The function of methyl-menaquinone-6 and polysul®de reductase
membrane anchor (PsrC) in polysul®de respiration
of
Wolinella succinogenes
Wiebke Dietrich and Oliver Klimmek
Institut fu
È
r Mikrobiologie, Johann Wolfgang Goethe-Universita
È
t, Frankfurt am Main, Germany
Wolinella suc cinogenes grows by oxidative phosphorylation
with polysul®de as terminal electron a cceptor and either H
2
or formate a s electron donor (polysul®de respiration). The
function of the respiratory chains catalyzing these reactions
was i nvestigated. Proteoliposomes containing polysul®de
reductase (Psr) and either hydrogenase or formate dehy-
drogenase i solated f rom the membrane fraction of Wolinella
succinogenes catalyzed polysul®de respiration, provided that
methyl-menaquinone-6 isolated from W. succinogenes was
also present. The speci®c activities of electron transport were
commensurate with those of the bacterial membrane frac-
tion. Using site-directed mutage nesis, certain residues were
substituted in P srC, t he membrane a nchor of polysul®de
reductase. Replacement of Y23, D76, Y159, D218, E225 or
R305 caused nearly full inhibition of polysul®de respiration
without aecting the activity of Psr, which was still bound to
the membrane. These residues are predicted to be located in
hydrophobic helices o f PsrC, or n ext to t hem. Substitution o f
13 other r esidues of PsrC e ither c aused p artial inh ibition
of polysul®de respiration or had no eect. The function of

2
±
+[S] ® CO
2
+HS
±
(b)
It was proposed that the electron transport chain catalyz-
ing reactions (a) or (b) consisted of the membrane bound
components polysul®de reductase (Psr) and either h ydro-
genase or formate dehydrogenase [3,6±10]. The catalytic
subunits of the three enzymes are orien ted to t he periplasmic
side of the m embrane [4,6,10,11]. The three enzymes were
isolated from the membrane fraction of W. succinogenes
[3,6±8,12,13], and the corresponding genes were sequenced
[9,12,14]. Hydrogenase (Hyd) and formate dehydrogenase
(Fdh) are identical with the enzymes involved in fumarate
respiration with H
2
and f ormate in W. succinogenes [7,8,15].
The cytochrome b subunits of the two enzymes ( HydC and
FdhC) which carry the sites of quinone reduction are similar.
Their four histidine residues c oordinating the two heme B
groups are predicted to be located at similar places on three
homologous membrane helices.
Proteoliposomes containing Psr and either hydrogenase or
formate dehydrogenase were reported to catalyze reaction (a)
or (b) [4,6±8]. The electron transport activities amounted to
maximally 5 % of those measured in the bacterial membrane
fraction. The activities were not higher in proteoliposomes

molybdo-oxidoreductases and is probably the catalytic
subunit of Psr, which carries molybdenum coordinated by
molybdopterin guanine dinucleotide. PsrA and PsrB are
predicted to carry one and four iron-sulfur-centers, respec-
tively. PsrC is a hydrophobic p rotein which anchors the
enzyme in the membrane [10].
Correspondence to O. Klimme k, Institut fu
È
r Mikrobiologie, Johann
Wolfgang Goethe-Universita
È
t, Marie-Curie-Str. 9, D-60439 Frankfurt
am Main, Germany. Fax: + 49 6 9 79829527,
Tel. + 49 69 79829509, E-mail:
Abbreviations: DMN, 2,3-dimethyl-1,4-naphthoquinone; Hyd,
hydrogenase; Fdh, formate dehydrogenase; MK, menaquinone; MK
6
,
menaquinone with a side chain o f six isoprene units; MM, 5- or
8-methyl-MK
6
;MM
b
,MMboundtoPsrC;MM
b
H
±
, quinol anion of
MM bound to PsrC; Ps r, polysul®de reductase; [S], sulfur atom in
polysul®de; TTFB, 4,5,6,7-tetrachloro-2-tri¯uoromethyl-

substituted by vitamin K
1
.
Here we report on our attempts to r estore polysul®de
respiration in liposomes using one of the menaquinones of
W. succinogenes. To elucidate the function of PsrC, w hich is
thought to bind quinone, certain amino-acid residues of t his
subunit w ere replaced using site-directed mutagenesis. The
resulting mutants were characterized by measuring their
speci®c a ctivities of polysul®de respiration [reactions (a) and
(b)] and of Psr [reactions (c) and (d)]. The mechanism of
polysul®de respiration is discussed in the light of the
experimental results.
MATERIALS AND METHODS
Growth of
W. succinogenes
W. succinogenes was grown with formate as electron donor
and either fumarate or nitrate as electron acceptor as
described previou sly [17,18]. The medium containing nitrate
was supplemented with b rain±heart infusion (1,3% w/v;
Gibco BRL). Kanamycin (25 mgáL
A1
) and chloramphen icol
(12,5 mgáL
A1
) were added to the medium when indicated.
Cell fractionation
Cells of W . succinogenes grown with fumarate w ere sus-
pended (10 g proteináL
A1

membrane fraction.
Proteoliposomes were prepared by freeze-thawing sonic
liposomes containing the quinone indicated (Table 1;
10 lmolág phospholipid
A1
) with Psr (or fumarate reductase)
and either hydrogenase or formate dehydrogenase [8,13,22].
Per g phospholipid, a total of 26 nmol Psr, 31 nmol
fumarate reductase, 1 78 nmol hydrogenase and 89 nmol
formate dehydrogenase were applied.
Quinones
MK
6
and MM were extracted from the membrane fraction
of W. succinogenes grown with fumarate using a mixture o f
petrol ether a nd methanol. The quinones in the e xtract were
separated by H PLC according to [23]. The quinones were
quanti®ed by HPLC using vitamin K
1
as the s tandard. MK
4
(Sigma; c at. no. V-937 8) and v itamin K
1
(Fluka; cat. no.
95271) are commercially available.
Activities of Psr and of polysul®de respiration
The activity of Psr was measured at 37 °C by photometric
recording of polysul®de reduction with BH
4
±

Quinone H
2
® [S] HCO
2
±
® [S] HCO
2
±
® fumarate
±25517
Methyl-MK
6
(MM) 370 175 35
MK
6
27 5 1490
MK
4
34 7 1455
VitK
1
25 5 1180
Ó FEBS 2002 Polysul®de respiration of W. succinogenes (Eur. J. Biochem. 269) 1087
Genetic techniques
Standard genetic procedures were used essentially
according to [26]. DNA was isolated from W. succino-
genes with the DNeasy Tissue Kit from Qiagen. PCR
was carried out using the Expand High Fidelity
PCR System (Roche) or the Expand Long Template
PCR System (Roche) with standard ampli®cation proto-

A1
). The genome
of several transformants was checked for t he presence of
the cat
GC
and the psrC gene by means of Southern blot
analysis using Bgl II restriction (Fig. 1). As expected, only
one BglII fragment (9.7 kbp) of mutant KpsrC
hybridized to the cat
GC
and the psrC probe. The
in-frame integration of the plasmid was con®rmed by
sequencing.
Construction of
W. succinogenes
psrC mutants
The psrC mutants of W. succinogenes (see Table 2) were
constructed by t ransforming W. succinogenes DpsrC with
derivatives of pKpsrC. The derivatives were synthesized
using the Quick Change site-directed mutagenesis kit
(Stratagene) with pKpsrC as template and speci®cally
synthesized oligonucleotides carrying the desired nucleotide
mismatches. Modi®ed pKpsrC plasmids were sequenced to
con®rm the m utations. Transformation o f W. succinogenes
DpsrC with modi®ed plasmids and selection of t ransfor-
mants was performed as described above.
Computer analysis
Database searches made use of the program
BLAST
[31].

than an order of magnitude greater than of those p repared
with MK
6
,MK
4
, vitamin K
1
, or w ithout quinone. These
Fig. 1. P hysical map of the psr locus of W. succinogenes DpsrC and KpsrC. Mutant KpsrC was obtained by integration of pKpsrC into the genome
of the DpsrC mutant.
1088 W. Dietrich and O. Klimmek (Eur. J. Biochem. 269) Ó FEBS 2002
Table 2. P roperties of psrC mutants grown with formate and fumarate. The presence o f PsrA was tested by Western blot and E LISA. The speci®c
activities of Psr (BH
4
±
® [S] a nd HS
±
® DMN) and of polysul®de respiration (H
2
® [S] and HCO
2
±
® [S]) refer to total cellular protein (cells)
or to the protein of the membrane fraction ( MF).
Strain Preparation
PsrA
present
Uámg protein
A1
BH

S94A Cells 1.6 1.1
MF + 13 7.0 0.8 0.8
Y106F Cells 1.8 1.4
MF + 19 9.3 0.8 0.7
E146Q Cells 1.8 1.0
MF + 14 4.6 0.6 0.4
Y159F Cells £ 0.01 £ 0.01
MF + 12 7.0 0.02 0.02
T160V Cells 0.2 0.2
MF + 17 9.3 0.1 0.1
N174D Cells 1.4 1.2
MF + 17 11 0.9 0.8
S185A Cells 0.9 0.8
MF + 12 6.0 0.4 0.4
S188A Cells 0.5 0.5
MF + 11 5.1 0.2 0.2
S192A Cells 2.1 1.7
MF + 19 11 0.8 0.7
E209Q Cells 2.3 2.1
MF + 19 13 1.2 1.1
D218N Cells £ 0.01 £ 0.01
MF + 15 7.9 0.02 0.02
E225Q Cells 0.03 0.03
MF + 11 4.9 0.03 0.03
E225D Cells 1.1 1.2
MF + 14 7.8 0.4 0.7
W261F Cells 2.0 1.3
MF + 21 10 0.7 0.6
R305F Cells 0.04 0.07
MF + 13 7.2 0.02 0.02

1
but not by MM (data not shown).
The turnover number of Psr in polysul®de respiration
with formate in the proteoliposomes is close to that
measured in the membrane fraction o f w ild-type W. suc-
cinogenes (see Table 2) w hich contains approximately
0.1 lmol Psr per g membrane protein [3,8]. The turnover
number with H
2
is 50% higher i n the proteoliposomes than
in the membrane fraction. This higher a ctivity is p robably
due to the higher a mount of hydro genase relative to Psr in
the proteoliposomes. In summary, functional electron
transport chains catalyzing reactions (a) or (b) at the
expected activities can be restored from the isolated enzymes
and MM. Hence no further components appear to be
required for the electron transport. MM is speci®cally
involved in polysul®de respiration and cannot be substituted
by MK
6
although this is also present in the membrane of
W. succinogenes.
In the experiment shown in Fig. 2, t he membrane fraction
of W. succinogenes was fused with sonic liposomes contain-
ing i ncreasing amounts of MM. The six d ifferent prepara-
tions so obtained contained equal amounts of phospholipids
from the membrane f raction and from the liposomes. The
activity of polysul®de respiration with H
2
increased hyper-

membrane fraction were not inhibited by TTFB (not
shown). Thus, the activity of polysul®de respiration appears
to be stimulated by Dp.
Characterization of psrC mutants
PsrC is predicted to form eight membrane-spanning helices
and to be s imilar to four hydrophobic subunits of other
electron transport enzymes which are likely to react
with quinones (Fig. 4). The nrfD genes of E. coli and
Haemophilus in¯uenzae are constituents of gene clusters
encoding Ôcytochrome c nitrite reductaseÕ.NrfAofE. coli is
known to b e the catalytic subunit of this enzyme [37]. The
gene product of nrfD was proposed to encode the m embrane
anchor of the enzyme and to carry the site of quinol
oxidation. Tetrathionate reductase (Ttr) of Salmonella
typhimurium is thought to catalyze the reduction of
Fig. 2. A ctivity of polysul®de respiration with H
2
as a function of the
MM content o f fusion particles. The maximum activity was assumed to
be measured with 57 lmo l MMág pho spho lipid
-1
.
Fig. 3. T he e ect of the protonophore TTFB o n the activities of poly-
sul®de respiration in cells of W. succinogenes. TTFB dissolved in
demethylsulfoxide was added 3 min before the electron transport was
started. The speci®c activity of polysul®de respiration with H
2
and
formate was 3.6 and 0.72 Uámg
A1

fraction of mutant DpsrC [10]. Integration of plasmid
pKpsrC into the genome of DpsrC resulted in strain KpsrC
which carried the intact psrABC operon including psrC
(Fig. 1). This s train had wild-type a ctivities of polysul®de
respiration with H
2
and with formate. As with the wild-type
strain, P srA and the activities of Psr were f ound in the
membrane fraction of strain KpsrC. These results show that
PsrC anchors Psr in the membrane a nd is required for
polysul®de respiration, but not for t he Psr activities.
The other psrC mutants listed in Table 2 were construc-
ted by integrating derivatives of pKpsrC with altered codons
into the genome of the DpsrC mutant. All the mutants so
obtained h ad PsrA bound to the membrane, and the speci®c
activities of Psr were similar to those of the wild-type s train.
Hence, the i ntegration of Psr into the membrane and its
enzymic activities were not affected by the mutations.
The mutants differ in their speci®c activities of polysul® de
respiration. Eight of the 23 mutan ts either did not catalyze
polysul®de respiration or their speci®c activities were less
than 5% of those of the wild-type strain. In these mutants a
residue was a ltered which is presumably located in one of
the eight hydrophobic segments of PsrC (Y23F, Y159F,
E225Q, R305F and R305K) or next to their ends (D76N,
D76L, and D218N). The replacement of another three
residues l ocated in hydrophobic stretches of PsrC had less
drastic effects; the a ctivities of T160 V, S185A and S 188A
amounted to approximately 10, 50 and 25%, respectively, of
those of the wild-type strain. Substitution of Y106, E146,

af®nity in the mutants was d ecreased by more t han two
orders of magnitude. With mutants showing partial inhibi-
tion of the electron transport, this activity was either
stimulated (E225D) or was not signi®cantly altered (S185A,
S188A) by the increased quinone content.
DISCUSSION
The function of MM
The standard redox potential of MK in organic s olution at
pH 7 (E
o
¢) was d etermined to be A74 mV [40,41]. A methyl
group in the aromatic ring of n apthoquinones was found to
lower the E
o
¢ by approximately 16 mV [41]. Therefore, the
E
o
¢ of MM in organic solution is assumed to be A90 mV
[reaction (2) in Table 3]. The same value is likely to apply for
MM in the bacterial membrane, as the E
o
¢ of MK in a
bacterial membrane was determin ed to be close to that in
organic solution [43]. It will be sho wn below that MMH
2
dissolved i n the membrane is not suf®ciently electro-
negative to serve as donor for polysul®d e reduction.
Tetrasul®de (
2À
4

(4) in Table 3] turns out to be nearly equal to that of
elemental sulfur; this also holds true for p entasul®de. These
potentials a re approximately 150 mV more n egative than
that of MM [reaction ( 2) in Table 3].
As a consequence, the reduction of polysul®de by MMH
2
[reaction (f)] is extremely ende rgonic. From the equilibrium
constant at pH 8 of reaction ( f) [5 ´ 10
A16
M
3
,from
reactions (2) and (4) in Table 3] it is calculated that t he
reaction becomes exergonic when the ratio MM : MMH
2
exceeds 2 ´ 10
A4
with the concentrations of tetrasul®de and
sul®de at 10
A4
M
and 10
A2
M
, respectively.
3MMH
2
+ 
2À
4

electron transport chain c atalyzing polysul®de reduction
by H
2
, the steady state concentration of MM would b e
below 0.6 ´ 10
A6
M
. The corresponding velocity of MM
reduction by H
2
would be much lower than that of the
overall electron transport f rom H
2
to polysul®de. There-
fore, the species of MM involved in polysul®de respiration
should h ave a much lower redox potential than that of
MM dissolved in the membrane. The redox potential at
pH 8 of the species involved in polysul®de respiration can
be estimated a ssumin g its equilibrium ratio o xidized/
reduced to be 1 (instead of 2 ´ 10
A4
) and the equilibrium
concentrations of 
2À
4
(10
A4
M
) and HS
±

(Fig. 5 ). MM
b
reduction is assumed to be coupled to proton
uptake from the cytoplasmic side of t he membrane, and
MM
b
H
±
oxidation to be c oupled to proton release at the
periplasmic side. MM
b
and MM
b
H
±
are thought to be
located in the hydrophobic part of PsrC. Therefore, the
uptake and release of protons is expected to be gu ided by
proton channels. The channel for proton release should be
in PsrC and t hat for proton uptake i n HydC. The H
+
/e
ratio of 0.5 predicted by the mechanism is half that
determined for fumarate respiration of W. succinogenes,in
agreement with the growth yields of polysul®de and
fumarate respiration [2,3,6].
The 
pH8

of MM

2
would
become 85 mV more electropositive o n i ts way f rom the
periplasmic side to MM
b
(Fig. 5 ). The simultaneous transfer
of a proton from the cytoplasmic side to MM
b
should a ffect
thefreeenergyofMM
b
reduction by H
2
in the same way.
Thus MM
b
reduction by H
2
should become 24 kJámol
A1
more endergonic, as two electrons (derived from H
2
)from
Table 3. R edox potentials of compounds involved in polysul®de r espi-
ration with H
2
. E
o
¢ of reaction (3) was taken from [42]. The values o f
reactions (2), ( 4), and (5) are de rived as described in the text.

® S+4H
+
+6e
±
A260 -300
(5) MM
b
H
±
® MM
b
+H
+
+2e
±
A230 A260
1092 W. Dietrich and O. Klimmek (Eur. J. Biochem. 269) Ó FEBS 2002
the periplasmic (positive) side and one proton from the
cytoplasmic side are transferred to MM
b
.MM
b
H
±
oxida-
tion should become 8 kJámol
A1
more exergonic, as it is
coupled to the transfer of two e lectrons and one proton
from the center of the membrane t o the periplasmic side.

by the amount of MM
b
H
±
in the absence of a Dp,andthat
MM
b
H
±
dissociates from PsrC (C) according to reaction (g),
where 


designates a proto n taken up from or released to
the cytoplasmic side of the membrane.
MM
b
H
±
+ 


® MMH
2
+C (g)
Assuming that MM
b
H
±
is located i n the center of the

H
±
by [S]
which is coupled to the release of a p roton to the periplasm.
Table 4. S tandard free energies at pH 8 of the reactions thought to make
up polysul®de respiration with H
2
.MM
b
and MM
b
H
±
designate MM
bound to PsrC in the oxidized and reduced state (Fig. 4). [S] designates
polysul®de sulfur. D
pH8

values were calculated from the 
pH8

of
reactions 1, 4, and 5 given in Table 3. Dw Designates the electrical
potential across t he membrane which i s generated by polysul®de
respiration [3,5].
D
pH8

(kJá mol
A1

dues replaced in PsrC by site-directed muta-
genesis are indicated. Residues i n bold letters
correspond to mutants with 5% or less of the
wild-type speci®c activities of poly sul®de res-
piration.
Ó FEBS 2002 Polysul®de respiration of W. succinogenes (Eur. J. Biochem. 269) 1093
nearly two-fold upon the incorporation of additional MM.
As the MM dissolved in the membrane is likely to be fully
reduced in the steady s tate of polysul®de respiration, the
amount of MM
b
H
±
should increase according to reaction
(g) as increasing amounts o f MM are incorporated into the
membrane. On the basis of these considerations, the
titration curve (Fig. 2) re¯ects the formation of MM
b
H
±
according to reaction ( g).
The function of PsrC
Like the iron-sulfur subunit of fumarate reductase [46], PsrB
is thought to serve as mediator of electron transfer between
the prosthetic group (MM
b
) of the membrane anchor
(PsrC) and the catalytic subunit ( PsrA) of Psr. Therefore,
PsrA is probably boun d to PsrB which in turn is bound to
PsrC on the periplasmic side of the membrane. Dissociation

proton transfer to and from the cytoplasmic side according
to reaction (g) w hich describes the binding of reduced MM
to PsrC and its dissociation. Consistent with this function,
substitution of D76 by asparagine o r leucine r esulted in
nearly inactive mutants, whereas D76H exhibited about
10% of the wild-type activity of polysul®de respiration. The
hydroxyl groups of Y23 and Y159 may form hydrogen
bounds to MM
b
and MM
b
H
±
.
W. succinogenes mutants with one of the four heme
ligands of HydC replaced were found to lack the activities of
electron transport form H
2
to polysul®de and to fumarate
[15]. The heme groups appear to be required for the
reduction of MM
b
and o f MK. However, i t i s not known
whether the two quinones are reduced at the same site of
Hyd C. Assuming that there is only one site for quinone
reduction on HydC (or FdhC), it has to be postulated that
MM
b
protrudes f rom its binding site on PsrC into the lipid
phase of the membrane to accept electrons form the heme

rmann for skilful technical assistance. The w ork was supported by
the Deutsche Forschungsgemeinschaft (SFB 472).
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