Reconstitution of coupled fumarate respiration in liposomesby incorporating the electron transport enzymes isolated Simone Biel1, Jo¨rg Simon1, Roland Gross1, Teresa Ruiz2, Maarten Ruite
Trang 1Reconstitution of coupled fumarate respiration in liposomes
by incorporating the electron transport enzymes isolated
Simone Biel1, Jo¨rg Simon1, Roland Gross1, Teresa Ruiz2, Maarten Ruitenberg3and Achim Kro¨ger1
1
Institut fu¨r Mikrobiologie, Johann Wolfgang Goethe-Universita¨t, Frankfurt am Main, Germany;2Max-Planck-Institut fu¨r Biophysik, Abteilung Strukturbiologie, Frankfurt am Main, Germany;3Max-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 enzymes were found to be
oriented solely to the outside of the resulting
proteolipo-somes The proteoliposomes catalyzed fumarate reduction
by H2which generated an electrical proton potential (Dw¼
0.19 V, negative inside) in the same direction as that
gen-erated by fumarate respiration in cells of W succinogenes
The H+/e ratio brought about by fumarate reduction with
H2in proteoliposomes in the presence of valinomycin and
external K+was approximately 1 The same Dw and H+/e
ratio was associated with the reduction of
2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes
con-taining menaquinone and hydrogenase with or without
fumarate reductase Proteoliposomes containing
menaqui-none and fumarate reductase with or without hydrogenase
catalyzed fumarate reduction by DMNH2 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 demonstrate for 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 H2or formate, but not to menaquinol oxidation 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 H2or formate oxidation on the periplasmic side This mechanism
is supported by the properties of two hydrogenase mutants
of W succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane
Keywords: fumarate respiration; Wolinella succinogenes; proteoliposomes; H+/ e ratio; hydrogenase
The electron transport chain catalyzing fumarate respiration
with H2 (reaction a) or formate (reaction b) in Wolinella
succinogenesconsists of fumarate reductase, menaquinone
(MK), and either hydrogenase or formate dehydrogenase
(Fig 1)
H2þ Fumarate ! Succinate ðaÞ HCOÿ2 þ Fumarate þ H2O! HCOÿ3 þ Succinate ðbÞ The enzymes were isolated and the corresponding genes were sequenced [2,3] Each of the three enzymes consists of two hydrophilic subunits and a di-heme cytochrome b which is integrated in the membrane [4–7] The iron–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 (MKH2) is oxidized at 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 DMNH2 oxidation by fumarate (reaction e) The site of DMNH2 oxidation is located
on FrdC [4]
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.: + 49 69 79829507,
E-mail: A.Kroeger@em.uni-frankfurt.de
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 membrane
(in volts); Dw, electrical proton potential across a membrane (in volts).
(Received 6 December 2001, revised 12 February 2002, accepted 21
February 2002)
Trang 2HCOÿ2 þ DMN þ Hþ! CO2þ DMNH2 ðdÞ
DMNH2þ 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 H2or formate at the
catalytic sites of the enzymes on HydB or FdhA are
probably released on the periplasmic side of the 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, H2is split into protons and electrons at the active
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 of
W succinogenesformate dehydrogenase [14] As seen from
its crystal structure, the subunits of the E coli enzyme are
arranged as depicted 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, CO2 and protons, are probably released
through the 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 to HydA of W succinogenes [1] The helix
is predicted to be absent in FdhB [7]
Cells of W succinogenes catalyzing fumarate respiration with H2(reaction a) or formate (reaction 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 H2 or formate oxidation with DMN (reaction c or d) [16] In contrast, DMNH2oxidation by fumarate (reaction e) was not coupled to Dw generation Inverted vesicles of the
W succinogenesmembrane catalyzed fumarate respiration with H2, which generated a Dw¼ 0.18 V (positive inside) [15] The corresponding H+/e ratio was close to 1 The reduction of DMN by H2 catalyzed by these vesicles generated a much lower Dw, and the H+/e ratio was below 0.5
Proteoliposomes containing fumarate reductase, vita-min K1, and either formate dehydrogenase or hydro-genase were found to catalyze fumarate reduction by formate or H2 at the expected specific activities [17–19] The two reactions were not coupled to Dp generation or 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 H2 or formate, by menaquinol oxidation with fumarate, or by both reactions; and (c) what is the mechanism of Dp generation
E X P E R I M E N T A L P R O C E D U R E S
Preparation of proteoliposomes Phosphatidylcholine was prepared from egg yolk according
to Singleton et al leaving out the chromatographic steps [20] Di-palmitoyl phosphatidate was purchased from Fluka MK was extracted from the membrane fraction of
W succinogenesand separated from methyl-MK by HPLC [21] MK and methyl-MK of W succinogenes carry a side chain with six isoprene units Phosphatidylcholine (50 mg) and phosphatidate (5 mg) were dissolved in a mixture of CHCl3and methanol (2 : 1, v/v) After the addition of MK (10 lmolÆg phospholipid)1), the solvents were evaporated, and the residue was sonicated at 0°C in 50 mM Hepes (adjusted to pH 7.5 with KOH) until minimum turbidity of the suspension The resulting 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)1in 50 mM 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 according to [6] and/or fumarate reductase [18] (0.18 gÆg phospholipid)1), stirring was continued for 1 h For removal of detergent, Bio-Beads SM-2 (Bio-Rad) (0.24 gÆmL)1) were added and stirring was continued for 1 h
Fig 1 Composition and orientation of the enzymes involved in fumarate
respiration of W succinogenes Fumarate reductase (FrdA, B, C) and
formate dehydrogenase (FdhA, B, C) are integrated in the membrane
by their di-heme cytochrome b subunits (FrdC and FdhC)
Hydro-genase (HydA, B, C) is integrated in the membrane by its di-heme
cytochrome b subunit (HydC) and the C-terminal hydrophobic stretch
of HydA [1] HydC and FdhC carry the sites of MK reduction MKH 2
is oxidized at FrdC Ni, catalytic site of hydrogenase; Mo,
molybde-num ion coordinated by molybdopterin guanine dinucleotide; Fe/S,
iron–sulfur centers; Cyt b, di-heme cytochrome b.
Trang 3Proteoliposomes 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 mM Hepes, 2 mM
malonate, and 1 mM azide After the addition of formate
dehydrogenase [18] (40 mgÆg phospholipid)1) and
fuma-rate reductase (0.16 gÆg phospholipid)1), the mixture was
frozen in liquid N2and then thawed at room temperature
Freeze-thawing was repeated twice The detergent
intro-duced with the enzyme preparations was removed by
stirring the mixture for 1 h with Bio-Beads SM-2
(0.5 gÆmL)1) The suspension was sonicated (Branson
sonifier equipped with a microtip) for 20 s at 0°C before
use
Enzymic activities of proteoliposomes and protein
The reduction of fumarate by H2 or formate was
recorded as the absorbance difference at 270 minus
290 nm (De¼ 0.45 mM )1Æcm)1) in a buffer (50 mM
Hepes, pH 7.5, 37°C) containing 2 mM fumarate and
flushed with H2 [or N2 when formate (10 mM) was used
as electron donor] DMN (0.2 mM) reduction by H2 or
formate (10 mM) as well as DMNH2 (0.2 mM) oxidation
by fumarate (1 mM) was recorded in the same buffer
using the same wavelength pair (De¼ 15.2 mM )1Æcm)1)
Methyl viologen reduction by H2 was recorded at
578 nm (e¼ 9.8 mM )1Æcm)1) in a H2-saturated buffer
(0.15M 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 mMHepes buffer (pH 7.5, 25°C) which was flushed
with H2(or N2when formate was used) The TPP+electrode
was calibrated by adding known amounts of TPP+before
the electron transport was started by the addition of the
substrates Dw was calculated from the TPP+concentrations
within the proteoliposomes (Ti) and in the medium (Te) using
the Nernst equation Tiwas calculated from the maximum
amount of TPP+(Ts, in molÆg phospholipid)1) taken up
from the medium in the steady state of electron transport
according to Eqn (1) [16,25]
ðTiÞnþ 1 ¼ Te þ Tsÿ ViðTiÞn
ðTiÞn
Te
ð1Þ
Vi(3.5 mLÆg 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 mM phosphate, after gel
filtration using a Sephacryl S-1000 SF (Pharmacia) column
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+ The internal TPP+ concentration (Ti)n+1 was
calculated from an assumed value of (Ti)n(Eqn 1) Using
the value so obtained, calculation was repeated until (Ti)n+1
was consistent with (T)
Measurement of H+/e ratios Proteoliposomes containing hydrogenase, MK, and fuma-rate reductase were prepared as described above, however, the preparation buffer contained 95 mMHepes (adjusted to
pH 7.3 by KOH) The suspension was dialyzed for 14 h against buffer C (50 lM Hepes, 45 mM KCl and 50 mM
sucrose, pH 7.3, 0°C), flushed with H2 After valinomycin (0.5 lmolÆg phospholipid)1) and phenol red (60 lM) had been added, the suspension (1 g phospholipidÆL)1) was mixed with fumarate (5 mM) or DMN (50–100 lM) in buffer C at 25°C A stop-flow spectrophotometer was used for mixing [26] Ten volumes of the suspension were mixed with one volume of substrate 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 filtration using a Sephadex G-25 column (Pharmacia) equilibrated with N2-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 mM) and DMN (50–100 lM) or formate (100 mM) at 25°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 lM trypsin was mixed with TAME (9.1 or 18.2 lM final concentration) in buffer C
at 25°C
Construction ofW 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 complementary prim-ers was used for each modification (forward primer used for mutant N128D: 5¢-(3642)–CTCAAAGGGGTT TACGATCCCGTTCAGCTAGC-3¢, and for mutant Q131L: 5¢-(3649)–GGGTTTACAATCCCGTTCTCCTA 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 mgÆL)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]
Trang 4R E S U L T S
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 to 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 H2to fumarate was measured in the various
preparations (Fig 2A) The activity increased with
increas-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 (VET) from the activities of hydrogenase
(H2 fi DMN) and fumarate reductase (DMNH2 fi
Fumarate), suggesting that only some of the enzyme
molecules were involved in electron transport (H2 fi
Fumarate) The activities measured with proteoliposomes
prepared at the critical or a higher ratio were close to the
theoretical ones
The activity of hydrogenase measured with DMN as
acceptor was nearly the same in the different preparations
(Fig 2A) In contrast, the activity of fumarate reductase
(DMNH2 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 activity reflected the accessibility of fumarate reductase
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 Triton 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 two 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 H2 in the different preparations
(Fig 2A) Methyl viologen does not penetrate the
mem-brane at a velocity commensurate with that of its reduction,
in contrast to H2and DMN [11,31] The activity of methyl
viologen reduction by H2was the same in the preparations
obtained at the critical or lower ratios, and was 70% and
65% of this activity in the proteoliposomes prepared at the
two highest ratios This suggests that hydrogenase is
completely exposed to the outside in the proteoliposomes
prepared at the critical or lower ratios, whereas 30–35% of
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 H2 to fumarate was highest with proteoliposomes prepared at the critical ratio and was lower with the other preparations (Fig 2B) A similar result was obtained for DMN reduction
by H Thus TPP+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 or fumarate was measured as shown 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 in the medium.
Trang 5coupled to the reduction 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 the inside (see Discussion)
In the following only proteoliposomes prepared at the
critical ratio are used, unless indicated otherwise
Gel filtration 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 H2 to fumarate were
about 10% of those in growing bacteria
Assuming that the proteoliposomes are spherical, their
average internal volume (3.5 mLÆg 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 between 50 nm and 70 nm (not shown)
The external surface of the vesicles was studded with
particles which probably represent fumarate reductase and
hydrogenase molecules [30] Assuming that 1 g
phospho-lipid corresponds to an outer membrane surface of
2.6· 106cm2 [32], a spherical proteoliposome of 100 nm
(or 50 nm) external diameter is calculated to carry 94 (or 23)
molecules of fumarate reductase (monomeric) and 12 (or 3)
molecules of hydrogenase As all the active enzyme
mole-cules appear to participate in electron transport from H2to
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 H2 (Fig 3) After the
addition of TPP+, its concentration was recorded 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 the presence of a protonophore (FCCP)
The experiment suggests that the electron transport from H2
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+ (Ti) was calculated from the
maximal amount of TPP+taken up in the steady state of
electron transport (Ts) Tiwas calculated according to the
method designed by Zaritsky et al (Eqn 1) [25] The value
of Tiso obtained corresponded to 33% of the amount of
TPP+ (T) taken up during fumarate respiration in the
experiment shown in Fig 3 The residual part of Ts is thought to be bound to the proteoliposomal membrane Dw was calculated from Ti and the corresponding external TPP+concentration (Te) according to the Nernst equation The Dw generated by fumarate respiration with H2in the experiment shown in Fig 3 was determined to be 0.19 V (Table 1) A Dw of the same direction and strength was generated by DMN reduction with H2 In contrast, no TPP+uptake was observed during fumarate reduction by DMNH2 This reaction also did not cause the uptake of tetraphenylboranate (TPB–) in a similar experiment per-formed with a TPB–electrode [16] (not shown) Proteolipo-somes containing MK and only hydrogenase catalyzed DMN reduction by H2which 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 Dw¼ 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 generated by DMN reduction with formate
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] Proteoliposomes containing hydro-genase, MK, and fumarate reductase suspended in a buffer (50 lM Hepes and 45 mM KCl) saturated with H2 were treated with valinomycin (0.5 lmol g)1phospholipid) The amount of valinomycin was just sufficient to prevent TPP+
uptake driven by the reduction of DMN or fumarate with
H After the addition of phenol red, DMN reduction by H
Fig 3 Recording of the external TPP+concentration in a suspension of proteoliposomes during fumarate reduction by H 2 Proteoliposomes containing hydrogenase, MK, and fumarate reductase were suspended (0.4 g phospholipidÆL)1) in an H 2 -saturated buffer (pH 7.5, 25 °C) The TPP + electrode was calibrated by three additions of 1 l M TPP + The electron transport was started by adding fumarate 20 lmol FCCP per g phospholipid was applied were indicated.
Trang 6was started by the addition of a small amount of DMN
(Fig 4A) The number of protons released into the external
medium within 1 s was proportional to the added amount
of DMN The H+/e ratio was calculated from the protons released and the amount of DMN added The time course
of proton release was consistent with that of DMN reduction by H2 The Kmfor DMN of this reaction was determined to be 15 lM(not shown) The release of protons did not occur with proteoliposomes which had been treated with a protonophore (FCCP, curve III)
The H+/e ratio of fumarate reduction by H2was measured 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 separate experiment in the absence of phenol 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 with a protonophore (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 N2, 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 When fumarate was added 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 H2 or formate obtained from various experi-ments were close to 1
HydC mutants
To understand the mechanism of the Dp generation which is coupled to quinone reduction by H2or formate, the site of 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 fumarate reductase in the H 2 -saturated suspension designated 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 the Experimental procedures Proteoliposomes treated with FCCP
(20 lmolÆg phospholipid)1) were used in experiments III and V The
concentration of DMN 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) in the absence of phenol
red (VI) The slopes of curves IV and VI were used for calculating the
H + /e ratio of fumarate reduction by H
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 experiments were performed as described
in Fig 3 However, the suspension was flushed with N 2 instead of H 2 when DMNH 2 (1 m M ) or formate (1 m M ) were used as donor DMN was applied at 1 m M concentrations T s represents the maximum amount of TPP + taken up by the proteoliposomes in the steady state of electron transport, and T e the corresponding TPP+concentration in the medium (see Fig 3) The internal TPP+concentration (T i ) was calculated according to Eqn (1) Dw was calculated from T e and T i according to the Nernst equation.
Donor Acceptor
Activity (UÆmg phospholipid)1)
T s
(lmolÆg phospholipid)1)
T i
(l M )
T e
(l M )
Dw (V)
DMNH 2 Fumarate 4.1 No TPP+uptake
Trang 7cytochrome b subunit (FdnI) of E coli formate
dehydro-genase-N [9] HydC is schematically drawn in Fig 5 based
on the structure of FdnI [14] The two heme groups of FdnI
are in electron transfer distance nearly on top of each other
when viewed along the membrane normal The proximal
(upper) heme group is in electron transfer distance to one of
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 connecting helices II and III 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 or leucine (Table 3) The
two mutants (N128D and Q131L) did not grow by fumarate
respiration with H2 When grown with formate and
fumarate, the mutant cells did not catalyze fumarate
reduction by H2, in contrast to the wild-type strain The
specific activities of DMN reduction by H2measured with
the membrane fraction of the mutants amounted to 6% or
less of the wild-type activity, whereas the activities of benzyl
viologen reduction by H2were close to 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 reduced upon H2addition to the Triton
X-100 extract of the oxidized membrane fraction was the
same with the mutants (0.3 lmolÆg protein)1) and with the
wild-type strain [29] Mutant H122A had wild-type
pro-perties with respect to growth and enzyme activities
Residue H122 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
D I S C U S S I O N
Energetics For technical reasons, the H+/e ratio of apparent proton translocation can only be measured at vanishing Dp It is generally thought that the same ratio is valid in the presence and absence of Dp As the amount of free energy conserved
by apparent proton translocation across the membrane cannot exceed that provided by the driving redox reaction, the H+/e ratio (nH +/ne) can be calculated from the redox potential difference (DE) and Dp according to Eqn (2), provided that the energetic efficiency (q) of the process is known
nHþ
ne ¼ qDE
Assuming q¼ 1, the theoretical maximum H+/e ratio of fumarate respiration with H2 (reaction a) is calculated to
be 2.6, using Dp¼ 0.17 V, and DE ¼ 0.45 V [from Eo¢ for
H+/H2 ()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 formate and HCO3 as its oxidation product (reaction b), as the corresponding value of DEo¢ is close to that obtained with H
Table 2 H + /e ratios measured with proteoliposomes The
proteolipo-somal preparation (A) contained MK, hydrogenase, and fumarate
reductase In preparation (B) hydrogenase was replaced by formate
dehydrogenase The upper two experiments were performed as
described in Fig 4A,B The H+/e ratio with formate and DMN was
measured as shown in Fig 4A However, the proteoliposomes were
suspended in a buffer flushed with N 2 instead of H 2 , and the
suspen-sion was mixed with a solution containing formate and DMN In the
experiment with DMNH 2 and fumarate, the anoxic proteoliposomal
suspension containing DMNH 2 (0.2 m M ) was mixed with fumarate
(5 m M ), and the absorbance of phenol red was recorded The reduction
by formate of the MK present in the proteoliposomes was observed at
270 nm when the proteoliposomes were mixed with formate in the
absence of fumarate and phenol red The corresponding H+/e ratio
was calculated using the velocity of MK reduction (De ¼ 12.0 m M )1
cm)1) n represents the number of measurements with different
pre-parations of proteoliposomes.
Preparation Donor Acceptor H+/e ratio n
A H 2 Fumarate 1.0 ± 0.1 6
A H 2 DMN 0.96 ± 0.04 10
A DMNH 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 is based on the crystal structure of E coli FdnI [14] The shaded squares represent the heme groups A molecule of HQNO is shown at the site of MK reduction which is confined by the axial ligand H200 of the distal heme group and by residues N128 and Q131 in the stretch connecting the hydrophobic parts of helices II and III.
Trang 8The theoretical maximum H+/e ratio of fumarate
reduction by the MKH2within the bacterial membrane is
calculated to be 0.6, assuming the redox potential of the
MK/MKH2couple to be equal to its standard potential in
organic solution (Eo¢ ¼)0.074 V [34]) This assumption is
consistent with the finding that the MK/MKH2ratio is not
far from 1 in the membrane of W succinogenes catalyzing
fumarate respiration [35] Furthermore, the standard
poten-tial of MK/MKH2in a bacterial membrane was measured
to be close to that in organic solution [36] Therefore, the
actual H+/e ratio of MKH2oxidation by fumarate should
be lower than 0.6
The site of MKH2oxidation
The site of MKH2oxidation on the cytochrome b subunit
(FrdC) of fumarate reductase is not known In the
crystallographic model of the oxidized enzyme, a cavity
was discovered which extends from the hydrophobic phase
of the membrane, close to the distal heme group of FrdC to
the periplasmic aqueous phase [37] The cavity could
accommodate a MKH2 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 MKH2 Replacement of E66 by a
glutamine residue resulted in a mutant (E66Q) which did
not catalyze DMNH2 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 mutant enzyme is due to the
absence of the carboxylate group of E66 In the wild-type
enzyme, E66 could facilitate quinol oxidation by accepting
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 the reaction is predicted to be 1 This value is
higher than the maximum possible value predicted by the
energetic calculation (see above) Furthermore, fumarate
reduction by DMNH2was not coupled to Dw generation in
cells, inverted vesicles [16], or in proteoliposomes (Table 1)
Mechanism of Dp generation
In the model mechanisms drawn in Fig 6, H2oxidation by
MK is assumed to 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 H2 oxidation on the outside (Fig 6A,B) MKH2 oxidation by fumarate is depicted as
an electroneutral process in Fig 6A, whereas 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 Fumarate) or with the membrane fraction of cells grown with formate and fumarate as described [29] The properties of mutant H122A were taken from [29].
Strain Doubling time (h)
UÆmg cell protein)1
H 2 fi Fumarate H 2 fi DMN H 2 fi Benzyl viologen
Fig 6 Hypothetical mechanisms of Dp generation in proteoliposomes (A and B) in cells of W succinogenes (C) The sites of MK reduction and of MKH 2 oxidation are drawn schematically in the center of the membrane These sites may actually be located closer to the membrane surfaces Equivalent mechanisms may apply with formate as electron donor instead of H 2 p, periplasmic side of the membrane; c, cyto-plasmic side.
Trang 9genic in Fig 6B In Fig 6A, the protons formed by MKH2
oxidation are released on the outside, where they balance
the protons consumed by fumarate reduction, and fumarate
respiration with H2is predicted to be electrogenic with a
H+/e ratio of 1 In contrast, fumarate respiration with H2is
predicted to be an electroneutral process in 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 H2
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 MKH2 has to be an
electroneutral process in the proteoliposomes This
conclu-sion is confirmed by the result that DMNH2oxidation by
fumarate was not coupled to the uptake of TPP+or TPB–
by the proteoliposomes DMNH2 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 H2
were found to accumulate SCN–, and the H+/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, the H+/e ratio should be 2 However, the H+/e
ratio of fumarate respiration is apparently not affected by
the orientation of the enzymes relative to each other and is
the same in inverted vesicles and proteoliposomes This
confirms the electroneutral operation of fumarate reductase
in the bacterial membrane (Fig 6C) as well as in the
proteoliposomes (Fig 6A)
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 of
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 H2 is inhibited in these mutants, suggesting
that the site of quinone reduction on HydC is located
close to the cytoplasmic surface of the membrane (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]
A C K N O W L E D G E M E N T S
This work was supported by grants of the Deutsche
Forschungsgeme-inschaft (SFB 472) and by the Fonds der Chemischen Industrie to
A Kro¨ger.
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