrates into the genome of
W. succinogenes by homologous recombination. Deriva-
tives of pHydcat were synthesized using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene, Heidelberg,
Germany) with the plasmid as template and specifi-
cally synthesized oligonucleotides carrying the desired
nucleotide mismatches. A pair of co mplementary prim-
ers was used for each modification (forward primer
used for mutant N128D: 5¢-(3642)–CTCAAAGGGGTT
TAC
GATCCCGTTCAGCTAGC-3¢, and for mutant
Q131L: 5¢-(3649)–GGGTTTACAATCCCGTT
CTCCTA
GCAGCCTATATGGG-3¢). Altered nucleotides are
printed in bold, and the corresponding codons are
underlined. The numbers in parentheses denote the
nucleotide positions [6]. Modified pHydcat plasmids were
isolated using Qiagen tips (Qiagen, Hilden, Germany)
and sequenced to confirm the mutations. Nitrate-grown
cells of W. succinogenes DhydABC were used for trans-

formation as described [28,29]. Transformants were
selected on plates with a medium containing formate
and nitrate as energy substrates, kanamycin (25 m gÆL
)1
),
and chlorampenicol (12.5 mgÆL
)1
). The integration of the
plasmids into the genome of W. succinogenes DhydABC
was confirmed by Southern blot analysis as described
previously [1].
1976 S. Biel et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Preparation and characterization of proteoliposomes
Rigaud and coworkers prepared proteoliposomes by incor-
porating bacteriorhodopsin into liposomes treated with
dodecylmaltoside and subsequent removal of the detergent
with Bio-Beads [ 22]. The liposomes were shown t o be stable
below a critical detergent/phospholipid ratio and to lyse at
higher ratios. The maximum Dp generated by light was
measured with proteoliposomes prepared at the critical
ratio.
In the experiment shown in Fig. 2, fumarate reductase
and hydrogenase isolated from W. succinogenes were
incorporated into sonic liposomes containing MK accord-
ing to the method described above. The detergent/phos-
pholipid ratio was varied, and the activity of electron
transport from H
2
to fumarate was measured in the various

preparations (Fig. 2A). The activity increased w ith i ncreas-
ing amounts of detergent until a maximum was reached at
the critical ratio of 0.8 g dodecylmaltoside per g phosphol-
ipid. At higher ratios the activity was lower. At subcritical
ratios, the electron transport activity was lower than
predicted (V
ET
) from the activities of hydrogenase
(H
2
fi DMN) and fumarate reductase (DMNH
2
fi
Fumarate), suggesting that only some of the enzyme
molecules were involved in electron transport (H
2
fi
Fumarate). The activities measured with proteoliposomes
prepared at the critical o r a higher ratio were close to the
theoretical ones.
The activity of hydrogenase measured with DMN as
acceptor w as ne arly the same i n t he different preparations
(Fig. 2 A). In contrast, the activity of fumarate reductase
(DMNH
2
fi Fumarate) was fairly constant up to the
critical ratio and decreased to approximately 70% and 60%
at the two highest dodecylmaltoside/phospholipid ratios.
The a ctivity reflected t he accessibility o f fumarate r eductase
in the preparations to external fumarate. This view was

confirmed by measuring the activity of fumarate reduction
with methyl viologen radical before and after lysis of the
proteoliposomes by the addition of Triton X-100 [11,19,30]
(not shown). There was no stimulation by Trito n X-100 in
the preparations obtained at the critical or lower ratios,
indicating that all the fumarate reductase molecules were
accessible to fumarate. The stimulation observed with
proteoliposomes prepared at the t wo highest ratios indica-
ted that 30–40% of the fumarate reductase molecules were
oriented towards the inside.
The orientation of the hydrogenase molecules in the
preparations is probably similar to that of fumarate
reductase. This is deduced from the activity of methyl
viologen reduction by H
2
in the different preparations
(Fig. 2 A). Methyl viologen does not penetrate the mem-
brane at a velocity commensurate w ith that of its reduction,
in contrast to H
2
and DMN [11,31]. The activity of methyl
viologen reduction by H
2
was the same in the preparations
obtained at the critical or lower ratios, and was 70% and
65% of this activity in the p roteoliposomes prepared at the
two highest ratios. This s uggests that hydrogenase is
completely exposed to the outside in the proteoliposomes
prepared at the c ritical or l ower ratios, w hereas 30–35% o f
the hydrogenase molecules are oriented to the inside of

proteoliposomes obtained at the highest ratios. It was not
possible to confirm the orientation of hydrogenase by
measuring its activity in the presence of Triton X-100, as the
turnover number of hydrogenase per se is inhibited upon the
addition of detergents.
The amount of TPP
+
taken up from the external medium
upon initiation of the electron transport from H
2
to
fumarate was highest with proteoliposomes prepared at
the critical r atio and was lower with t he other preparations
(Fig. 2 B). A similar result was obtained for DMN reduction
by H
2
.ThusTPP
+
uptake appears to be most efficiently
Fig. 2. Properties of proteoliposomal preparations obtained at various
dodecylmaltoside/phospholipid ratios. The various preparations were
obtained according to the method described for proteoliposomes
containing hydrogenase, MK, and fumarate reductase (see Experi-
mental procedures). However, the amount of dodecylmaltoside
applied was varied. The values of theoretical electron transport activity
(V
ET
) were calculated from those of DMN reduction by H
2
(H

2
fi DMN, V
Hyd
) and of fumarate reduction by DMNH
2
(DMNH
2
fi Fumarate, V
Frd
) according to: V
ET
¼ V
Hyd
ÆV
Frd
/
(V
Hyd
+ V
Frd
) [17]. TPP
+
uptake during electron transport from
H
2
to DMN o r fumarate was measured as show n in Fig. 3. T
s
repre-
sents the maximum amount of TPP
+

taken up by the proteoliposomes
in the steady state of electron transport (see Table 1), and T
e
the
corresponding TPP
+
concentration i n the medium.
Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1977
coupled to the r eduction of fumarate or DMN in proteo-
liposomes prepared at the critical detergent/phospholipid
ratio. All the enzyme molecules appear to participate in
electron transport, and all the enzyme molecules are
apparently oriented towards the outside in these proteo-
liposomes. The less efficient TPP
+
uptake by proteolipo-
somes prepared with amounts of detergent above the critical
ratio can be explained by the orientation of part of the
hydrogenase molecules towards th e i nside (see D iscussion).
In the following only proteoliposomes prepared at the
critical ratio are used, unless indicated otherwise.
Gel fi ltration with Sephacryl S-1000 SF indicated that all
the fumarate reductase (1.3 lmolÆg phospholipid
)1
)and
hydrogenase (0.16 lmolÆg phospholipid
)1
) used for prepa-
ration was incorporated into the proteoliposomes [30] (data
not shown). The molar ratio of the two enzymes was close

to that of the bacterial membrane. The enzyme contents
based on phospholipid were approximately six times those
in the bacterial membrane. The turnover numbers of the
enzymes in electron transport from H
2
to fumarate were
about 10% of those in growing bacteria.
Assuming that the proteoliposomes are spherical, their
average internal volume (3.5 mL Æg phospholipid
)1
) would
correspond to average values of the internal and external
diameter of 81 nm and 95 nm, respectively. In electron
micrographs after negative staining the proteoliposomes
appeared as mono-layered vesicles, most of which had
external diameters b etween 50 nm and 7 0 nm (not shown ).
The external surface of the vesicles was studded with
particles which probably represent fumarate reductase a nd
hydrogenase molecules [30]. Assuming that 1 g phospho-
lipid corresponds to an outer membrane surface of
2.6 · 10
6
cm
2
[32], a spherical proteoliposome of 100 nm
(or 50 nm) external diameter is calculated to carry 94 (or 23)
molecules o f fumarate reductase (monomeric) a nd 12 (or 3)
molecules of hydrogenase. As all the active enzyme mole-
cules a ppear to participate i n e lectron transport f rom H
2

to
fumarate (Fig. 2A), they are likely to be randomly distrib-
uted among the proteoliposomes.
Determination of Dw
A suspension of proteoliposomes (0.4 g phospholipidÆL
)1
)
containing MK, hydrogenase and fumarate reductase was
stirred under an atmosphere of H
2
(Fig. 3 ). After the
addition of TPP
+
, its concentration was rec orded using a
TPP
+
electrode. Upon initiation of electron transport by
fumarate addition, most of the external TPP
+
was taken up
by the proteoliposomes, and was released into the medium
again after consumption of fumarate. The cycle could be
repeated by a second addition of fumarate. TPP
+
uptake
was abolished by t he presence of a p rotonophore (FCCP).
The experiment suggests that the electron transport from H
2
to fumarate creates a Dw (negative inside) across the
proteoliposomal membrane which causes accumulation of

TPP
+
within the proteoliposomes.
Determination of the Dw required that the internal
concentration of TPP
+
(T
i
) was calc ulated from t he
maximal amount of TPP
+
taken u p i n t he steady state of
electron transport ( T
s
). T
i
was calculated according to the
method designed by Zaritsky et al. (Eqn 1) [25]. The value
of T
i
so obtained corresponded to 33% of the amount of
TPP
+
(T
s
) taken up during fumarate respiration in the
experiment shown in Fig. 3 . The residual part of T
s
is
thought to be bound to the proteoliposomal membrane. Dw

was calculated from T
i
and the corresponding external
TPP
+
concentration (T
e
) according t o the Nernst equation.
The Dw generated by fumarate respiration with H
2
in the
experiment shown in Fig. 3 was determined to be 0.19 V
(Table 1). A Dw of the same direction and strength was
generatedbyDMNreductionwithH
2
. In contrast, no
TPP
+
uptake was observed during f umarate r eduction by
DMNH
2
. This reaction also did not cause the uptake of
tetraphenylboranate (TPB
–
) in a similar experiment per-
formed w ith a T PB
–
electrode [16] (not shown). Proteolipo-
somes containing MK and only hydrogenase catalyzed
DMN reduction by H

2
which generated a Dw with a similar
value as measured in proteoliposomes containing both
enzymes (not shown).
Fumarate reduction by formate did not generate a Dw
in proteoliposomes prepared according to the method
described above with formate dehydrogenase instead of
hydrogenase. However, a Dw ¼ 0 .13 V (negative inside)
was found to be generated by the electron transport from
formate to fumarate using proteoliposomes prepared
according to the alternative method described in the
Experimental procedures (Table 1). The same Dw was
generatedbyDMNreductionwithformate.
Determination of H
+
/e ratios
H
+
/e ratios were measured with proteoliposomes using an
external pH indicator (phenol red) and a stop-flow
spectrophotometer [26]. P roteoliposomes containing hydro-
genase, MK, and fumarate reductase suspended in a buffer
(50 l
M
Hepes and 45 m
M
KCl) saturated with H
2
were
treated with valinomycin (0.5 lmol g

)1
phospholipid). The
amount of valinomycin was just s ufficient to prevent TPP
+
uptake driven b y the reduction of DMN or fumarate w ith
H
2
. After the addition of phenol red, DMN reduction by H
2
Fig. 3. Recording of the external TPP
+
concentration in a suspension of
proteoliposomes during fumarate reduction by H
2
. Proteolipos omes
containing hydrogenase, MK, and fumarate reductase were suspended
(0.4 g phospho lipid ÆL
)1
)inanH
2
-saturated buffer (pH 7.5 , 25 °C).
The TPP
+
electrode was calibrated by three addition s of 1 l
M
TPP
+
.
The electron transport was started by adding fumarate. 20 lmol
FCCP per g phospholipid was applied w ere indicated.

1978 S. Biel et al. (Eur. J. Biochem. 269) Ó FEBS 2002
was started by the addition of a small amount of DMN
(Fig. 4 A). The number of p rotons released in to the external
medium within 1 s was proportional to the ad ded amount
of DM N. T he H
+
/e ratio was calculated from the p rotons
released and the amount of DM N added. The time course
of proton release was consistent with that of DMN
reduction by H
2
.TheK
m
for DMN of this reaction was
determined to be 15 l
M
(not shown). The release of protons
did not occur with proteoliposomes which had been treated
with a protonophore (FCCP, curve III).
The H
+
/e ratioof f umarate r eduction by H
2
was m easured
with proteoliposomes treated in the same way as described
above. The reaction was started by the addition of fumarate
instead of DMN, and the consumption of fumarate was
recorded in a se parate experiment in the absenc e of ph enol
red (Fig. 4B, curve VI). The H
+

/e ratio was determined from
the ratio of the velocities of acidification (curve IV) and of
fumarate reduction. The velocity of proton release was
approximately twice that of fumarate reduction in the first
second after fumarate addition and became slower at longer
reaction times. Proton release did not occur with proteo-
liposomes treated w ith a protonop hore (curve V).
The H
+
/e ratio of DMN reduction by formate was
measured in the same way as in the experiment shown in
Fig. 4A (Table 2). Proteoliposomes containing formate
dehydrogenase instead of hydrogenase in buffer flushed
with N
2
, were allowed to react with a solution containing
DMN and formate. The H
+
/e ratio of fumarate reduction
by formate could not be measured. When formate was
added before the proteoliposomes were mixed with fuma-
rate, a drastic inhibition of formate dehydrogenase was
observed. W hen fumarate was a dded before the suspension
was mixed with formate, the reduction of the MK present in
the proteoliposomes interfered with the measurement of
fumarate reduction. Therefore, the velocity of MK reduc-
tion was recorded at 270 nm upon mixing of the proteo-
liposomes with formate. The H
+
/e ratio of MK reduction

by formate was calculated form the velocities of MK
reduction and of acidification measured with phenol red in a
second experiment. As seen from Table 2, the average H
+
/e
ratios with H
2
or formate obtained from various experi-
ments were close to 1.
HydC mutants
To understand the mechanism of the Dpgenerationwhichis
coupled to quinone reduction by H
2
orformate,thesiteof
quinone reduction on HydC or FdhC of W. succinogenes
should be elucidated. The sequences of these di-heme
cytochromes b are similar to that of the di-heme
Fig. 4. Proton release coupled to the reduction of DMN (A) and of
fumarate ( B) by H
2
. Proteoliposomes containing hydrogenase, MK,
and f umarate reductase in the H
2
-saturated suspension des ignated in
the Experimental procedures were mixed with a solution of DMN (A)
or fumarate (B), and the absorbance of phenol red was recorded
(experiments I–V). Phenol red absorbance was calibrated as described
in t he Experimental p roce dures. Proteoliposomes t re ated with FCCP
(20 lmolÆg p hosp holip id
)1

)wereusedinexperimentsIIIandV.The
concentration o f D MN in the reaction mixture at reaction time zero
was 5.2 l
M
(I) and 9.2 l
M
(II and III). Fumarate reduction by H
2
was
recorded at 270 nm (e ¼ 0.55 m
M
)1
Æcm
)1
) i n the abse nce of phe nol
red (VI). The slopes of curves IV and VI were used for calculating the
H
+
/e ratio o f fumarate reduction b y H
2
.
Table 1. TPP
+
accumulation by proteoliposomes in the steady state of electron transport. Proteoliposomes containing hydrogenase (formate
dehydrogenase) and fumarate reductase were used with H
2
or DMNH
2
(formate) as electron donor. The experime nts were performed as described
in Fig. 3. However, the suspension w as flushed with N

2
instead of H
2
when DMNH
2
(1 m
M
) or formate (1 m
M
) were used a s donor. DMN was
applied at 1 m
M
concentrations. T
s
represents t he m axim um am ount of TPP
+
taken up by t he p roteoliposomes in the steady state o f electron
transport, and T
e
the corresponding TPP
+
concentration in t he med ium (see F ig. 3). The internal TPP
+
concentration ( T
i
) was ca lcul ated
according to E qn (1). Dw was c alculated from T
e
and T
i

according t o the Nernst equation.
Donor Acceptor
Activity
(UÆmg phospholipid
)1
)
T
s
(lmolÆg phospholipid
)1
)
T
i
(l
M
)
T
e
(l
M
)
Dw
(V)
H
2
Fumarate 2.6 7.1 666 0.39 )0.19
H
2
DMN 6.5 4.8 456 0.25 )0.19
DMNH

2
Fumarate 4.1 No TPP
+
uptake
Formate Fumarate 1.4 3.4 240 1.7 )0.13
Formate DMN 3.6 2.4 171 1.2 )0.13
Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1979
cytochrome b subunit (FdnI) of E. coli formate dehydro-
genase-N [9]. HydC is schematically drawn i n Fig. 5 based
on the structure of FdnI [14]. T he two heme groups of FdnI
are i n e lectron transfer distance nearly on t op of each other
when viewed along the membrane normal. The proximal
(upper) heme group is i n electron transfer distance to one o f
the iron–sulfur centers of the iron–sulfur subunit FdnH (not
shown). The site of quinone reduction is thought to be
occupied by a molecule of HQNO which is located on the
cytoplasmic side of the distal heme group. HQNO is in close
proximity to the axial heme ligand on helix IV and to an
asparagine (N110) and a glutamine residue (Q113) within
the hydrophilic stretch c onnecting helices II and I II. These
three residues are conserved in HydC (H200, N128, and
Q131 in Fig. 5) and in FdhC of the W. succinogenes
enzymes [9].
Mutants were constructed in which N128 or Q131 of
HydC was replaced by aspartate o r l eucine (Table 3). The
two mutants (N128D and Q131L) did not grow by fumarate
respiration with H
2
. When grown with formate and
fumarate, the mutant cells did not catalyze fumarate

reduction by H
2
, in contrast to the wild-type strain. The
specific activities o f DMN reduction by H
2
measured with
the membrane fractio n of the mutan ts amounted to 6% or
less of the wild-type a ctivity, whereas the activities of benzyl
viologen reduction by H
2
were close t o that of the wild-type
strain. The deficiency in quinone reduction was not due to
any loss of the heme groups from HydC. The amount of
heme B which was r educed upo n H
2
addition to the Triton
X-100 extract of the oxidized membrane fraction was the
same with the m utants (0.3 lmolÆgprotein
)1
)andwiththe
wild-type strain [29]. Mutant H122A had wild-type pro-
perties with r espect to growth and enzyme activities.
Residue H 122 is also located in the stretch connecting helix
II and III of HydC, but is not conserved in FdnI and FdhC.
The results suggest that the quinone reactivity of HydC is
specifically affected in mutants N128D and Q131L, in
agreement with the view that the site of quinone reduction is
located close to the cytoplasmic surface of the membrane.
DISCUSSION
Energetics

For technical reasons, the H
+
/e ratio of apparent proton
translocation can only be measured at vanishing Dp.Itis
generally thought that the same ratio is valid in the presence
and a bsence of Dp. As the am ount of free energy conserved
by apparent proton translocation across the membrane
cannot exceed that provided by the driving redox reac tion,
the H
+
/e ratio (n
H
+
/n
e
) can be calculated from the redox
potential difference (DE)andDp according to Eqn (2),
provided that the energetic efficiency (q) of the process is
known.
n
H
þ
n
e
¼ q
DE
Dp
ð2Þ
Assuming q ¼ 1, the t heoretical maximum H
+

/e ratio of
fumarate respiration with H
2
(reaction a) is calculated to
be 2.6, using Dp ¼ 0.17 V, and DE ¼ 0.45 V [from E
o
¢ for
H
+
/H
2
()0.42 V) and for fumarate/succinate (+0.03 V
[33])]. If the actual H
+
/e ratio was 1 or 2, the energetic
efficiency of fumarate respiration would be 0.38 or 0.76.
Nearly the same numbers apply for fumarate respiration
with fo rma te a nd HCO
3
–
as its oxidation product ( reaction
b), as the corresponding value of DE
o
¢ is close to that
obtained with H
2
.
Table 2 . H
+
/e ratios measured with p roteoliposomes. The proteolipo-

somal preparation (A) contained MK, hydrogenase, and fumarate
reductase. In preparation (B) hydrogen ase w as replaced by formate
dehydrogenase. The upper two experiments were performed as
described in Fig. 4A,B. The H
+
/e ratio with f ormate and DM N was
measured as sh own in Fig. 4A. However, the p roteoliposo mes were
suspended in a buffer fl ushe d with N
2
instead o f H
2
, a nd t he s uspen-
sion was mixed with a s olution containing formate and DMN. In the
experiment with DMNH
2
and fumarate, the anoxic proteoliposomal
suspension containing DMNH
2
(0.2 m
M
) was m ixed with fumarate
(5 m
M
), and the absorban ce of phenol red was recorded. The reduction
by formate of the MK present in the proteoliposomes was observed at
270 nm w hen t he prote oliposomes we re m ixed w ith formate in the
absence of fumarate and phenol red. The corresponding H
+
/e ratio
was c alculated using the velocity of MK red uction (De ¼ 12.0 m

M
)1
cm
)1
). n represents th e number of m easurem ents with different pre-
parations of p roteoliposomes.
Preparation Donor Acceptor H
+
/e ratio n
AH
2
Fumarate 1.0 ± 0.1 6
AH
2
DMN 0.96 ± 0.04 10
ADMNH
2
Fumarate 0.0 4
B Formate DMN 0.98 ± 0.12 9
B Formate MK 0.95 ± 0.12 4
Fig. 5. Hypothetical arrangement of the four predicted membrane-
spanning helices of W. succinogenes HydC. The scheme i s base d on
the crystal structure of E. coli FdnI [14]. The shaded squares represent
the heme groups. A molecule of H QNO is s hown at t he site of M K
reduction which is confined by the axial ligand H200 of the distal heme
group and by re sid ues N128 and Q 131 i n t he stretch c onnectin g the
hydrophobic parts of helices II and III.
1980 S. Biel et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The theoretical maximum H
+

/e ratio of fumarate
reduction by the M KH
2
within the bacterial me mbrane is
calculated to be 0.6, assuming the redox potential of the
MK/MKH
2
couple to be equal to its standard potential in
organic solution (E
o
¢ ¼ )0.074 V [34]). This assumption is
consistent with the finding that the MK/MKH
2
ratio i s not
far f rom 1 in the membrane of W. succinogenes catalyzing
fumarate respiration [35]. Furthermore, the standard poten-
tial of MK/MKH
2
in a bacterial membrane was measured
to be close to that in organic solution [36]. T herefore, the
actual H
+
/e ratio of MKH
2
oxidation by fumarate should
be lower than 0.6.
The site of MKH
2
oxidation
The site of MKH

2
oxidation on the cytochrome b subunit
(FrdC) of fumarate reductase is not kn own. In the
crystallographic model of the oxidized enzyme, a cavity
was discovered which extends from the hydrophobic phase
of the m embrane, close to t he distal heme group of Fr dC to
the periplasmic aqueous phase [37]. The cavity could
accommodate a MKH
2
molecule after minor structural
alterations. A glutamate residue (E66) lines the cavity and is
a possible acceptor of a hydrogen bond from one of the
hydroxyl groups of MKH
2
. Replacement of E66 by a
glutamine residue resulted in a mutant (E66Q) which did
not catalyze DMNH
2
oxidation by fumarate. In contrast,
the activity of fumarate reduction by benzyl viologen radical
as well as the crystal structure of the enzyme and the
midpoint potentials of the heme groups were not affected by
the mutation. These results suggest that the inhibition of
quinol oxidation activity in the m utant enzyme is due to the
absence of the carboxylate g roup of E66. In the wild-type
enzyme, E66 could f acilitate quinol oxidation by a ccepting
one of the protons liberated by quinol oxidation which
could then be released on the periplasmic side of the
membrane via the cavity. As a consequence, quinol
oxidation by fumarate should be electrogenic, and the

H
+
/e ratio of t he reaction is predicted t o be 1 . T his value is
higher than the maximum possible value predicted by the
energetic calculation (see above). Furthermore, fumarate
reduction by D MNH
2
was not coupled to Dw generation in
cells, inverted vesicles [16], o r in proteoliposomes ( Table 1).
Mechanism of Dp generation
In the model m echanisms d rawn in Fig. 6, H
2
oxidation b y
MK is assumed t o be electrogenic with a H
+
/e ratio of 1.
The protons consumed in MK reduction are taken up from
the inside of the proteoliposomes, and simultaneously
protons are released by H
2
oxidation on the outside
(Fig. 6 A,B). MKH
2
oxidation by fumarate is depicted as
an electroneutral process in Fig. 6A, w hereas it is electro-
Table 3. Growth and enzymic activities of hydC mutants of W. succinogenes. The doubling times of growth with H
2
and fumarate, and the enzymic
activities were measured in cells (H
2

fi Fumarat e) or with the memb rane fraction of cells g rown with formate a nd fumarate as d escribed [29]. The
properties of mutant H 122A were taken f rom [29].
Strain Doubling time (h)
UÆmg cell protein
)1
H
2
fi Fumarate H
2
fi DMN H
2
fi Benzyl viologen
Wild-type 1.8 3.5 4.5 2.1
N128D / 6 0.05 0.25 2.5
Q131L / 6 0.05 0.08 2.0
H122A 1.8 3.6 4.5 2.3
Fig. 6. Hypothetical mechanisms of Dp generation in proteoliposomes
(AandB)incellsofW. su ccinogenes (C). The sites o f MK reduction
and of MKH
2
oxidation a re drawn s chematically in the center o f the
membrane. These sites may actually be located closer to the membrane
surfaces. E quivalent mechanisms m ay apply with formate as electron
donor instead of H
2
. p, periplasmic side of the membrane; c, cyto-
plasmic s ide.
Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1981
genic i n Fig. 6B. In Fig. 6A, t he protons formed by MKH
2

oxidation are released on the outside, where they balance
the protons consumed by fumarate reduction, and fumarate
respiration with H
2
is predicted to be electrogenic with a
H
+
/e ratio of 1. In contrast, fumarate respiration with H
2
is
predicted to be an electroneutral process i n the proteolipo-
somes according to the mechanism of Fig. 6B.
The experimental results obtained with the proteolipo-
somes are in agreement with the mechanism depicted in
Fig. 6A and contradict that of Fig. 6B. The reduction by H
2
of quinone and of fumarate was found to be electrogenic,
and the H
+
/e ratio was 1 for both processes. As a
consequence, fumarate reduction by MKH
2
has to be an
electroneutral process in the proteoliposomes. This conclu-
sion is confirmed b y the result that DMNH
2
oxidation b y
fumarate was not coupled to the uptake of TPP
+
or TPB

–
by the proteoliposomes. DMNH
2
oxidation by fumarate
was previously also found to be electroneutral in cells and in
inverted vesicles of W. succinogenes [16]. Furthermore,
inverted vesicles catalyzing fumarate respiration with H
2
were found to accumulate SCN
–
,andtheH
+
/e ratio was
measured to be close to 1 [15]. In these vesicles, hydrogenase
is oriented to the inside and fumarate reductase to the
outside. Therefore, if fumarate reductase operated electro-
genically, t he H
+
/e ratio should be 2. However, the H
+
/e
ratio of f umarate r espiration is apparently not affected by
the orientatio n of the e nzymes rela tive t o each ot her and is
the same in inverted vesicles and proteoliposomes. This
confirms the e lectroneutral operation of fumarate red uctase
in the bacterial membrane (Fig. 6C) as well as in the
proteoliposomes (Fig. 6 A).
The view that the protons consumed in MK or DMN
reduction are taken up from the inside of the proteolipo-
somes (Fig. 6A,B) or from the cytoplasmic side of cells o f

W. succinogenes (Fig. 6C) is supported by the properties
of the hydC mutants N128D and Q131L. As expected on
the basis of the structure of E. coli FdnI, quinone
reduction by H
2
is inhibited in these mutants, suggesting
that the site of quinone reduction on HydC is located
close t o the cytoplasmic surface of the m embrane (Fig. 5).
This is likely to hold true also for FdhC of W. succino-
genes formate dehydrogenase, where residues equivalent
to N128 and Q131 of HydC are conserved. The arrange-
ment of the heme groups and the quinone binding site of
FdhC is expected to resemble that of HydC and of E. coli
FdnI [9].
ACKNOWLEDGEMENTS
This work was supported by grants of t he Deutsche Forschungsgeme-
inschaft (SFB 472) and by the Fonds der Chemischen Industrie to
A. Kro
¨
ger.
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