Menaquinone-dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential

Transcription

1 Eur. J. Biochem. 257, (1998) FEBS 1998 Menaquinone-dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential Jan SCHIRAWSKI and Gottfried UNDEN Institut für Mikrobiologie und Weinforschung, Universität Mainz, Germany (Received 6 April 1998) EJB /6 Succinate dehydrogenases from bacteria and archaea using menaquinone (MK) as an electron acceptor (succinate/menaquinone oxidoreductases) contain, or are predicted to contain, two heme-b groups in the membrane-anchoring protein(s), located close to opposite sides of the membrane. All succinate/ubiquinone oxidoreductases, however, contain only one heme-b molecule. In Bacillus subtilis and other bacteria that use MK as the respiratory quinone, the succinate oxidase activity (succinate O 2 ), and the succinate/ menaquinone oxidoreductase activity were specifically inhibited by uncoupler (CCCP, carbonyl cyanide m-chlorophenylhydrazone) or by agents dissipating the membrane potential (valinomycin). Other parts of the respiratory chains were not affected by the agents. Succinate oxidase or succinate/ubiquinone oxidoreductase from bacteria using ubiquinone as an acceptor were not inhibited. We propose that the endergonic electron transport from succinate (E 30 mv) to MK (E 80 mv) in succinate/menaquinone oxidoreductase includes a reversed electron transport across the cytoplasmic membrane from the inner (negative) to the outer (positive) side via the two heme-b groups. The reversed electron transport is driven by the proton or electrical potential, which provides the driving force for MK reduction. Keywords: succinate dehydrogenase; succinate/quinone oxidoreductase; reversed electron transport; proton potential; Bacillus subtilis. Succinate dehydrogenase (Sdh, or succinate/quinone oxidoreductase) catalyzes succinate oxidation in the citric-acid cycle and transfers the electrons to the respiratory quinones in the membrane (for review see [1, 2]). The enzyme consists of a peripheral portion with the active site for succinate and a membrane portion with the active site for the quinone. The hydrophilic portion, which protrudes into the cytoplasmic space, consists of a flavoprotein (SdhA subunit) with the active site for succinate, and an iron sulfur protein (SdhB) carrying three iron sulfur clusters which are involved in the electron transfer from SdhA to the membrane subunit(s). The SdhA and SdhB subunits from diverse sources, including Gram-positive and Gram-negative bacteria, archaea, and the mitochondria from eucarya are very much alike with regard to the prosthetic groups, function and protein sequences [1, 2]. The membraneous subunit(s), however, show a large variation in their sequences, subunit composition and heme-b content [3]. These proteins have in common the presence of five or six transmembrane helices, which are derived from one (SdhC) or from two subunits (SdhC and SdhD), depending on the organism, and one or two heme- B groups are found. No plausible explanation has been supplied Correspondence to G. Unden, Universität Mainz, Institut für Mikrobiologie und Weinforschung, Becherweg 15, D Mainz, Germany Phone: Fax: Unden@mail.uni-mainz.de Abbreviations. CCCP, carbonyl cyanide m-chlorophenylhydrazone; Cl 2 Ind, 2,6-dichlorophenol indophenol; DMN, 2,3-dimethyl-1,4-naphthoquinone; MK, menaquinone; MKH 2, reduced menaquinone (menaquinol); Q, ubiquinone; QH 2, reduced ubiquinone (ubiquinol); Sdh, succinate dehydrogenase. Enzyme. Succinate/quinone oxidoreductase (EC ). for the differences in subunit and prosthetic-group compositions of the membrane anchor. Sdh is taken as a prototype of a non-coupling enzyme in the respiratory chain of mitochondria and bacteria [2, 4, 5] in contrast to respiratory enzymes, which couple redox reactions to proton translocation, such as the bc 1 complex and cytochrome c oxidases. In mitochondria and many Gram-negative bacteria, ubiquinone (Q, E 110 mv) is used as the electron acceptor for Sdh [5 7]. Other bacteria, in particular Gram-positive bacteria, use the electronegative menaquinone (MK, E 80 mv) as the electron acceptor [8]. From the class of succinate/menaquinone oxidoreductases, only the Sdh of Bacillus subtilis has been studied in detail so far [2]. Due to its electronegative redox potential, MK is a poor electron acceptor from succinate (E 30 mv), which is 110 mv more electronegative. Electron transfer from succinate to O 2 was known to depend on intact membranes and a proton potential in B. subtilis. It was suggested that this reflects the endergonic nature of the reaction [8, 9]. The enzyme from B. subtilis contains one membranous subunit (SdhC) with five transmembrane helices, which provide four conserved His residues as the axial ligands for two heme-b groups [3, 10, 11]. A similar enzyme from Paenibacillus macerans was described recently [12]. Here, we studied the question, why succinate oxidase activity in Bacillus and related bacteria depends on the membrane potential. We show that MK-dependent Sdh enzymes catalyze a reversed electron transport across the cytoplasmic membrane and that the membrane potential provides the energy for the endergonic reduction of MK by succinate. Structural predictions for the membrane anchor protein (SdhC) suggest that the two heme- B molecules enable the transport of the electrons across the membrane down the electrochemical gradient to a MK site close

2 Schirawski and Unden (Eur. J. Biochem. 257) 211 Table 1. Effect of an uncoupler (CCCP, 10 µm) or valinomycin (Val, 9 µm) on succinate and glucose-oxidase activity by bacterial cells containing menaquinone (MK) or ubiquinone (Q) as the respiratory quinones. The activities were measured with the O 2 electrode in cell suspensions or EDTA-treated cells (E. coli). Succ, succinate; Gluc, glucose. n.d., not determined. Quinone type Succ O 2 Gluc O 2 CCCP Val CCCP U/g Bacillus subtilis W23 MK B. licheniformis MK 74 3 n.d Paenibacillus macerans MK 2 2 n.d. 165 a 165 a Corynebacterium glutamicum MK 48 1 n.d Pseudomonas stutzeri Q n.d. n.d. n.d. Paracoccus denitrificans Q n.d. n.d. n.d. Escherichia coli AN387 Q MK a Glycerol O 2 activity (U/g protein). to the positive outside. Thus, Sdh represents a new chemiosmotic site in the respiratory chain of these bacteria. MATERIALS AND METHODS Bacteria and growth. Paenibacillus (formerly Bacillus) macerans (DSM24) [13], B. subtilis (DSM10), B. licheniformis (DSM 13) and Pseudomonas stutzeri (DSM5190) were obtained from the German collection of microorganisms (DSM), B. subtilis W23 from Dr Kröger (Frankfurt), Corynebacterium glutamicum (ATCC 13032) from Dr Burkowski (Köln) and Paracoccus denitrificans (ATCC 17741) from Dr Ludwig (Frankfurt). B. licheniformis and P. macerans were grown at 37 C in modified White medium [14, 15] containing 31 mm Na 2 HPO 4, 18 mmkh 2 PO 4, 15 mm (NH 4 ) 2 SO 4, 0.2 mm MgSO 4, 0.02 mm MnCl 2, 0.01 mm FeSO 4, ph 7.0, with the addition of yeast extract (no , Gibco) to 0.1% (mass/vol.), acid-hydrolyzed casein (no , Serva) to 0.1% (mass/vol.), and L- tryptophan to 0.01% (mass/vol.). Pc. denitrificans was grown in yeast-tryptone medium [16] at 30 C, Ps. stutzeri in DSM1 medium at 37 C. The other strains were grown in Luria-Bertani medium [16] at 37 C (Escherichia coli wild-type AN387 [7]) or at 30 C (B. subtilis, B. subtilis W23 and C. glutamicum). For growth under aerobic conditions, the media were supplemented with 20 mm succinate (or 5 mm glycerol for P. macerans). Growth was performed in 300-ml Erlenmeyer flasks containing 30 ml medium in an incubation shaker at 200 rpm. Cell suspensions. The bacteria were sedimented by centrifugation, washed twice in 50 mm sodium potassium phosphate, ph 7.4, containing 2 mm MgCl 2, resuspended in the same buffer to A 578 of and used for oxygen-uptake measurements. EDTA-treated bacteria (E. coli AN387) were obtained as described by Engel et al. [17]. After harvesting, the bacteria were washed and suspended in 120 mm Tris/HCl, ph 8.0, to A 578 of 3 4. Then, 1 mm EDTA, ph 7.0, was added and the bacterial suspension was shaken gently for 15 min. The bacteria were washed twice with 200 mm sodium Hepes, ph 7.5, resuspended in the same buffer to A 578 of and used for oxygen-uptake measurements. Enzyme activities. Oxygen uptake (succinate O 2 ) by intact cells was measured using a Clarke-type electrode at the growth temperature of the bacteria in 50 mm sodium potassium phosphate, ph 7.4, containing 2 mm MgCl 2. All samples were equilibrated with air before substrate (50 mm succinate, 20 mm glucose or 10 mm glycerol) and uncoupler CCCP (10 µm final concentration, from a 1 mm stock solution in ethanol) or valinomycin (9 µm final concentration from a 0.9 mm solution in ethanol) was added. Sdh activity was assayed as the succinate-dependent reduction of 2,6-dichlorophenol indophenol (Cl 2 Ind). The assay was performed in anoxic cuvettes containing 50 mm potassium/sodium phosphate, 2 mm MgCl 2, 0.25 mm Cl 2 Ind, 0.4 mm phenazine methosulphate, 20 mm succinate, ph 7.4, and µl of the cell suspensions or cell-free extracts [15]. The reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by succinate (Succ DMN activity) was measured as the velocities of the difference of extinction changes at nm ( ε mm 1 cm 1 ) [8]. For the measurement, the bacterial cells were incubated in anoxic potassium/sodium phosphate (50 mm), ph 7.4, containing 2 mm MgCl 2 at 30 C with 0.2 mm DMN. The reaction was started by the addition of 20 mm succinate. One unit of activity (U) corresponded to the oxidation of 1 µmol succinate by 2 e /min. Protein concentrations were determined by the Biuret method with KCN [18]. Cell-free extracts were prepared as described [15]. Transport measurements. Uptake of [ 14 C]succinate into the bacteria was measured as described [17]. After harvesting, the bacteria were washed once with buffer A (0.1 M NaH 2 PO 4 / K 2 HPO 4, 1 mm MgCl 2, 100 µg/ml chloramphenicol, ph 7.4) and resuspended in the same buffer to A 578 of 3 4. The bacteria were energized by incubation for 10 min in buffer A containing 20 mm glucose at their growth temperature. In an anaerobic chamber [2,3-14 C]succinate (100 µm; 44.0 MBq/mmol succinate) was added to the bacterial cells, and samples (100 µl) were withdrawn after intervals of 30 s to 30 min. Intracellular contents of labeled dicarboxylates were determined after silicone-oil centrifugation. For anaerobically grown cells, buffer A contained 1 mm dithiothreitol and was degassed as described by Engel et al. [19]. For calculation of the dry mass of the bacterial cell suspensions it was assumed that A corresponds to 280 mg dry mass/l. RESULTS Inhibition of MK-dependent succinate oxidase by membrane de-energization. Various bacteria were assayed for succinate oxidase activity after growth under aerobic conditions (Table 1). The procaryotes contained either MK, Q or a mixture of both in the membrane. With the exception of P. macerans, cell suspensions of the procaryotes contained high succinate oxidase activities, irrespective of the quinone involved. P. macerans is not able to use external succinate, due to the lack of a succinateuptake system under these conditions as shown below. When the

3 212 Schirawski and Unden (Eur. J. Biochem. 257) Fig. 1. Succinate 2,3-dimethyl-1,4-naphthoquinone (DMN) activity of cell suspensions of B. subtilis W23. Succinate-dependent reduction of DMN was followed in the dual-wavelength photometer at nm, as the decrease in absorbance ( A) under anoxic conditions. The addition of succinate (20 mm) and of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 µm) is indicated. Table 2. Effect of uncoupler (CCCP) and cell disintegration (cell homogenate) on succinate-dehydrogenase activity of B. subtilis W23. Treatment of cell Succinate DMN Succinate Cl 2 Ind suspension U/g protein No addition CCCP (10 µm) Cell homogenate succinate oxidase activity was measured in the presence of an uncoupler (CCCP), which degrades the proton potential, the oxidase activities of the MK-containing bacteria B. subtilis, B. licheniformis and Corynebacterium glutamicum dropped by more than 90% (Table 1). The same decrease in succinate-oxidase activity was found with the addition of the ionophor valinomycin, which degrades the membrane potential. In Pseudomonas stutzeri, Paracoccus denitrificans and E. coli, however, the succinate-oxidase activity was not significantly affected by addition of the uncoupler (Table 1). The Sdh of the latter group of bacteria differs from that of the former in the type of quinone that is used as electron acceptor: Ps. stutzeri, Pc. denitrificans and E. coli Sdh all operate exclusively with Q as the acceptor, while the Sdh of B. subtilis, B. licheniformis and C. glutamicum transfer their electrons to MK. In contrast, glucose or glyceroloxidase activity did not respond to the presence of uncoupler in any of the bacteria, irrespective of the quinone type present. With glucose and glycerol as substrates, the NADH and glycerol-3-phosphate dehydrogenases are the primary dehydrogenase(s) which transfer reducing equivalents to the respiratory chain or the quinones, respectively. Thus, for the different substrates glucose, glycerol and succinate, the respiratory chains differ only by the primary dehydrogenases, whereas the remaining part of the respiratory chains is the same. This suggests that the uncoupler and the ionophor specifically inhibited the succinate/menaquinone oxidoreductases. Specific inhibition of succinate/menaquinone oxidoreductase by an uncoupler. For direct studies of the effects of an uncoupler on succinate/menaquinone oxidoreductase, the activity of the enzyme was assayed in B. subtilis cells with DMN as the acceptor (Fig. 1). DMN is, in structure and midpoint potential (E 80 mv), very similar to MK and can be used as a Fig. 2. Uptake of [ 14 C]succinate into cells of B. subtilis ( ) and of P. macerans ( ). Uptake was measured in cell suspensions (1.44 g dry mass/l) obtained from bacteria grown with 20 mm (B. subtilis) or 10 mm glycerol (P. macerans) under aerobic conditions. Uptake was measured with 100 µm [ 14 C]succinate by silicone-oil centrifugation. The cells were energized by 10-min incubation in the presence of 20 mm glucose at their growth temperature prior to measurement. Table 3. Succinate oxidation and uptake by aerobically grown B. subtilis, P. macerans and E. coli. Succinate oxidation with O 2 and succinate uptake were measured in cell suspension, succinate oxidation with dichlorophenol indophenol (Cl 2 Ind) in cell homogenates. For succinateoxidase activity, the decrease in succinate is given based on the following reaction : 1 succinate 3.5 O 2 4CO 2 3H 2 O. Uptake of succinate into the cells was determined by silicone-oil centrifugation. For calculation of uptake rates, it was assumed that the periplasmic and cytoplasmic volumes correspond to 1 µl and 2 µl/mg dry mass, respectively. [ 14 C] Succinate Succinate O 2 Succinate uptake (cells) (cells) Cl 2 Ind (cell homogenate) µmol/(mg dry µmol succinate/ cells)/min (mg protein)/min B. subtilis a 310 P. macerans E. coli AN b 70 a B. subtilis W23. b EDTA cells. water-soluble substrate analog. The succinate DMN activity could be determined with intact bacteria, but the activity was completely lost on addition of the uncoupler (Fig. 1 and Table 2). In a similar way, the activity was lost after breaking the bacterial cells (Table 2) or adding valinomycin. Therefore, the activities were lost by largely different treatments, all of which have in common the degradation of the proton or membrane potential. This suggests that this may be the reason for the effects, and other potential explanations, such as changes in the MK/reduced menaquinone (MKH 2 ) ratio, are very unlikely. In addition, the activity of Sdh was assayed with the artificial acceptor dichlorophenol indophenol (Cl 2 Ind), which does not require the membrane anchor protein or the quinone site for reactivity [20]. In contrast to the MK-dependent activity, succinate Cl 2 Ind activity was not inhibited by breaking the cells or adding either the uncoupler (Table 2) or valinomycin. Obviously, the enzyme itself was not inhibited by the various treatments, and the inhibition cannot be due to decreased succinate uptake. Taken together, the experiments show that succinate/menaquinone oxidoreductase is the p-requiring step in succinate-oxidase activity.

4 Schirawski and Unden (Eur. J. Biochem. 257) 213 Table 4. Correlation of the number of (supposed) heme-liganding His residues in the membrane-anchor proteins of succinate dehydrogenases (Sdhs) to the type of quinone used as the electron acceptor in various organisms (A, archaeon; B, bacterium; E, mitochondrium in eucaryotic organism). The numbers of transmembrane helices (TM) and conserved His residues refers to the membrane-anchor proteins (SdhC or SdhCD, respectively). Source Quinone TM Con- Heme Referhelices served ence His Escherichia coli (B) Q [34] Paracoccus denitrificans (B) Q [35] Bos taurus (E) Q [36] Bacillus subtilis (B) MK [1, 10] Paenibacillus macerans (B) MK a [12] Thermoplasma acidophilum (A) MMK b a [37] Natronobacterium pharaonis (A) MK a [38] Fig. 3. Model for the transmembrane helices and the location of the heme-b groups of SdhC of P. macerans [12]. The model shows the binding of two heme-b molecules in SdhC and their relative positions to the membrane surface, according to the suggestion for B. subtilis and W. succinogenes [3, 11, 32]. b L and b H stand for low (L) and high (H) potential heme B. a Number derived from the number of conserved His residues. b Thermoplasmaquinone, methylated MK with low midpoint potential similar to MK [1]. c EMBL database, accession no. Y Succinate uptake as a prerequisite for succinate oxidation in B. subtilis and P. macerans. Uptake of [ 14 C]succinate into the bacteria was measured by silicone-oil centrifugation (Fig. 2). B. subtilis accumulated succinate in a time-dependent manner and, after 5 10 min, maximal accumulation was achieved. The rates were typical for this bacterium [21], but lower than those measured previously for E. coli [17, 19]. The uptake rates, in particular those for B. macerans, were distinctly lower than the succinate-oxidase activities (see Table 2). This can be partially explained by the use of resting cells for the uptake measurements and the difficulties in measuring initial uptake rates (v max ). P. macerans, however, did not accumulate [ 14 C]succinate and the transport rates were by a factor of more than 150 lower than those of B. subtilis (Table 3). Therefore, aerobically grown P. macerans is not able to accumulate and use external succinate. When the bacteria were tested for growth on succinate under aerobic conditions, only B. subtilis and E. coli, but not P. macerans were able to grow (not shown). Cell suspensions of B. subtilis and E. coli showed high succinate-oxidase activities (Table 3). In P. macerans cells, succinate oxidase was not detected, although in cell homogenates high activities of Sdh were present, comparable to the activities from B. subtilis and E. coli. Taken together, the experiments make it clear that P. macerans contains an active Sdh, but is not able to use externally supplied succinate due to the lack of an uptake system. Succinate, however, which is produced by catabolic reactions and the citricacid cycle, is metabolized, since substrates such as glucose or glycerol are oxidized mainly to CO 2, demonstrating a complete citric-acid cycle (not shown). Therefore, it is clear that the active site for succinate of the succinate/menaquinone oxidoreductases is located in the cytoplasm as expected from its function in the citric-acid cycle and the lack of export signal sequences on the sdha and sdhb genes [12, 22]. Two (predicted) heme-binding sites in the membrane-anchor subunits of MK-dependent Sdh enzymes. The membrane-anchor subunits of various Sdh enzymes show a great variation in their sequences and the number of bound heme-b groups or the Fig. 4. Model for the topology of menaquinone (MK)-dependent and ubiquinone (Q)-dependent succinate dehydrogenases in the cytoplasmic membrane and implications on reaction energetics. The topology of the active sites for succinate and the supposed orientation of the quinone sites for H accession are shown. In addition, the midpoint potentials for the fumarate/succinate ( 30 mv), Q/QH 2 ( 110 mv) and MK/MKH 2 ( 80 mv) couples are given. The squares represent the heme-b groups. For p ( 160 mv) and Ψ ( 140 mv), the values for aerobic growth of E. coli were used [33]. number of putative His ligands (for review see [1, 3]). The enzymes carry one or two heme groups and two or four conserved His residues, which serve as the axial ligands for the heme(s). Table 4 gives the number of conserved His ligands and of bound heme-b molecules for a selection of MK-dependent and Q-dependent Sdh enzymes. All of the succinate/ubiquinone oxidoreductases contain two conserved His residues, which can bind one heme-b molecule, irrespective of the source which can be bacterial, archaeal or eucaryotic. The succinate/menaquinone oxidoreductases, however, all contain four conserved His residues and, correspondingly, two heme-b groups. Again, this applies to enzymes from bacteria and from archaea (Thermoplasma acidophilum and Natronobacterium pharaonis), operating with electronegative quinones. The same correlation is found for all other Sdh enzymes, not shown in Table 4, for which this information is available. Thus, there is a clear correlation between the type (or redox potential) of the quinone and the number of

5 214 Schirawski and Unden (Eur. J. Biochem. 257) heme-b molecules (or conserved His residues) in the membraneanchor subunits of the Sdh enzymes (Table 4). DISCUSSION Potential transmembrane arrangement of two heme-b molecules in the membrane anchor of succinate/menaquinone oxidoreductases. Recently, mainly based on the redox chemistry of the prosthetic groups and the quinone used, Sdh enzymes have been classified into succinate/ubiquinone oxidoreductases and succinate/menaquinone oxidoreductases [1]. The experiments shown here demonstrate that both enzymes have substantial functional differences. Obviously, all succinate/menaquinone oxidoreductases contain two heme-b groups and/or four conserved His residues as their ligands, whereas the succinate/ ubiquinone oxidoreductases contain one heme-b and, accordingly, two conserved His residues. The presence of two heme-b molecules in the succinate/menaquinone oxidoreductases appears to be essential for their function. The His residues for the low potential heme B, which are supposed as the active site for MK reduction, are close to the outer aspect, the ligands for the high potential heme B to the inner aspect of the membrane, as concluded from the position of the His residues in the predicted transmembrane helices (Fig. 3) [11, 12, 23, 24]. The strict conservation of the four His ligands in succinate/menaquinone oxidoreductases from phylogenetically distant procaryotes, but not in succinate/ubiquinone oxidoreductases, suggests their specific requirement for the former group of enzymes. MK-dependent succinate oxidation: an endergonic reaction driven by reversed, transmembrane electron transport. From the midpoint potentials of the succinate/fumarate (E 30 mv) and MKH 2 /MK (E 80 mv) redox couples, it follows that E for the reaction of succinate/menaquinone oxidoreductase is negative and G is strongly endergonic ( G 21.2 kj/mol). For Q-dependent Sdh (QH 2 /Q, E 110 mv), however, succinate oxidation is exergonic ( G 15.4 kj/mol). The experiments shown here demonstrate that only succinate/menaquinone oxidoreductase, but not succinate/ubiquinone oxidoreductase, requires p or Ψ as the driving force for activity. A model for Q-dependent and MKdependent Sdh enzymes can be made (Fig. 4), which explains the requirement for Ψ or p by the latter. The model combines the structural predictions for the membrane-anchor proteins, in particular the number and location of the heme-b molecules, and the dependence on p or Ψ. The model assumes that the active site for MK reduction and the site for the accession of the protons (quinone 2e 2H quinol) is on the outer aspect of the membrane. Thus, the electrons for MK reduction are delivered from the inner (negative) side of the membrane and move along an electrical gradient ( Ψ), which is about 140 mv in aerobic bacteria. By this means, E for the overall reaction becomes positive ( E 30 mv) and G exergonic ( G 7.7 kj/mol). The assumption that p or Ψ is required for driving the flow of the electrons over the membrane is supported by the midpoint potentials of the heme-b groups. Heme B H on the inner aspect of the membrane has an E m,7.4 of 65 mv, and heme B L on the outer aspect of the membrane an E m,7.4 of 95 mv [10]. This directly suggests that the transmembrane Ψ (or p) is used to move the electrons uphill from heme B H to heme B L. It has to be concluded that Sdh from procaryotes using MK or other electronegative quinones is a coupling enzyme, depending on reversed electron transport. In the succinate/ubiquinone oxidoreductase, however, there is no need for a transmembrane electron transfer and the active site for Q reduction could be on the cytoplasmic aspect of the membrane. Significance of endergonic succinate oxidation in aerobic and anaerobic bacteria. The experiments and structural predictions shown here demonstrate that Sdh enzymes need specific prerequisites for the use of MK as an electron acceptor. These are not present in Q-dependent enzymes. Due to these structural and mechanistic deficiencies the (Q-dependent) Sdh from E. coli, which contains both types of quinones, is not able to operate with MK [6, 25]. It can be predicted that all bacteria and archaea, such as Halobacterium containing only MK, have a succinate/menaquinone oxidoreductase of the type shown here. There is a large group of strictly anaerobic bacteria, for example, Desulfuromonas acetoxidans, Desulfobacter postgatei and Syntrophobacter pfennigii, which oxidize acetate and other volatile fatty acids, such as propionate and butyrate, using elemental S or sulfate as the terminal electron acceptors [26 30]. These bacteria play an important role in mineralization of organic matter in anoxic biotopes, such as marine or freshwater sediments. The oxidation of the fatty acids occurs via a modified citric-acid cycle or the methylmalonyl-coa pathway and involves the function of Sdh using MK as an electron acceptor. Succinate oxidation with S is strongly endergonic (succinate S fumarate H 2 S; G kj/mol S) and was the first endergonic catabolic reaction clearly shown to depend on reversed electron transport [26, 31]. It was suggested that the endergonic reaction from menaquinol to S (S/HS ; E 260 mv) requires reversed electron transport for function [26, 27]. As shown here, the same problem arises for the succinate/ menaquinone reduction and we propose that these anaerobic bacteria also contain a coupling succinate/menaquinone oxidoreductase. This suggests that the coupling succinate/menaquinone oxidoreductases described here are of widespread and general significance for aerobic and anaerobic bacteria. 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