Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes Simone Biel 1 ,Jo¨ rg Simon 1 , Roland Gross 1 , Teresa Ruiz 2 , Maarten Ruitenberg 3 and Achim Kro¨ ger 1 1 Institut fu ¨ r Mikrobiologie, Johann Wolfgang Goethe-Universita ¨ t, Frankfurt am Main, Germany; 2 Max-Planck-Institut fu ¨ r Biophysik, Abteilung Strukturbiologie, Frankfurt am Main, Germany; 3 Max-Planck-Institut fu ¨ r Biophysik, Abteilung Biophysikalische Chemie, Frankfurt am Main, Germany Hydrogenase and fumarate reductase isolated from Woli- nella succinogenes were incorporated into liposomes con- taining menaquinone. The two e nzymes were found to be oriented solely to the outside of the resulting proteolipo- somes. The proteoliposomes catalyzed fumarate reduction by H 2 which generated an electrical p roton potential (Dw ¼ 0.19 V, negative inside) i n the same direction as that gen- erated by fumarate respiration in cells of W. succinogenes. The H + /e ratio brought abo ut by f umarate reduction with H 2 in proteoliposomes in the presence of v alinomycin and external K + was a pproximately 1 . T he same Dw and H + /e ratio was associated with the r eduction of 2,3-dimethyl-1,4- naphthoquinone (DMN) by H 2 in proteoliposomes con- taining menaquinone and h ydrogenase with or without fumarate reductase. Proteoliposomes containing menaqui- none and fumarate reductase with o r without hydrogenase catalyzed fumarate reduction by DMNH 2 which did not generate a Dw. Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Dw of 0.13 V ( negative inside). The H + /e ratio of formate oxidation by menaquinone or DMN was close to 1. The results d emonstrate f or the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Dw generation is coupled to menaquinone reduction by H 2 or formate, but not to menaquinol oxid ation by fumarate. Dw generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H 2 or formate o xidation on the p eriplasmic side. This mechanism is supported b y t he properties o f t wo hydrogenase m utants of W. succinogenes which indicate that the s ite of quinone reduction is close to the cytoplasmic surface of the membra ne. Keywords: fumarate respiration; Wolinella succinogenes; proteoliposomes; H + / e ratio; hydrogenase. The electron transport chain catalyzing fumarate respiration with H 2 (reaction a) or formate (reaction b) in Wolinella succinogenes consists of fumarate reductase, menaquinone (MK), and either hydrogenase or formate dehydrogenase (Fig. 1 ). H 2 þ Fumarate ! Succinate ðaÞ HCO À 2 þ Fumarate þ H 2 O ! HCO À 3 þ Succinate ðbÞ The enzymes were isolated and the corresponding genes were sequenced [2,3]. Each of the t hree enzymes consists of two hydrophilic subunits and a di-heme cytochrome b which is integrated in the membrane [4–7]. The i ron–sulfur subunits (HydA, FdhB, FrdB) mediate electron transfer from the catalytic subunits to the cytochromes b or vice versa [8]. The di-heme cytochromes b of hydrogenase and of formate dehydrogenase carry the sites of MK reduction, and are similar in their sequences [6,9,10]. Menaquinol (MKH 2 ) is oxidized a t the di-heme cytochrome b of fumarate reductase [4,5]. The dehydrogenases (hydrogenase and formate dehy- drogenase) catalyze the reduction of the water soluble MK analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) by their respective substrates (reaction c and d). The site of DMN reduction is located on HydC [6]. Fumarate reductase catalyzes DMNH 2 oxidation by fumarate (reaction e). The site of DMNH 2 oxidation is located on FrdC [4]. H 2 þ DMN ! DMNH 2 ðcÞ Correspondence to A. Kro ¨ ger, Institut fu ¨ r Mikrobiologie, Johann Wolfgang Goethe-Universita ¨ t, Marie- Curie-Str. 9, D-60439 Frankfurt am Main, Germany. Fax: + 49 69 79829527, Tel.: + 4 9 6 9 79829507, E-mail: 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 membr ane (in volts); Dw, e lectrical proton potential across a membrane (in volts). (Received 6 December 2001, r evised 12 February 2002, accepted 21 February 20 02) Eur. J. Biochem. 269, 1974–1983 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02842.x HCO À 2 þ DMN þ H þ ! CO 2 þ DMNH 2 ðdÞ DMNH 2 þ Fumarate ! DMN þ Succinate ðeÞ The substrate sites of hydrogenase and of formate dehydrogenase are exposed to the bacterial periplasm, whereas that of fumarate reductase faces the cytoplasm (Fig. 1) [1,11]. From the crystal structure of fumarate reductase it is obvious that the protons consumed by fumarate reduction at the catalytic site of FrdA are taken up from the cytoplasmic side of the membrane [8]. The protons liberated by the oxidation of H 2 or formate a t the catalytic sites of the enzymes on HydB or FdhA are probably released on the periplasmic s ide of t he membrane. This is suggested by the crystal structures of related enzymes. The periplasmic Nickel hydrogenases isolated from sulfate reducing bacteria consist of two subunits which are similar to HydA and HydB of W. succinogenes hydrogenase. As suggested by the structures of those enzymes, H 2 is split into protons and electrons at the a ctive site [12,13]. The protons are released at the surface of the catalytic subunit. The electrons are passed by three consecutive iron–sulfur centers to a cytochrome c which binds to the surface of the iron–sulfur subunit. The corresponding electron acceptor in the case of W. succin- ogenes hydrogenase is the di-heme cytochrome b HydC which is a subunit of the enzyme. Escherichia coli formate dehydrogenase-N consists of three different subunits whose sequences resemble those o f W. s uccinogenes formate dehydrogenase [14]. A s seen from its crystal structure, the subunits of the E. coli enzyme are arranged as depict ed in Fig. 1 [ 14]. A cavity in the catalytic subunit of E. coli formate dehydrogenase-N extends from the surface to the molybdenum ion where formate is oxidized. The electrons derived from formate are likely to be passed to the iron–sulfur center close to the molybdenum. The products, CO 2 and proton s, are probably released through t he cavity. A similar mechanism is likely to apply for W. succinogenes formate dehydrogenase. The C-termi- nus of the iron–sulfur subunit (FdnH) of E. coli formate dehydrogenase-N forms a membrane-spanning helix [14]. This applies also t o HydA of W. succinogenes [1]. The helix is predicted to be absent in FdhB [7]. Cells of W. succinogenes ca talyzing fumarate respiration with H 2 (reaction a) or formate (re action b) were found to develop a Dw of 0.14 or 0.16 V (negative inside) [15,16]. The corresponding DpH across the membrane was found to be negligible. A similar Dw was generated in cells by H 2 or formate oxidation with DMN (reaction c or d) [16]. In contrast, DMNH 2 oxidation by fumarate (reaction e) was not coupled to Dw generation. Inverted vesicles of the W. su ccinogenes membrane catalyzed fumarate respiration with H 2 , which generated a Dw ¼ 0.18 V (positive inside) [15]. The corresponding H + /e ratio was close to 1. The reduction of DMN by H 2 catalyzed by these vesicles generated a much lower Dw,andtheH + /e ratio was below 0.5. Proteoliposomes containing fumarate reductase, vita- min K 1 , and either formate dehydrogenase or hydro- genase were found to catalyze fumarate reduction by formate or H 2 at the expected specific activities [17–19]. The two reactions were not coupled to Dpgenerationor the Dp generated was very low [19]. In this paper, we address the following questions: (a) can coupled fumarate respiration be restored by incorporating the isolated enzymes into liposomes containing menaquinone; (b) is the Dp generated by menaquinone reduction with H 2 or formate, by menaquinol oxidation with fumarate, or by both reactions; and (c) what is the mechanism of Dp generation. EXPERIMENTAL PROCEDURES Preparation of proteoliposomes Phosphatidylcholine w as prepared from egg yolk a ccording to Singleton et al. leaving out the chromatographic steps [20]. Di-palmitoyl phosphatidate was purchased from Fluka. MK w as extracted f rom the membrane fraction of W. su ccinogenes and separated from methyl-MK by H PLC [21]. MK and methyl-MK o f W. succinogenes carry a side chain with six isoprene units. Phosphatidylcholine (50 mg) and phosphatidate (5 mg) were dissolved in a mixture of CHCl 3 and methanol ( 2 : 1, v/v). A fter the a ddition of MK (10 lmolÆg phospholipid )1 ), the solvents were evaporated, and the residue was sonicated at 0 °Cin50m M Hepes (adjusted t o pH 7.5 with KOH) until minimum t urbidity of the s uspension. The r esulting suspension of sonic liposomes contained 10 g phospholipidÆL )1 . Proteoliposomes containing hydrogenase and fumarate reductase were prepared according to a procedure previously described [22]. Dodecyl- b- D -maltoside (0.8 gÆg phospho- lipid )1 ) was added to a suspension of sonic liposomes containing MK (1 g phospholipidÆL )1 in 50 m M Hepes at pH 7.5), and the mixture was stirred for at least 3 h at room temperature. After the addition of hydrogenase (20 mgÆg phospholipid )1 ) prepared a ccording to [ 6] and/or fumarate reductase [18] (0.18 g Æg phospholipid )1 ), stirring was continued for 1 h. For removal of detergent, B io-Beads SM-2 ( Bio-Rad) (0.24 gÆmL )1 ) w ere added a nd stirring was continued for 1 h. Fig. 1. Composition and orientation of the enzymes involved in fumarate respiration o f W. succinogenes. Fuma rate reductase (FrdA, B, C) and formate d ehydrogenase (FdhA, B , C) are i ntegrated in t he membrane by th eir di-heme cytochrome b subunits (FrdC and F dhC). Hydro- genase (HydA, B, C) is integrated in the membrane by its di-heme cytochrome b sub unit (HydC) and the C -terminal hydrop hobic stretch of HydA [1]. HydC and FdhC carry the sites of MK reduction. MKH 2 is oxidized at FrdC. Ni, catalytic site of h ydrogenase; M o, molybde- num ion coordinated by molybd opterin guanine dinu cleotide; Fe/S, iron–sulfur centers; Cyt. b,di-hemecytochromeb. Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1975 Proteoliposomes containing formate dehydrogenase and fumarate reductase were prepared using sonic liposomes with MK (10 g phospholipidÆL )1 ) in a buffer (adjusted to pH 7.3 with KOH) containing 95 m M Hepes, 2 m M malonate, and 1 m M azide. After the addition of formate dehydrogenase [18] (40 mgÆg p hospholipid )1 )andfuma- rate reductase (0.16 g Æg phospholipid )1 ), the mixture was frozen in liquid N 2 and then thawed a t room temperature. Freeze-thawing was repeated twice. The detergent intro- duced with the enzyme preparations was removed by stirring the m ixture for 1 h with Bio-Beads SM-2 (0.5 g ÆmL )1 ). The suspension was sonicated (Branson sonifier equipped with a microtip) for 20 s at 0 °Cbefore use. Enzymic activities of proteoliposomes and protein The reduction of fumarate by H 2 or formate was recorded as the absorbance difference at 270 minus 290 nm (De ¼ 0.4 5 m M )1 Æcm )1 ) in a buffer (50 m M Hepes, pH 7.5, 37 °C) containing 2 m M fumarate and flushed with H 2 [or N 2 when formate (10 m M )wasused as electron donor]. DMN (0.2 m M ) reduction by H 2 or formate (10 m M )aswellasDMNH 2 (0.2 m M ) oxidation by fumarate (1 m M ) was recorded in the same buffer using the same wavelength pair (De ¼ 15.2 m M )1 Æcm )1 ). Methyl viologen reduction by H 2 was recorded at 578 nm (e ¼ 9.8 m M )1 Æcm )1 )inaH 2 -saturated buffer (0.15 M glycine, pH 9.5, 37 °C). The unit of activity (U) corresponds to the transfer of 2 lmol electronsÆmin )1 . Protein was determined using the Biuret method with KCN [23]. Determination of Dw The TPP + electrode was constructed according to [24]. Proteoliposomes were suspended (0.4 g phospholipidÆL )1 ) in 50 m M Hepes buffer (pH 7.5, 25 °C) which was flushed with H 2 (or N 2 when formate w as used). The T PP + electrode was calibrated by adding known a mounts of TPP + before the electron transport was started by the addition of the substrates . Dw wa s calculated from t he TPP + concentrations within the proteoliposomes ( T i ) and in the medium (T e )using the Nernst equation. T i was calculated from the maximum amount of TPP + (T s ,inmolÆg phospholipid )1 )takenup from the medium i n the steady state o f electron t ransport according t o E qn (1) [ 16,25]. ðT i Þ n þ 1 ¼ T e þ T s À V i ðT i Þ n K ln ðT i Þ n T e ð1Þ V i (3.5 mL Æg phospholipid )1 ) represents the average inter- nal volume of the proteoliposomes which was obtained from the amount of phosphate retained by proteoliposomes prepared in the presence of 50 m M phosphate, after gel filtration using a Sephacryl S-1000 SF (Pharmacia) c olumn. The binding constant K (53 mLÆg phospholipid )1 )was calculated from the amount of TPP + absorbed by the proteoliposomal membrane at various concentrations of TPP + .TheinternalTPP + concentration (T i ) n+1 was calculated from an assumed value of (T i ) n (Eqn 1). Using the value so obtained, calculation was repeated until (T i ) n+1 was consistent with (T i ) n . Measurement of H + /e ratios Proteoliposomes containing hydrogenase, MK, and fuma- rate reductase were prepared as described a bove, however, the preparation buffer contained 95 m M Hepes (adjusted to pH 7.3 by KOH). The suspension was dialyzed for 14 h against buffer C (50 l M Hepes, 45 m M KCl and 50 m M sucrose, pH 7.3, 0 °C), flushed with H 2 . After valinomycin (0.5 lmolÆg phospholipid )1 ) and phenol red (60 l M )had been added, the suspension (1 g phospholipidÆL )1 )was mixed with fumarate (5 m M ) or DMN (50–100 l M )in buffer C at 25 °C. A stop-flow spectrophotometer was used for mixing [26]. Ten volumes o f the suspension were mixed with one volume of sub strate. The amount of protons released was calculated from the absorbance change of phenol red at 550 nm. For buffer exchange, proteoliposomes containing for- mate dehydrogenase and fumarate reductase were subjected to gel fi ltration using a Sephadex G -25 column (Pharmacia) equilibrated with N 2 -flushed buffer C at room temperature. After the addition of valinomycin and phenol red (see above), the suspension was mixed with buffer C containing either formate (100 m M ) and DMN (50–100 l M )orformate (100 m M )at25°C. Phenol red absorbance at 550 nm was calibrated using tryptic hydrolysis of N-a-tosyl- L -arginyl-O-methylester (TAME) according to [27]. In this reaction, one proton is released per mol of substrate. The proteoliposomal suspension containing 5 l M trypsin was mixed with TAME (9.1 or 18.2 l M final concentration) in buffer C at 25 °C. Construction of W. succinogenes hydC mutants The hydC mutants of W. succinogenes were constructed by transforming the deletion mutant DhydABC with derivatives of pHydcat [1]. Plasmid pHydcat contains the entire hydABC operon and integrates into the genome of W. succinogenes by homologous recombination. Deriva- tives of pHydcat were synthesized using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany) with the plasmid as template and specifi- cally synthesized oligonucleotides carrying the desired nucleotide mismatches. A pair of co mplementary prim- ers was used for each modification (forward primer used for mutant N128D: 5¢-(3642)–CTCAAAGGGGTT TAC GATCCCGTTCAGCTAGC-3¢, and for mutant Q131L: 5¢-(3649)–GGGTTTACAATCCCGTT CTCCTA GCAGCCTATATGGG-3¢). Altered nucleotides are printed in bold, and the corresponding codons are underlined. The numbers in parentheses denote the nucleotide positions [6]. Modified pHydcat plasmids were isolated using Qiagen tips (Qiagen, Hilden, Germany) and sequenced to confirm the mutations. Nitrate-grown cells of W. succinogenes DhydABC were used for trans- formation as described [28,29]. Transformants were selected on plates with a medium containing formate and nitrate as energy substrates, kanamycin (25 m gÆL )1 ), and chlorampenicol (12.5 mgÆL )1 ). The integration of the plasmids into the genome of W. succinogenes DhydABC was confirmed by Southern blot analysis as described previously [1]. 1976 S. Biel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 RESULTS Preparation and characterization of proteoliposomes Rigaud and coworkers prepared proteoliposomes by incor- porating bacteriorhodopsin into liposomes treated with dodecylmaltoside and subsequent removal of the detergent with Bio-Beads [ 22]. The liposomes were shown t o be stable below a critical detergent/phospholipid ratio and to lyse at higher ratios. The maximum Dp generated by light was measured with proteoliposomes prepared at the critical ratio. In the experiment shown in Fig. 2, fumarate reductase and hydrogenase isolated from W. succinogenes were incorporated into sonic liposomes containing MK accord- ing to the method described above. The detergent/phos- pholipid ratio was varied, and the activity of electron transport from H 2 to fumarate was measured in the various preparations (Fig. 2A). The activity increased w ith i ncreas- ing amounts of detergent until a maximum was reached at the critical ratio of 0.8 g dodecylmaltoside per g phosphol- ipid. At higher ratios the activity was lower. At subcritical ratios, the electron transport activity was lower than predicted (V ET ) from the activities of hydrogenase (H 2 fi DMN) and fumarate reductase (DMNH 2 fi Fumarate), suggesting that only some of the enzyme molecules were involved in electron transport (H 2 fi Fumarate). The activities measured with proteoliposomes prepared at the critical o r a higher ratio were close to the theoretical ones. The activity of hydrogenase measured with DMN as acceptor w as ne arly the same i n t he different preparations (Fig. 2 A). In contrast, the activity of fumarate reductase (DMNH 2 fi Fumarate) was fairly constant up to the critical ratio and decreased to approximately 70% and 60% at the two highest dodecylmaltoside/phospholipid ratios. The a ctivity reflected t he accessibility o f fumarate r eductase in the preparations to external fumarate. This view was confirmed by measuring the activity of fumarate reduction with methyl viologen radical before and after lysis of the proteoliposomes by the addition of Triton X-100 [11,19,30] (not shown). There was no stimulation by Trito n X-100 in the preparations obtained at the critical or lower ratios, indicating that all the fumarate reductase molecules were accessible to fumarate. The stimulation observed with proteoliposomes prepared at the t wo highest ratios indica- ted that 30–40% of the fumarate reductase molecules were oriented towards the inside. The orientation of the hydrogenase molecules in the preparations is probably similar to that of fumarate reductase. This is deduced from the activity of methyl viologen reduction by H 2 in the different preparations (Fig. 2 A). Methyl viologen does not penetrate the mem- brane at a velocity commensurate w ith that of its reduction, in contrast to H 2 and DMN [11,31]. The activity of methyl viologen reduction by H 2 was the same in the preparations obtained at the critical or lower ratios, and was 70% and 65% of this activity in the p roteoliposomes prepared at the two highest ratios. This s uggests that hydrogenase is completely exposed to the outside in the proteoliposomes prepared at the c ritical or l ower ratios, w hereas 30–35% o f the hydrogenase molecules are oriented to the inside of proteoliposomes obtained at the highest ratios. It was not possible to confirm the orientation of hydrogenase by measuring its activity in the presence of Triton X-100, as the turnover number of hydrogenase per se is inhibited upon the addition of detergents. The amount of TPP + taken up from the external medium upon initiation of the electron transport from H 2 to fumarate was highest with proteoliposomes prepared at the critical r atio and was lower with t he other preparations (Fig. 2 B). A similar result was obtained for DMN reduction by H 2 .ThusTPP + uptake appears to be most efficiently Fig. 2. Properties of proteoliposomal preparations obtained at various dodecylmaltoside/phospholipid ratios. The various preparations were obtained according to the method described for proteoliposomes containing hydrogenase, MK, and fumarate reductase (see Experi- mental procedures). However, the amount of dodecylmaltoside applied was varied. The values of theoretical electron transport activity (V ET ) were calculated from those of DMN reduction by H 2 (H 2 fi DMN, V Hyd ) and of fumarate reduction by DMNH 2 (DMNH 2 fi Fumarate, V Frd ) according to: V ET ¼ V Hyd ÆV Frd / (V Hyd + V Frd ) [17]. TPP + uptake during electron transport from H 2 to DMN o r fumarate was measured as show n in Fig. 3. T s repre- sents the maximum amount of TPP + taken up by the proteoliposomes in the steady state of electron transport (see Table 1), and T e the corresponding TPP + concentration i n the medium. Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1977 coupled to the r eduction of fumarate or DMN in proteo- liposomes prepared at the critical detergent/phospholipid ratio. All the enzyme molecules appear to participate in electron transport, and all the enzyme molecules are apparently oriented towards the outside in these proteo- liposomes. The less efficient TPP + uptake by proteolipo- somes prepared with amounts of detergent above the critical ratio can be explained by the orientation of part of the hydrogenase molecules towards th e i nside (see D iscussion). In the following only proteoliposomes prepared at the critical ratio are used, unless indicated otherwise. Gel fi ltration with Sephacryl S-1000 SF indicated that all the fumarate reductase (1.3 lmolÆg phospholipid )1 )and hydrogenase (0.16 lmolÆg phospholipid )1 ) used for prepa- ration was incorporated into the proteoliposomes [30] (data not shown). The molar ratio of the two enzymes was close to that of the bacterial membrane. The enzyme contents based on phospholipid were approximately six times those in the bacterial membrane. The turnover numbers of the enzymes in electron transport from H 2 to fumarate were about 10% of those in growing bacteria. Assuming that the proteoliposomes are spherical, their average internal volume (3.5 mL Æg phospholipid )1 ) would correspond to average values of the internal and external diameter of 81 nm and 95 nm, respectively. In electron micrographs after negative staining the proteoliposomes appeared as mono-layered vesicles, most of which had external diameters b etween 50 nm and 7 0 nm (not shown ). The external surface of the vesicles was studded with particles which probably represent fumarate reductase a nd hydrogenase molecules [30]. Assuming that 1 g phospho- lipid corresponds to an outer membrane surface of 2.6 · 10 6 cm 2 [32], a spherical proteoliposome of 100 nm (or 50 nm) external diameter is calculated to carry 94 (or 23) molecules o f fumarate reductase (monomeric) a nd 12 (or 3) molecules of hydrogenase. As all the active enzyme mole- cules a ppear to participate i n e lectron transport f rom H 2 to fumarate (Fig. 2A), they are likely to be randomly distrib- uted among the proteoliposomes. Determination of Dw A suspension of proteoliposomes (0.4 g phospholipidÆL )1 ) containing MK, hydrogenase and fumarate reductase was stirred under an atmosphere of H 2 (Fig. 3 ). After the addition of TPP + , its concentration was rec orded using a TPP + electrode. Upon initiation of electron transport by fumarate addition, most of the external TPP + was taken up by the proteoliposomes, and was released into the medium again after consumption of fumarate. The cycle could be repeated by a second addition of fumarate. TPP + uptake was abolished by t he presence of a p rotonophore (FCCP). The experiment suggests that the electron transport from H 2 to fumarate creates a Dw (negative inside) across the proteoliposomal membrane which causes accumulation of TPP + within the proteoliposomes. Determination of the Dw required that the internal concentration of TPP + (T i ) was calc ulated from t he maximal amount of TPP + taken u p i n t he steady state of electron transport ( T s ). T i was calculated according to the method designed by Zaritsky et al. (Eqn 1) [25]. The value of T i so obtained corresponded to 33% of the amount of TPP + (T s ) taken up during fumarate respiration in the experiment shown in Fig. 3 . The residual part of T s is thought to be bound to the proteoliposomal membrane. Dw was calculated from T i and the corresponding external TPP + concentration (T e ) according t o the Nernst equation. The Dw generated by fumarate respiration with H 2 in the experiment shown in Fig. 3 was determined to be 0.19 V (Table 1). A Dw of the same direction and strength was generatedbyDMNreductionwithH 2 . In contrast, no TPP + uptake was observed during f umarate r eduction by DMNH 2 . This reaction also did not cause the uptake of tetraphenylboranate (TPB – ) in a similar experiment per- formed w ith a T PB – electrode [16] (not shown). Proteolipo- somes containing MK and only hydrogenase catalyzed DMN reduction by H 2 which generated a Dw with a similar value as measured in proteoliposomes containing both enzymes (not shown). Fumarate reduction by formate did not generate a Dw in proteoliposomes prepared according to the method described above with formate dehydrogenase instead of hydrogenase. However, a Dw ¼ 0 .13 V (negative inside) was found to be generated by the electron transport from formate to fumarate using proteoliposomes prepared according to the alternative method described in the Experimental procedures (Table 1). The same Dw was generatedbyDMNreductionwithformate. Determination of H + /e ratios H + /e ratios were measured with proteoliposomes using an external pH indicator (phenol red) and a stop-flow spectrophotometer [26]. P roteoliposomes containing hydro- genase, MK, and fumarate reductase suspended in a buffer (50 l M Hepes and 45 m M KCl) saturated with H 2 were treated with valinomycin (0.5 lmol g )1 phospholipid). The amount of valinomycin was just s ufficient to prevent TPP + uptake driven b y the reduction of DMN or fumarate w ith H 2 . After the addition of phenol red, DMN reduction by H 2 Fig. 3. Recording of the external TPP + concentration in a suspension of proteoliposomes during fumarate reduction by H 2 . Proteolipos omes containing hydrogenase, MK, and fumarate reductase were suspended (0.4 g phospho lipid ÆL )1 )inanH 2 -saturated buffer (pH 7.5 , 25 °C). The TPP + electrode was calibrated by three addition s of 1 l M TPP + . The electron transport was started by adding fumarate. 20 lmol FCCP per g phospholipid was applied w ere indicated. 1978 S. Biel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 was started by the addition of a small amount of DMN (Fig. 4 A). The number of p rotons released in to the external medium within 1 s was proportional to the ad ded amount of DM N. T he H + /e ratio was calculated from the p rotons released and the amount of DM N added. The time course of proton release was consistent with that of DMN reduction by H 2 .TheK m for DMN of this reaction was determined to be 15 l M (not shown). The release of protons did not occur with proteoliposomes which had been treated with a protonophore (FCCP, curve III). The H + /e ratioof f umarate r eduction by H 2 was m easured with proteoliposomes treated in the same way as described above. The reaction was started by the addition of fumarate instead of DMN, and the consumption of fumarate was recorded in a se parate experiment in the absenc e of ph enol red (Fig. 4B, curve VI). The H + /e ratio was determined from the ratio of the velocities of acidification (curve IV) and of fumarate reduction. The velocity of proton release was approximately twice that of fumarate reduction in the first second after fumarate addition and became slower at longer reaction times. Proton release did not occur with proteo- liposomes treated w ith a protonop hore (curve V). The H + /e ratio of DMN reduction by formate was measured in the same way as in the experiment shown in Fig. 4A (Table 2). Proteoliposomes containing formate dehydrogenase instead of hydrogenase in buffer flushed with N 2 , were allowed to react with a solution containing DMN and formate. The H + /e ratio of fumarate reduction by formate could not be measured. When formate was added before the proteoliposomes were mixed with fuma- rate, a drastic inhibition of formate dehydrogenase was observed. W hen fumarate was a dded before the suspension was mixed with formate, the reduction of the MK present in the proteoliposomes interfered with the measurement of fumarate reduction. Therefore, the velocity of MK reduc- tion was recorded at 270 nm upon mixing of the proteo- liposomes with formate. The H + /e ratio of MK reduction by formate was calculated form the velocities of MK reduction and of acidification measured with phenol red in a second experiment. As seen from Table 2, the average H + /e ratios with H 2 or formate obtained from various experi- ments were close to 1. HydC mutants To understand the mechanism of the Dpgenerationwhichis coupled to quinone reduction by H 2 orformate,thesiteof quinone reduction on HydC or FdhC of W. succinogenes should be elucidated. The sequences of these di-heme cytochromes b are similar to that of the di-heme Fig. 4. Proton release coupled to the reduction of DMN (A) and of fumarate ( B) by H 2 . Proteoliposomes containing hydrogenase, MK, and f umarate reductase in the H 2 -saturated suspension des ignated in the Experimental procedures were mixed with a solution of DMN (A) or fumarate (B), and the absorbance of phenol red was recorded (experiments I–V). Phenol red absorbance was calibrated as described in t he Experimental p roce dures. Proteoliposomes t re ated with FCCP (20 lmolÆg p hosp holip id )1 )wereusedinexperimentsIIIandV.The concentration o f D MN in the reaction mixture at reaction time zero was 5.2 l M (I) and 9.2 l M (II and III). Fumarate reduction by H 2 was recorded at 270 nm (e ¼ 0.55 m M )1 Æcm )1 ) i n the abse nce of phe nol red (VI). The slopes of curves IV and VI were used for calculating the H + /e ratio o f fumarate reduction b y H 2 . Table 1. TPP + accumulation by proteoliposomes in the steady state of electron transport. Proteoliposomes containing hydrogenase (formate dehydrogenase) and fumarate reductase were used with H 2 or DMNH 2 (formate) as electron donor. The experime nts were performed as described in Fig. 3. However, the suspension w as flushed with N 2 instead of H 2 when DMNH 2 (1 m M ) or formate (1 m M ) were used a s donor. DMN was applied at 1 m M concentrations. T s represents t he m axim um am ount of TPP + taken up by t he p roteoliposomes in the steady state o f electron transport, and T e the corresponding TPP + concentration in t he med ium (see F ig. 3). The internal TPP + concentration ( T i ) was ca lcul ated according to E qn (1). Dw was c alculated from T e and T i according t o the Nernst equation. Donor Acceptor Activity (UÆmg phospholipid )1 ) T s (lmolÆg phospholipid )1 ) T i (l M ) T e (l M ) Dw (V) H 2 Fumarate 2.6 7.1 666 0.39 )0.19 H 2 DMN 6.5 4.8 456 0.25 )0.19 DMNH 2 Fumarate 4.1 No TPP + uptake Formate Fumarate 1.4 3.4 240 1.7 )0.13 Formate DMN 3.6 2.4 171 1.2 )0.13 Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1979 cytochrome b subunit (FdnI) of E. coli formate dehydro- genase-N [9]. HydC is schematically drawn i n Fig. 5 based on the structure of FdnI [14]. T he two heme groups of FdnI are i n e lectron transfer distance nearly on t op of each other when viewed along the membrane normal. The proximal (upper) heme group is i n electron transfer distance to one o f the iron–sulfur centers of the iron–sulfur subunit FdnH (not shown). The site of quinone reduction is thought to be occupied by a molecule of HQNO which is located on the cytoplasmic side of the distal heme group. HQNO is in close proximity to the axial heme ligand on helix IV and to an asparagine (N110) and a glutamine residue (Q113) within the hydrophilic stretch c onnecting helices II and I II. These three residues are conserved in HydC (H200, N128, and Q131 in Fig. 5) and in FdhC of the W. succinogenes enzymes [9]. Mutants were constructed in which N128 or Q131 of HydC was replaced by aspartate o r l eucine (Table 3). The two mutants (N128D and Q131L) did not grow by fumarate respiration with H 2 . When grown with formate and fumarate, the mutant cells did not catalyze fumarate reduction by H 2 , in contrast to the wild-type strain. The specific activities o f DMN reduction by H 2 measured with the membrane fractio n of the mutan ts amounted to 6% or less of the wild-type a ctivity, whereas the activities of benzyl viologen reduction by H 2 were close t o that of the wild-type strain. The deficiency in quinone reduction was not due to any loss of the heme groups from HydC. The amount of heme B which was r educed upo n H 2 addition to the Triton X-100 extract of the oxidized membrane fraction was the same with the m utants (0.3 lmolÆgprotein )1 )andwiththe wild-type strain [29]. Mutant H122A had wild-type pro- perties with r espect to growth and enzyme activities. Residue H 122 is also located in the stretch connecting helix II and III of HydC, but is not conserved in FdnI and FdhC. The results suggest that the quinone reactivity of HydC is specifically affected in mutants N128D and Q131L, in agreement with the view that the site of quinone reduction is located close to the cytoplasmic surface of the membrane. DISCUSSION Energetics For technical reasons, the H + /e ratio of apparent proton translocation can only be measured at vanishing Dp.Itis generally thought that the same ratio is valid in the presence and a bsence of Dp. As the am ount of free energy conserved by apparent proton translocation across the membrane cannot exceed that provided by the driving redox reac tion, the H + /e ratio (n H + /n e ) can be calculated from the redox potential difference (DE)andDp according to Eqn (2), provided that the energetic efficiency (q) of the process is known. n H þ n e ¼ q DE Dp ð2Þ Assuming q ¼ 1, the t heoretical maximum H + /e ratio of fumarate respiration with H 2 (reaction a) is calculated to be 2.6, using Dp ¼ 0.17 V, and DE ¼ 0.45 V [from E o ¢ for H + /H 2 ()0.42 V) and for fumarate/succinate (+0.03 V [33])]. If the actual H + /e ratio was 1 or 2, the energetic efficiency of fumarate respiration would be 0.38 or 0.76. Nearly the same numbers apply for fumarate respiration with fo rma te a nd HCO 3 – as its oxidation product ( reaction b), as the corresponding value of DE o ¢ is close to that obtained with H 2 . Table 2 . H + /e ratios measured with p roteoliposomes. The proteolipo- somal preparation (A) contained MK, hydrogenase, and fumarate reductase. In preparation (B) hydrogen ase w as replaced by formate dehydrogenase. The upper two experiments were performed as described in Fig. 4A,B. The H + /e ratio with f ormate and DM N was measured as sh own in Fig. 4A. However, the p roteoliposo mes were suspended in a buffer fl ushe d with N 2 instead o f H 2 , a nd t he s uspen- sion was mixed with a s olution containing formate and DMN. In the experiment with DMNH 2 and fumarate, the anoxic proteoliposomal suspension containing DMNH 2 (0.2 m M ) was m ixed with fumarate (5 m M ), and the absorban ce of phenol red was recorded. The reduction by formate of the MK present in the proteoliposomes was observed at 270 nm w hen t he prote oliposomes we re m ixed w ith formate in the absence of fumarate and phenol red. The corresponding H + /e ratio was c alculated using the velocity of MK red uction (De ¼ 12.0 m M )1 cm )1 ). n represents th e number of m easurem ents with different pre- parations of p roteoliposomes. Preparation Donor Acceptor H + /e ratio n AH 2 Fumarate 1.0 ± 0.1 6 AH 2 DMN 0.96 ± 0.04 10 ADMNH 2 Fumarate 0.0 4 B Formate DMN 0.98 ± 0.12 9 B Formate MK 0.95 ± 0.12 4 Fig. 5. Hypothetical arrangement of the four predicted membrane- spanning helices of W. succinogenes HydC. The scheme i s base d on the crystal structure of E. coli FdnI [14]. The shaded squares represent the heme groups. A molecule of H QNO is s hown at t he site of M K reduction which is confined by the axial ligand H200 of the distal heme group and by re sid ues N128 and Q 131 i n t he stretch c onnectin g the hydrophobic parts of helices II and III. 1980 S. Biel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The theoretical maximum H + /e ratio of fumarate reduction by the M KH 2 within the bacterial me mbrane is calculated to be 0.6, assuming the redox potential of the MK/MKH 2 couple to be equal to its standard potential in organic solution (E o ¢ ¼ )0.074 V [34]). This assumption is consistent with the finding that the MK/MKH 2 ratio i s not far f rom 1 in the membrane of W. succinogenes catalyzing fumarate respiration [35]. Furthermore, the standard poten- tial of MK/MKH 2 in a bacterial membrane was measured to be close to that in organic solution [36]. T herefore, the actual H + /e ratio of MKH 2 oxidation by fumarate should be lower than 0.6. The site of MKH 2 oxidation The site of MKH 2 oxidation on the cytochrome b subunit (FrdC) of fumarate reductase is not kn own. In the crystallographic model of the oxidized enzyme, a cavity was discovered which extends from the hydrophobic phase of the m embrane, close to t he distal heme group of Fr dC to the periplasmic aqueous phase [37]. The cavity could accommodate a MKH 2 molecule after minor structural alterations. A glutamate residue (E66) lines the cavity and is a possible acceptor of a hydrogen bond from one of the hydroxyl groups of MKH 2 . Replacement of E66 by a glutamine residue resulted in a mutant (E66Q) which did not catalyze DMNH 2 oxidation by fumarate. In contrast, the activity of fumarate reduction by benzyl viologen radical as well as the crystal structure of the enzyme and the midpoint potentials of the heme groups were not affected by the mutation. These results suggest that the inhibition of quinol oxidation activity in the m utant enzyme is due to the absence of the carboxylate g roup of E66. In the wild-type enzyme, E66 could f acilitate quinol oxidation by a ccepting one of the protons liberated by quinol oxidation which could then be released on the periplasmic side of the membrane via the cavity. As a consequence, quinol oxidation by fumarate should be electrogenic, and the H + /e ratio of t he reaction is predicted t o be 1 . T his value is higher than the maximum possible value predicted by the energetic calculation (see above). Furthermore, fumarate reduction by D MNH 2 was not coupled to Dw generation in cells, inverted vesicles [16], o r in proteoliposomes ( Table 1). Mechanism of Dp generation In the model m echanisms d rawn in Fig. 6, H 2 oxidation b y MK is assumed t o be electrogenic with a H + /e ratio of 1. The protons consumed in MK reduction are taken up from the inside of the proteoliposomes, and simultaneously protons are released by H 2 oxidation on the outside (Fig. 6 A,B). MKH 2 oxidation by fumarate is depicted as an electroneutral process in Fig. 6A, w hereas it is electro- Table 3. Growth and enzymic activities of hydC mutants of W. succinogenes. The doubling times of growth with H 2 and fumarate, and the enzymic activities were measured in cells (H 2 fi Fumarat e) or with the memb rane fraction of cells g rown with formate a nd fumarate as d escribed [29]. The properties of mutant H 122A were taken f rom [29]. Strain Doubling time (h) UÆmg cell protein )1 H 2 fi Fumarate H 2 fi DMN H 2 fi Benzyl viologen Wild-type 1.8 3.5 4.5 2.1 N128D / 6 0.05 0.25 2.5 Q131L / 6 0.05 0.08 2.0 H122A 1.8 3.6 4.5 2.3 Fig. 6. Hypothetical mechanisms of Dp generation in proteoliposomes (AandB)incellsofW. su ccinogenes (C). The sites o f MK reduction and of MKH 2 oxidation a re drawn s chematically in the center o f the membrane. These sites may actually be located closer to the membrane surfaces. E quivalent mechanisms m ay apply with formate as electron donor instead of H 2 . p, periplasmic side of the membrane; c, cyto- plasmic s ide. Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1981 genic i n Fig. 6B. In Fig. 6A, t he protons formed by MKH 2 oxidation are released on the outside, where they balance the protons consumed by fumarate reduction, and fumarate respiration with H 2 is predicted to be electrogenic with a H + /e ratio of 1. In contrast, fumarate respiration with H 2 is predicted to be an electroneutral process i n the proteolipo- somes according to the mechanism of Fig. 6B. The experimental results obtained with the proteolipo- somes are in agreement with the mechanism depicted in Fig. 6A and contradict that of Fig. 6B. The reduction by H 2 of quinone and of fumarate was found to be electrogenic, and the H + /e ratio was 1 for both processes. As a consequence, fumarate reduction by MKH 2 has to be an electroneutral process in the proteoliposomes. This conclu- sion is confirmed b y the result that DMNH 2 oxidation b y fumarate was not coupled to the uptake of TPP + or TPB – by the proteoliposomes. DMNH 2 oxidation by fumarate was previously also found to be electroneutral in cells and in inverted vesicles of W. succinogenes [16]. Furthermore, inverted vesicles catalyzing fumarate respiration with H 2 were found to accumulate SCN – ,andtheH + /e ratio was measured to be close to 1 [15]. In these vesicles, hydrogenase is oriented to the inside and fumarate reductase to the outside. Therefore, if fumarate reductase operated electro- genically, t he H + /e ratio should be 2. However, the H + /e ratio of f umarate r espiration is apparently not affected by the orientatio n of the e nzymes rela tive t o each ot her and is the same in inverted vesicles and proteoliposomes. This confirms the e lectroneutral operation of fumarate red uctase in the bacterial membrane (Fig. 6C) as well as in the proteoliposomes (Fig. 6 A). The view that the protons consumed in MK or DMN reduction are taken up from the inside of the proteolipo- somes (Fig. 6A,B) or from the cytoplasmic side of cells o f W. succinogenes (Fig. 6C) is supported by the properties of the hydC mutants N128D and Q131L. As expected on the basis of the structure of E. coli FdnI, quinone reduction by H 2 is inhibited in these mutants, suggesting that the site of quinone reduction on HydC is located close t o the cytoplasmic surface of the m embrane (Fig. 5). This is likely to hold true also for FdhC of W. succino- genes formate dehydrogenase, where residues equivalent to N128 and Q131 of HydC are conserved. The arrange- ment of the heme groups and the quinone binding site of FdhC is expected to resemble that of HydC and of E. coli FdnI [9]. 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Liebl, U ., Pezennec, S., Riedel, A., Kellner, E. & Nitschke, W. (1992) The Rieske Fe/S center from t he gr am-positive b acterium PS3 and its interaction with the menaquin one pool stud ied by EPR. J. Biol. Chem. 267, 14068–14072. 37. Lancaster, C.R.D., Gross, R., H aas, A., Ritter, M., Ma ¨ ntele, W., Simon, J. & Kro ¨ ger, A. (2000) Essential role of Glu-66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase. Proc.NatlAcd.Sci.USA97, 13051–13056. Ó FEBS 2002 Coupled fumarate respiration in proteoliposomes (Eur. J. Biochem. 269) 1983 . Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes Simone. coupled fumarate respiration be restored by incorporating the isolated enzymes into liposomes containing menaquinone; (b) is the Dp generated by menaquinone