The function of methyl-menaquinone-6 and polysul®de reductase membrane anchor PsrC in polysul®de respiration Wiebke Dietrich and Oliver Klimmek Institut fuÈr Mikrobiologie, Johann Wolfga
Trang 1The function of methyl-menaquinone-6 and polysul®de reductase membrane anchor (PsrC) in polysul®de respiration
Wiebke Dietrich and Oliver Klimmek
Institut fuÈr Mikrobiologie, Johann Wolfgang Goethe-UniversitaÈt, Frankfurt am Main, Germany
Wolinella succinogenes grows by oxidative phosphorylation
with polysul®de as terminal electron acceptor and either H2
or formate as electron donor (polysul®de respiration) The
function of the respiratory chains catalyzing these reactions
was investigated Proteoliposomes containing polysul®de
reductase (Psr) and either hydrogenase or formate
dehy-drogenase isolated from 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 mutagenesis, certain residues were
substituted in PsrC, the membrane anchor of polysul®de
reductase Replacement of Y23, D76, Y159, D218, E225 or R305 caused nearly full inhibition of polysul®de respiration without aecting the activity of Psr, which was still bound to the membrane These residues are predicted to be located in hydrophobic helices of PsrC, or next to them Substitution of
13 other residues of PsrC either caused partial inhibition
of polysul®de respiration or had no eect The function of methyl-menaquinone-6, which is thought to be bound to PsrC, is discussed
Keywords: methyl-menaquinone; polysul®de respiration; sulfur respiration; hydrogenase; formate dehydrogenase
Wolinella succinogenes grows at the expense of polysul®de
([S]) respiration with H2[reaction (a)] or formate [reaction
(b)] as electron donor [1±3] Reactions (a) (DGo¢ )31
kJámol Hÿ 1
2 ) and (b) (DGo¢ ) 30 kJ mol)1formate) are
coupled to the generation of an electrochemical proton
potential (Dp 0.17 V) across the bacterial membrane
which drives ATP synthesis [3±6]
H2+ [S] ® HS±+ H+ (a)
HCO2 + [S] ® CO2+ 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
hydro-genase or formate dehydrohydro-genase [3,6±10] The catalytic
subunits of the three enzymes are oriented to the periplasmic
side of the membrane [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 H2and formate 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 coordinating the two heme B groups are predicted to be located at similar places on three homologous membrane helices
Proteoliposomes containingPsr and either hydrogenase or formatedehydrogenasewerereportedtocatalyzereaction(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 additionally containing vitamin K1 which is known to substitute for menaquinone in fumarate respiration The isolated Psr was found to consist of the three subunits predicted by the psrABC operon [3,6,8] It contained molybdenum (1 mol per mol enzyme), molybdopterine guanine dinucleotide, free iron and sul®de, and menaqui-none Heme, ¯avin and other heavy metals were absent The enzyme catalyzed the reduction of polysul®de by BH4 [reaction (c)] and the oxidation of sul®de by 2,3-dimethyl-1,4-naphthoquinone (DMN) [reaction (d)]
BH4 + [S] ® BH3+ HS± (c)
HS±+ DMN + H+ ® [S] + DMNH2 (d) PsrA is similar to the catalytic subunits of several 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 protein which anchors the enzyme in the membrane [10]
Correspondence to O Klimmek, Institut fuÈr Mikrobiologie, Johann
Wolfgang Goethe-UniversitaÈt, Marie-Curie-Str 9, D-60439 Frankfurt
am Main, Germany Fax: + 49 69 79829527,
Tel + 49 69 79829509, E-mail: klimmek@em.uni-frankfurt.de
Abbreviations: DMN, 2,3-dimethyl-1,4-naphthoquinone; Hyd,
hydrogenase; Fdh, formate dehydrogenase; MK, menaquinone; MK 6 ,
menaquinone with a side chain of six isoprene units; MM, 5- or
8-methyl-MK 6 ; MM b , MM bound to PsrC; MM b H ± , quinol anion of
MM bound to PsrC; Psr, polysul®de reductase; [S], sulfur atom in
polysul®de; TTFB,
4,5,6,7-tetrachloro-2-tri¯uoromethyl-benzimidazol;
Dp, electrochemical proton potential across a membrane; Dw, electric
term of Dp; Ttr, tetrathionate reductase.
(Received 25 June 2001, revised 1 November 2001, accepted
7 November 2001)
Trang 2When the membrane fraction of W succinogenes was
fused to liposomes containing menaquinone isolated from
W succinogenes, the electron transport activities [reactions
(a) and (b)] decreased as a function of the degree of dilution
of the membrane proteins by phospholipid [6,16] This
effect was interpreted to indicate that the electron transfer
from the dehydrogenases to Psr requires diffusion and
collision of the enzyme molecules within the membrane
Consistent with this interpretation, the electron transport
activity with fumarate was not decreased upon dilution of
the membrane proteins In this case the electron transfer
from the dehydrogenases to fumarate reductase is
accom-plished by menaquinone (MK) whose diffusion velocity is
two orders of magnitude higher than that of the much
larger enzymes
When the membrane fraction was fused to liposomes
containing either no quinone or vitamin K1 instead of
menaquinone from W succinogenes, the activities of
poly-sul®de respiration decreased more markedly as the
phos-pholipid content increased [6,16] This suggested that one of
the two menaquinones of W succinogenes was speci®cally
required for polysul®de respiration and could not be
substituted by vitamin K1
Here we report on our attempts to restore polysul®de
respiration in liposomes using one of the menaquinones of
W succinogenes To elucidate the function of PsrC, which is
thought to bind quinone, certain amino-acid residues of this
subunit were replaced using site-directed mutagenesis The
resulting mutants were characterized by measuring their
speci®c activities 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
M A T E R I A L S A N D M E T H O D S
Growth ofW succinogenes
W succinogenes was grown with formate as electron donor
and either fumarate or nitrate as electron acceptor as
described previously [17,18] The medium containing nitrate
was supplemented with brain±heart infusion (1,3% w/v;
Gibco BRL) Kanamycin (25 mgáL)1) and chloramphenicol
(12,5 mgáL)1) were added to the medium when indicated
Cell fractionation
Cells of W succinogenes grown with fumarate were
sus-pended (10 g proteináL)1) in 50 mM Tris/HCl, pH 8.0 at
0 °C The suspension was passed through a French press at
130 Mpa and 10 mLámin)1¯ow The resulting cell
homo-genate was centrifuged for 40 min at 100 000 g to yield the
soluble cell fraction (supernatant) and the membrane
fraction The membrane fraction was resuspended in the
same buffer (10 g proteináL)1)
SDS/PAGE, Western blotting and ELISA
SDS/PAGE was carried out according to [19] Protein was
transferred to nitrocellulose sheets by electroblotting in a
discontinuous buffer system [20] PsrA was visualized by
indirect ELISA using the corresponding antiserum and goat
anti-(rabbit IgG) Ig conjugated to peroxidase [9]
Fusion particles and proteoliposomes Sonic liposomes containing quinone were prepared from a mixture of egg phospholipid (95%, w/w) [21] and phospha-tidylethanolamine (5%, Fluka; cat no 60650) as described previously [13,22] Fusion particles (see below) were pre-pared by freeze±thawing a mixture of sonic liposomes containing 5- or 8-methyl-MK6(MM) and bacterial mem-brane fraction [16] The fusion particles contained equal amounts of phospholipid from the liposomes and from the membrane fraction
Proteoliposomes were prepared by freeze-thawing sonic liposomes containing the quinone indicated (Table 1;
10 lmolág phospholipid)1) 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, 178 nmol hydrogenase and 89 nmol formate dehydrogenase were applied
Quinones
MK6and MM were extracted from the membrane fraction
of W succinogenes grown with fumarate using a mixture of petrol ether and methanol The quinones in the extract were separated by HPLC according to [23] The quinones were quanti®ed by HPLC using vitamin K1as the standard MK4 (Sigma; cat no V-9378) and vitamin K1 (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 BH4 [24] or by photometric recording of DMN reduction with sul®de [2] Polysul®de respiration with H2or formate (electron trans-port) was recorded photometrically at 37 °C as described previously [2,8,16] The unit of activity (U) corresponded to consumption of 1 lmol substrate per min
Protein Protein was determined after precipitation with trichloro-acetic acid using the Biuret method with KCN [25]
Table 1 Activities of polysul®de respiration of proteoliposomes con-taining dierent naphthoquinones Polysul®de respiration with H 2
(H 2 ® [S]) or with formate (HCO 2 ® [S]) was measured in prote-oliposomes containing Psr and either hydrogenase or formate dehydrogenase isolated from W succinogenes Fumarate respiration with formate (HCO 2 ® fumarate) was measured in proteoliposomes containing fumarate reductase and formate dehydrogenase as described previously [22] The activities are given as substrate turnovers
of Psr or fumarate reductase at 37 °C All values are in units of s )1 Quinone H 2 ® [S] HCO 2 ® [S] HCO 2 ® fumarate
Methyl-MK 6 (MM) 370 175 35
VitK 1 25 5 1180
Trang 3Genetic 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-cols and a Hybaid Omnigene Thermocycler (MWG
Biotech) Southern blotting to nylon membranes was
performed as described previously [27] DNA probes
were generated with the PCR DIG Probe Synthesis Kit
(Roche), and hybrids were visualized using the DIG/
Luminescent Detection Kit (Roche)
Construction ofW succinogenes KpsrC
Mutant KpsrC was constructed by integrating plasmid
pKpsrC into the genome of mutant DpsrC [10] (Fig 1)
Plasmid pKpsrC was constructed from pT7-6 [28] by
inserting the catGC gene excised from pDF4a [29] using
BamHI restriction The orientation of catGCwas con®rmed
by restriction analysis Subsequently, a fragment comprising
most of psrB (521 bp), psrC, and 60 bp of the 3¢ end of rhpR
was inserted using HindIII and XbaI This fragment was
synthesized from genomic DNA by PCR with primers
carrying at their 5¢ ends suitable restriction sites for cloning
The identity of the cloned PCR fragment was con®rmed by
sequencing
Cells of W succinogenes DpsrC were transformed with
the plasmid as described previously [30] Transformants
were selected on agar plates containing the nitrate
medium, 2.6% (w/v) brain-heart-infusion agar (Gibco
BRL) and chloramphenicol (12.5 mgáL)1) The genome
of several transformants was checked for the presence of
the catGCand the psrC gene by means of Southern blot
analysis using BglII restriction (Fig 1) As expected, only
one BglII fragment (9.7 kbp) of mutant KpsrC
hybridized to the catGC and the psrC probe The
in-frame integration of the plasmid was con®rmed by
sequencing
Construction ofW succinogenes psrC mutants The psrC mutants of W succinogenes (see Table 2) were constructed by transforming 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 mutations Transformation of W succinogenes DpsrC with modi®ed plasmids and selection of transfor-mants was performed as described above
Computer analysis Database searches made use of the program BLAST [31] Search for membrane-spanning helices was performed using theTMPREDprogram [32] Multiple sequences were aligned using the programCLUSTALW[33]
R E S U L T S
Reconstitution of polysul®de respiration
W succinogenes grown with polysul®de or with fumarate catalyzes polysul®de respiration [reactions (a) and (b)] as well as fumarate respiration with H2or formate [16] These bacteria contain equal amounts (approximately 3 lmol per
g phospholipid) of MK6and of MM [34] The methyl group
in the aromatic ring of MM is at position 5 or 8 Like MK,
MM appears to be dissolved in the lipid phase of the membrane and is not tightly bound to membrane proteins Both quinones can be extracted from the membrane with a mixture of petrol ether and methanol
In the experiment shown in Table 1, Psr and either hydrogenase or formate dehydrogenase were incorporated into liposomes containing one of the quinones indicated (10 lmolág phospholipid)1) The activities of polysul®de respiration of proteoliposomes containing MM were more than an order of magnitude greater than of those prepared with MK6, MK4, vitamin K1, or without quinone These
Fig 1 Physical 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.
Trang 4Table 2 Properties of psrC mutants grown with formate and fumarate The presence of PsrA was tested by Western blot and ELISA The speci®c activities of Psr (BH 4 ® [S] and 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 PsrApresent
Uámg protein )1
BH 4 ® [S] HS ± ® DMN H 2 ® [S] HCO 2 ® [S]
a The cell homogenate was used.
Trang 5low activities were probably due to the low level of MM
present in the enzyme preparations used for preparing the
proteoliposomes Proteoliposomes prepared with fumarate
reductase and formate dehydrogenase catalyzed fumarate
respiration with formate at high activities, if MK6, MK4or
vitamin K1were also incorporated However, if MM was
incorporated instead, the activity was as low as that of
proteoliposomes prepared without added quinone In
liposomes containing fumarate reductase and hydrogenase,
fumarate respiration with H2was restored by MK6, MK4or
vitamin K1but 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 of wild-type W
suc-cinogenes (see Table 2) which contains approximately
0.1 lmol Psr per g membrane protein [3,8] The turnover
number with H2is 50% higher in the proteoliposomes than
in the membrane fraction This higher activity is probably
due to the higher amount of hydrogenase 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 MK6although this is also present in the membrane of
W succinogenes
In the experiment shown in Fig 2, the membrane fraction
of W succinogenes was fused with sonic liposomes
contain-ing increascontain-ing amounts of MM The six different
prepara-tions so obtained contained equal amounts of phospholipids
from the membrane fraction and from the liposomes The
activity of polysul®de respiration with H2increased
hyper-bolically with the amount of MM The activity was 50 and
80% of the maximum activity with 2.5 and 10 lmol MM
per g phospholipid, respectively Thus the activity is
considerably enhanced by the incorporation of additional
MM into the membrane fraction which normally contains
approximately 3 lmol MM per g phospholipid As noted
in the Discussion section, the titration curve (Fig 2)
may re¯ect binding of reduced MM to PsrC If this
assumption is valid, the dissociation constant would be 2.5 lmol MMág phospholipid)1
The effect of Dp on the electron transport activity The speci®c activities of Psr in the membrane fraction are about twice those found in cells of W succinogenes (see Table 2) This is consistent with the location of Psr in the membrane which contains half of the total cellular protein
In contrast, the speci®c activities of polysul®de respiration are higher in cells than in the membrane fraction (see Table 2) This is probably due to the Dp generated across the membrane of cells by polysul®de respiration [5] The Dp
is lost upon cell disruption The addition of a protonophore
to cells also caused inhibition of polysul®de respiration (Fig 3) The protonophore 4,5,6,7-tetrachloro-2-tri¯uoro-methylbenzimidazol (TTFB) which is known to dissipate the Dp [5,35] was found to cause up to 70% inhibition of polysul®de respiration in cells Polysul®de respiration in the membrane fraction or fumarate respiration in cells or in the 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 similar 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 NrfA of E coli is known to be the catalytic subunit of this enzyme [37] The gene product of nrfD was proposed to encode the membrane 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 Activity of polysul®de respiration with H 2 as a function of the
MM content of fusion particles The maximum activity was assumed to
be measured with 57 lmol MMág phospholipid -1
Fig 3 The eect of the protonophore TTFB on 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 )1 cell protein, respectively at 37°C.
Trang 6tetrathionate to thiosulfate by a quinol [39] The catalytic
subunit (TtrA) is predicted to carry molybdenum and an
iron±sulfur center TtrB is predicted to harbour four iron±
sulfur centers TtrC was proposed to anchor the enzyme in
the membrane and to carry the quinol site TtrC is larger
than PsrC and probably contains an additional
hydropho-bic helix at its C-terminus A molybdo-iron±sulfur enzyme
which is similar to Psr and tetrathionate reductase is possibly
encoded by a gene cluster of Archaeoglobus fulgidus This
cluster includes orf 2386 which is predicted to code for a
hydrophobic protein resembling PsrC The majority of
residues conserved among the ®ve proteins are located in the
hydrophobic stretches or close to them Conserved residues
that may be essential for electron or proton transfer are
presented in highlighted letters in Fig 4 The corresponding
residues of PsrC were replaced by other residues, and the
resulting mutants were characterized (Table 2)
The psrC mutants were constructed from a mutant
(DpsrC) of W succinogenes carrying the kan gene instead of
psrC (Fig 1) Cells of this mutant grown with fumarate did
not catalyze polysul®de respiration with H2or formate, in
contrast to the wild-type strain (Table 2) PsrA and the
enzymic activities of Psr [reactions (c) and (d)] were found in
cells of the mutant, but were missing in the membrane
fraction PsrA and the activity of reaction (d) were
previously found to be located in the periplasmic cell
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 strain had wild-type activities of polysul®de
respiration with H2and with formate As with the wild-type
strain, PsrA and the activities of Psr were found in the
membrane fraction of strain KpsrC These results show that
PsrC anchors Psr in the membrane and is required for
polysul®de respiration, but not for the 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 had PsrA bound to the membrane, and the speci®c activities of Psr were similar to those of the wild-type strain Hence, the integration 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 mutants 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 altered 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 located in hydrophobic stretches of PsrC had less drastic effects; the activities of T160V, S185A and S188A amounted to approximately 10, 50 and 25%, respectively, of those of the wild-type strain Substitution of Y106, E146, S192 and of W261 which are located in hydrophobic stretches had no effect on the electron transport activities Wild-type activities were also measured with mutants W16F, H82A, S94A, N174D, E209Q, and Y310F These residues are predicted to be located in the hydrophilic loops
of PsrC In summary, the properties of the mutants (Table 2) suggest that certain residues of PsrC are essential for electron transfer from the dehydrogenases to polysul®de, indicating that PsrC serves speci®c functions in addition to that of the membrane anchor of Psr
The activity of polysul®de respiration in the membrane fraction of the wild-type strain was found to be considerably enhanced by increasing the amount of MM (Fig 2) Therefore, it was feasible that higher amounts of MM would restore the electron transport in the mutants lacking this activity To test this possibility, the membrane fraction
Fig 4 Sequence alignment of PsrC of W succinogenes (W s.) to four proteins predicted from DNA sequences Residues which were replaced in PsrC
by site-directed mutagenesis are highlighted These residues are shown in white on black background, if the corresponding mutants showed 5% or less of the wild-type activities of polysul®de respiration (Table 2) Putative membrane spanning segments are boxed A f., Archaeoglobus fulgidus [36]; E c., Escherichia coli [37]; H i., Haemophilus in¯uenzae [38]; S t., Salmonella typhimurium [39].
Trang 7of these mutants (Y23F, Y159F, D218N, E225Q, R305F
and R305K) was fused to liposomes containing high
concentrations of MM as in the experiment shown in
Fig 2 Electron transport activity was not restored in these
fusion particles which contained 20-fold the amount of MM
present in the membrane fraction (not shown) This suggests
that the lack of electron transport activity was not caused by
a decreased af®nity of the putative MM binding site for
MM However, a stimulation would not be seen if this
af®nity in the mutants was decreased by more than 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
D I S C U S S I O N
The function of MM
The standard redox potential of MK in organic solution at
pH 7 (Eo¢) was determined to be )74 mV [40,41] A methyl
group in the aromatic ring of napthoquinones was found to
lower the Eo¢ by approximately 16 mV [41] Therefore, the
Eo¢ of MM in organic solution is assumed to be )90 mV
[reaction (2) in Table 3] The same value is likely to apply for
MM in the bacterial membrane, as the Eo¢ of MK in a
bacterial membrane was determined to be close to that in
organic solution [43] It will be shown below that MMH2
dissolved in the membrane is not suf®ciently
electro-negative to serve as donor for polysul®de reduction
Tetrasul®de (S2ÿ
4 ) and pentasul®de (S2ÿ
5 ) are the only polysul®de species occurring at signi®cant concentrations in
the solutions (10)2 M HS±, pH 8) used for measuring
polysul®de respiration [44] The concentration of
pentasul-®de is about half that of tetrasulpentasul-®de The redox potential of
tetrasul®de [reaction (4) in Table 3] was evaluated from that
of elemental sulfur [reaction (3) in Table 3] using the
equilibrium constant of reaction (e) (3.6 ´ 10)9 M; [2,44])
3/8 S8+ HS± ® S2ÿ
4 + H+ (e) The standard potential of tetrasul®de at pH 8 [reaction
(4) in Table 3] turns out to be nearly equal to that of
elemental sulfur; this also holds true for pentasul®de These
potentials are approximately 150 mV more negative than
that of MM [reaction (2) in Table 3]
As a consequence, the reduction of polysul®de by MMH2
[reaction (f)] is extremely endergonic From the equilibrium
constant at pH 8 of reaction (f) [5 ´ 10)16 M3, from
reactions (2) and (4) in Table 3] it is calculated that the
reaction becomes exergonic when the ratio MM : MMH2
exceeds 2 ´ 10)4with the concentrations of tetrasul®de and sul®de at 10)4 Mand 10)2 M, respectively
3MMH2+ S2ÿ
4 ® 3MM + 4HS±+ 2H+ (f) The ratio corresponds to a concentration of MM within the membrane (0.6 ´ 10)6 M) which is more than an order
of magnitude below the Km of hydrogenase for DMN (15 ´ 10)6 M) The Kmfor this water soluble menaquinone analogue is thought to re¯ect those for the quinones within the membrane on the basis that the content of 3 lmol qui-noneág phospholipid)1corresponds to 3 ´ 10)3 Mquinone concentration If MM/MMH2 was a component of the electron transport chain catalyzing polysul®de reduction
by H2, the steady state concentration of MM would be below 0.6 ´ 10)6 M The corresponding velocity of MM reduction by H2 would be much lower than that of the overall electron transport from H2 to polysul®de There-fore, the species of MM involved in polysul®de respiration should have 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 assuming its equilibrium ratio oxidized/ reduced to be 1 (instead of 2 ´ 10)4) and the equilibrium concentrations of S2ÿ
4 (10)4 M) and HS± (10)2 M) used above The value so obtained [EpH8
o )260 mV, reaction (5) in Table 3] is regarded as the upper limit of redox potential of the quinol suited as donor of polysul®de reduction
Hypothetical mechanism of polysul®de respiration
It is envisaged that the species of MM serving in polysul®de respiration is bound to PsrC in its oxidized (MMb) and its reduced form (MMbH±) which is the quinol anion MMbis thought to be reduced to MMbH± by accepting electrons from the cytochrome b subunit (HydC) of hydrogenase (or FdhC of formate dehydrogenase) upon collision with PsrC (Fig 5) MMbreduction is assumed to be coupled to proton uptake from the cytoplasmic side of the membrane, and
MMbH±oxidation to be coupled to proton release at the periplasmic side MMb and MMbH± are thought to be located in the hydrophobic part of PsrC Therefore, the uptake and release of protons is expected to be guided by proton channels The channel for proton release should be
in PsrC and that for proton uptake in 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 EpH8
o of MMb/MMbH±is assumed to be )260 mV [reaction (5) in Table 3] Using this value, the free energy (DGpH8
o ) of MMbreduction by H2and of MMbH±oxidation
by polysul®de is calculated (Table 4, Dw 0) The free energy values are expected to be altered by the
Dw 170 mV across the membrane of growing cells Assuming MMband MMbH± to be located in the center
of the membrane, each electron derived from H2 would become 85 mV more electropositive on its way from the periplasmic side to MMb(Fig 5) The simultaneous transfer
of a proton from the cytoplasmic side to MMbshould affect the free energy of MMbreduction by H2in the same way Thus MMb reduction by H2 should become 24 kJámol)1 more endergonic, as two electrons (derived from H2) from
Table 3 Redox potentials of compounds involved in polysul®de
respi-ration with H 2 E o ¢ of reaction (3) was taken from [42] The values of
reactions (2), (4), and (5) are derived as described in the text.
E o ¢ (mV) E pH8
o
(1) H 2 ® 2H + + 2e ± )420 )480
(2) MMH 2 ® MM + 2H + + 2e ± )90 )150
(3) HS ± ® 1/8 S 8 + H + + 2e ± )275 )305
(4) 4HS ± ® S + 4H + + 6e ± )260 -300
(5) MM b H ± ® MM b + H + + 2e ± )230 )260
Trang 8the periplasmic (positive) side and one proton from the
cytoplasmic side are transferred to MMb MMbH±
oxida-tion should become 8 kJámol)1 more exergonic, as it is
coupled to the transfer of two electrons and one proton
from the center of the membrane to the periplasmic side
The free energy conserved in the Dp generated by polysul®de
respiration is given by the difference in DGpH8
o of polysul®de reduction by H2in the absence and in the presence of the Dw (16 kJámol Hÿ1
2 ), and is exclusively conserved from MMb reduction by H2(Table 4) This value is consistent with the formation of 0.33 mol ATP per mol H2at a phosphoryla-tion potential of 44 kJámol ATP)1 [45] This ATP yield would be half that of fumarate respiration, in agreement with the growth yields [2,3,6]
The effect of Dp on electron transport activity The effect of Dp on the activity of electron transport (Fig 3) can be explained if it is assumed that the activity of polysul®de respiration in the membrane fraction is limited
by the amount of MMbH±in the absence of a Dp, and that
MMbH±dissociates from PsrC (C) according to reaction (g), where H
i designates a proton taken up from or released to the cytoplasmic side of the membrane
MMbH±+ H
i ® MMH2+ C (g) Assuming that MMbH± is located in the center of the membrane, its amount should be increased by a Dp across the membrane according to reaction (g), whose velocity could be lower than the activity of polysul®de respiration The view that polysul®de respiration is limited by the amount of MMbH±in the membrane fraction in the absence
of a Dp is supported by the experiment shown in Fig 2
In this experiment, polysul®de respiration was stimulated
Fig 5 Hypothetical mechanism of polysul®de respiration with H 2
MM b , MM bound to PsrC; MM b H ± , hydroquinone anion of MM
bound to PsrC The dotted and the striped areas designate Hyd and
Psr, respectively Hyd catalyses the reduction of MM b by H 2 , probably
at one of the hemes This reaction is coupled to the uptake of a proton
from the cytoplasm Psr catalyses the oxidation of MM b H ± by [S]
which is coupled to the release of a proton to the periplasm.
Table 4 Standard 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 DG pH8
o values were calculated from the E pH8
o of reactions 1, 4, and 5 given in Table 3 Dw Designates the electrical potential across the membrane which is generated by polysul®de respiration [3,5].
DG pH8
o (kJá mol )1 )
Dw 0 mV Dw 170 mV
H 2 + MM b ® MM b H ± + H + )43 )19
MM b H ± + [S] ® MM b + HS ± +8 0 Total: H 2 + [S] ® HS ) + H + )35 )19
Fig 6 Hypothetical topology of PsrC
Resi-dues replaced in PsrC by site-directed
muta-genesis are indicated Residues in bold letters
correspond to mutants with 5% or less of the
wild-type speci®c activities of polysul®de
res-piration.
Trang 9nearly two-fold upon the incorporation of additional MM.
As the MM dissolved in the membrane is likely to be fully
reduced in the steady state of polysul®de respiration, the
amount of MMbH±should increase according to reaction
(g) as increasing amounts of MM are incorporated into the
membrane On the basis of these considerations, the
titration curve (Fig 2) re¯ects the formation of MMbH±
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 (MMb) of the membrane anchor
(PsrC) and the catalytic subunit (PsrA) of Psr Therefore,
PsrA is probably bound to PsrB which in turn is bound to
PsrC on the periplasmic side of the membrane Dissociation
of PsrA from the membrane was not observed in any of the
psrC mutants listed in Table 2 (mutant DpsrC excepted)
The activities of Psr are not impaired in the mutants listed
in Table 2 This also refers to the mutants lacking at least
95% of the activities of polysul®de respiration The residues
substituted in these mutants (in bold type in Fig 6) are
either charged at neutral pH or are tyrosines, the phenolic
hydroxyl groups of which appear to be essential for
polysul®de respiration The function of these residues may
be explained on the basis of the hypothetical mechanism
depicted in Fig 5 R305 is well suited for binding and
stabilizing the postulated quinol anion of MM (MMbH±)
which is thought to be bound to PsrC in a hydrophobic
environment The mere positive charge of R305 is
appar-ently not suf®cient for its function, as the smaller lysine
residue did not substitute for R305 D218 and E225 may
serve in guiding the proton formed by the oxidation of
MMbH±to the periplasmic side of the membrane
Consis-tent with this function, the negative charge of E225 appears
to be essential, as mutant E225Q was nearly inactive, while
E225D showed more than 25% of the wild-type activity
D76 as well as Y23 and Y159 may possibly involved in the
proton transfer to and from the cytoplasmic side according
to reaction (g) which describes the binding of reduced MM
to PsrC and its dissociation Consistent with this function,
substitution of D76 by asparagine or leucine resulted 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 MMband MMbH±
W succinogenes mutants with one of the four heme
ligands of HydC replaced were found to lack the activities of
electron transport form H2to polysul®de and to fumarate
[15] The heme groups appear to be required for the
reduction of MMband of MK However, it is 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
MMbprotrudes from its binding site on PsrC into the lipid
phase of the membrane to accept electrons form the heme
group(s) together with a proton as depicted in Fig 5
Alternatively, MMH2 transiently bound to the
cyto-chrome b might transfer a hydride to MMb Consistent
with the latter mechanism, hydrogenase was found to
catalyze MM reduction by H2 which is coupled to the
generation of a Dw across the membrane of liposomes
containing hydrogenase (S Biel, J Simon & R Gross, Institut fuÈr Mikrobiologie, Johann Wolfgang Goethe-UniversitaÈt Frankfurt-am-Main, personal communication)
In fumarate respiration, MK may be reduced at the same site on HydC In this case, the resulting MKH2would be free to diffuse to fumarate reductase
A C K N O W L E D G E M E N T S The authors are indebted to A KroÈger for helpful discussion and to O SchuÈrmann for skilful technical assistance The work was supported by the Deutsche Forschungsgemeinschaft (SFB 472).
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