Anaerobic respiration with elemental sulfur and with disul des

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1 FEMS Microbiology Reviews 22 (1999) 353^381 Anaerobic respiration with elemental sulfur and with disul des Reiner Hedderich a; *, Oliver Klimmek b, Achim Kroëger b, Reinhard Dirmeier c, Martin Keller c, Karl O. Stetter c a Max-Planck-Institut fuër terrestrische Mikrobiologie and Laboratorium fuër Mikrobiologie des Fachbereichs Biologie der Philipps-Universitaët, Karl-von-Frisch-StraMe, D Marburg, Germany b Institut fuër Mikrobiologie der Johann-Wolfgang-Goethe-Universitaët Frankfurt am Main, Marie-Curie-StraMe 9, D Frankfurt am Main, Germany c Lehrstuhl fuër Mikrobiologie, Universitaët Regensburg, UniversitaëtsstraMe 31, D Regensburg, Germany Abstract Received 24 June 1998; received in revised form 21 October 1998; accepted 21 October 1998 Anaerobic respiration with elemental sulfur/polysulfide or organic disulfides is performed by several bacteria and archaea, but has only been investigated in a few organisms in detail. The electron transport chain that catalyzes polysulfide reduction in the Gram-negative bacterium Wolinella succinogenes consists of a dehydrogenase (formate dehydrogenase or hydrogenase) and polysulfide reductase. The enzymes are integrated in the cytoplasmic membrane with the catalytic subunits exposed to the periplasm. The mechanism of electron transfer from formate dehydrogenase or hydrogenase to polysulfide reductase is discussed. The catalytic subunit of polysulfide reductase belongs to the family of molybdopterin-dinucleotide-containing oxidoreductases. From the hyperthermophilic archaeon Pyrodictium abyssi isolate TAG11 an integral membrane complex has been isolated which catalyzes the reduction of sulfur with H 2 as electron donor. This enzyme complex, which is composed of a hydrogenase and a sulfur reductase, contains heme groups and several iron-sulfur clusters, but does not contain molybdenum or tungsten. In methanogenic archaea, the heterodisulfide of coenzyme M and coenzyme B is the terminal electron acceptor of the respiratory chain. In methanogens belonging to the order Methanosarcinales, this respiratory chain is composed of a dehydrogenase, the membrane-soluble electron carrier methanophenazine, and heterodisulfide reductase. The catalytic subunit of heterodisulfide reductase contains only iron-sulfur clusters. An iron-sulfur cluster may directly be involved in the reduction of the disulfide substrate. z 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Sulfur respiration; Disul de respiration; Polysul de reductase; Heterodisul de reductase; Wolinella succinogenes; Pyrodictium abyssi; Methanogenic archaea Contents 1. Introduction Biology of sulfur and disul de respiration Biology of sulfur respiration Biology of disul de respiration * Corresponding author. Tel.: +49 (6421) ; Fax: +49 (6421) ; hedderic@mailer.uni-marburg.de / 99 / $19.00 ß 1999 Published by Elsevier Science B.V. PII: S (98)

2 354 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ Chemistry of elemental sulfur, polysul de, and organic disul des Polysul de as a possible intermediate of sulfur respiration Sulfur respiration of Wolinella succinogenes Bioenergetic data Electron transport enzymes Mechanism of electron transfer from hydrogenase to polysul de reductase The function of the Sud protein Mechanism of vp generation Sulfur respiration in hyperthermophilic archaea Sulfur reduction in fermentative hyperthermophiles Sulfur respiration in species of Pyrodictium Disul de respiration in methanogenic archaea Disul de respiration in Methanosarcina species Heterodisul de reductase Hydrogenases F 420 H 2 dehydrogenase Methanophenazine Composition of the di erent respiratory chains and mechanisms of vp generation Disul de respiration in Methanobacteriales, Methanococcales, Methanopyrales, and Methanomicrobiales Other heterodisul de-generating reactions Heterodisul de reductase ^ mechanistic considerations Heterodisul de-reductase-related proteins in non-methanogens Conclusions Acknowledgments References Introduction Many microorganisms can utilize a variety of organic and inorganic compounds as terminal electron acceptors of anaerobic respiration. Among these electron acceptors, sulfur compounds (sulfate, sul te, thiosulfate, organic sulfoxides, elemental sulfur, polysul de, and organic disul des) may play important roles [1,2]. This article will focus on anaerobic respiration with elemental sulfur, with polysul de, and with organic disul des. In Section 2 the biology of some relevant organisms will be brie y discussed, while Sections 3 and 4 will deal with the chemistry of elemental sulfur, polysul de and organic disul des. In Sections 5 and 6, a bacterial (Wolinella succinogenes) and an archael system (Pyrodictium) of sulfur respiration will be described in detail. Section 7 covers the disul de respiration involved in catabolism by methanogenic archaea. Sulfur respiration has been reviewed previously in [3^5] and methanogenesis in [6^11]. 2. Biology of sulfur and disul de respiration 2.1. Biology of sulfur respiration The ability to reduce sulfur using H 2 or organic substrates as electron donors is widespread among bacteria and archaea (Table 1). Most of these organisms are hyperthermophilic and belong to the archaeal domain. Water-containing volcanic areas such as terrestrial solfataric elds and hot springs, and shallow and abyssal submarine hydrothermal systems harbor hyperthermophilic archaea and bacteria, which grow optimally above 80³C [55]. Recently, hyperthermophiles have also been discovered in oilbearing, deep-subterranean rocks, about 4000 m below the Earth's surface [56]. Within volcanic environments, sulfur may be formed in variable concentrations at the surface by oxidation of H 2 S escaping from the depths. In their hot biotopes, hyperthermophiles form complex ecosystems consisting of a variety of primary producers and decomposers of organ-

3 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ Table 1 Archaeal and bacterial genera harboring members able to reduce elemental sulfur to H 2 S T opt (³C) ph opt Electron donors Reference Archaea Crenarchaeota: Acidianus 70^90 1.5^2.0 H 2 [12] Stygiolobus ^3.0 H 2 [13] Pyrobaculum H 2, peptone, extracts of meat and yeast, [14] bacterial and archaeal cell homogenates Thermo lum 85^90 5.0^6.0 Peptides [15,16] Thermoproteus 85^90 5.0^6.5 H 2, peptides, maltose, formate, fumarate, [17,18] ethanol, malate, methanol, glycogen, starch, amylopectin, formamide Desulfurococcus 85^90 6.0^6.4 Peptides, starch, pectin, glycogen, yeast extract, [19,20] casein hydrolysate Igneococcus ^6.0 H 2 Huber et al., unpublished results Pyrodictium ^6.0 H 2 [21,22] Stetteria H 2 [23] Thermodiscus H 2, yeast extract [17,24] Thermosphaera Yeast extract, peptone [25] Staphylothermus Peptone, extracts of meat and yeast [26] Hyperthermus 95^ Tryptone, peptone [27] Euryarchaeota: Pyrococcus 96^ ^7.0 Complex substrates, amino acids, starch, [28,29] maltose, pyruvate Thermococcus 75^88 5.8^9.0 Peptides, amino acids, sugars, starch, chitin, [30,31] pyruvate Caldococcus Peptides [32] Thermoplasma ^2.0 Extracts of yeast, meat, and bacteria [33] Methanopyrus H 2 [34] Methanobacterium 37^ H 2 [34] Methanothermus H 2 [34] Methanococcus 85^ H 2, formate [34] Bacteria Aquifex H 2, sulfur, thiosulfate [35] Ammonifex H 2 [36] Desulfurobacterium H 2 [37] Desulfuromonas Acetate, pyruvate, ethanol [38] Desulfuromusa ^7.0 Acetate, propionate [39] Desulfurella Acetate [40,41] Desulfovibrio Organic acids, alcohols [42] Fervidobacterium 65^70 6.5^7.0 Sugars, pyruvate, yeast extract [43,44] Geobacter ^7.0 Acetate [45] Pelobacter ^7.0 H 2, ethanol [46] Shewanella ^7.0 Lactate [47] Sulfospirillum ^7.5 H 2, formate [48,49] Thermotoga 66^80 6.5^7.5 Sugars, peptone, yeast extract, bacterial and [50,51] archaeal cell homogenates Thermosipho 70^75 6.5^7.5 Yeast extract, brain heart infusion, peptone, [52,53] tryptone Wolinella H 2, formate [54]

4 356 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 ic matter [55,57,58]. Mesophilic and thermophilic sulfur reducers, mostly from the bacterial domain [59], have been isolated from environments such as anoxic marine or brackish sediments, fresh water sediments, bovine rumen, hot water pools from solfataric elds, and volcanic hot springs. Among sulfur-reducing archaea and bacteria, members of the genera Acidianus, Stygiolobus, Thermoproteus, Pyrobaculum, Igneococcus, Pyrodictium, Wolinella, Desulfuromonas, Ammonifex, and Desulfurobacterium are able to gain ATP by lithotrophic sulfur respiration. In contrast, members of the archaeal genera Desulfurococcus, Staphylothermus, Hyperthermus, Thermococcus, and Pyrococcus and of the bacterial genera Thermotoga, Thermosipho, and Fervidobacterium are strictly fermentative sulfur reducers [55,57^59]. The hyperthermophilic bacterium Aquifex pyrophilus, although an aerobic chemolithoautotroph that uses sulfur in addition to hydrogen and thiosulfate as electron donor to reduce oxygen and nitrate, forms high levels of H 2 S from S 0 and H 2 in the late exponential growth phase [35]. In the presence of sulfur, also methanogenic archaea, especially thermophilic and hyperthermophilic members of the genera Methanopyrus, Methanobacterium, Methanothermus, and Methanococcus, produce substantial amounts of H 2 S, while methanogenesis is signi cantly reduced [34]. In some heterotrophs, such as Pyrococcus furiosus and Thermotoga maritima, sulfur is thought to serve as an additional electron sink, but in many organisms, e.g., Aquifex pyrophilus and the methanogens, the metabolic function of sulfur reduction is still uncertain Biology of disul de respiration The ability to use a disul de substrate as an electron acceptor for organotrophic or lithotrophic growth has been reported only for a small number of microorganisms, all of which are sulfur-reducing bacteria or archaea. Desulfuromonas acetoxidans grows not only with sulfur, but also with cystine and oxidized glutathione as electron acceptor and acetate as electron donor [38]. Pyrobaculum islandicum can grow with cystine or oxidized glutathione as electron acceptor and complex media as electron donor [14]. The sulfur-reducing bacteria W. succinogenes and Sulfospirillum deleyianum cannot grow with these disul des as electron acceptors [3,60]. The enzymes that catalyze disul de reduction in D. acetoxidans and P. islandicum have not been investigated. The enzyme responsible for disul de reduction in D. acetoxidans apparently di ers from the enzyme that reacts with polysul de or sulfur, as suggested by the observation that the membrane fraction of D. acetoxidans catalyzes the reduction of sulfur with NADH, but not the reduction of disul des [61]. In contrast to these organisms, which use an external disul de as electron acceptor for respiration, methanogenic archaea generate a disul de in the - nal step of methanogenesis. This disul de is then used as the terminal electron acceptor of the respiratory chain. 3. Chemistry of elemental sulfur, polysul de, and organic disul des The solubility of elemental sulfur in water at 25³C is very low (5 Wg l 31 ) [62]. The solubility at higher temperatures is not known. Polysul de is formed by dissolving sulfur ower in an aqueous sul de solution (Reaction (a)) [63]. ns 0 HS 3! S 23 n 1 H : a The S 8 -ring is cleaved by nucleophilic attack of HS 3. The amount of sulfur that can maximally be dissolved in a sul de solution at ph 8 and 37³C is nearly equivalent to the sul de content [63,64]. Much less polysul de is formed at ph values below the pk of H 2 S (Table 2). Tetrasul de (S 23 4 ) and pentasul de (S 23 5 ) are the predominant species of polysul de at ph s 6. The pk of proton dissociation of HS 3 4 and HS 3 5 are well below 7. Tetrasul de and pentasul de dismutate rapidly according to Reaction (b) [63]. Table 2 Proton dissociation constants of compounds involved in polysul- de reduction Reaction Temperature (³C) pk Reference H 2 SCHS 3 +H [65] HS 3 CS 23 +H 25 s 17 [65] HS 3 4!S23 4 H [66] HS 3 5!S23 5 H [66]

5 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ S 23 5 HS 3 3 4S 23 4 H : b As a consequence of the velocity of Reaction (b), it is not known whether S 23 4 or S 23 5 is the preferred substrate of polysul de reductase. For the same reason, the product of polysul de reduction is not known. It is assumed that only one sulfur atom is cleaved from the polysul de chain during catalysis (Reaction (c)). H 2 S 23 n! HS 3 S 23 n31 H vg 0 ˆ 331 kj=mol H 2 : c The redox potential of polysul de can be estimated from that of elemental sulfur and the equilibrium constant of Reaction (a) assuming that only one species of polysul de is formed in this reaction. The value given in Table 3 refers to the reduction of S 23 4 to HS 3 and is only 15 mv more positive than that of elemental sulfur reduction to HS 3. Similar to polysul de reduction, an S-S bond is cleaved in disul de reduction (reaction (d)). H 2 R-S-S-R! 2R-SH: d The redox potentials of the disul des reduced in methanogenic archaea (CoM-S-S-CoB) is not known. In Table 3, the redox potential for the cysteine/cystine couple (R-S-S-R/2R-SH) is given and is approximately 50 mv more positive than that of elemental sulfur. Hence, from an energetic standpoint, disul des should be the better electron acceptors. 4. Polysul de as a possible intermediate of sulfur respiration Table 3 Redox potentials pertinent to sulfur and disul de respiration Redox couple 0 E 0 (mv) Reference H /H [67] HCO 3 3 =HCO [67] S 0 /HS [67] S 23 4 =HS See text R-S-S-R/2 R-SH 3220 [68] Menaquinone in ethanol 374 [69] Elemental sulfur is not well suited as a substrate of bacterial sulfur respiration because of its low solubility in water. However, `hydrophilic' or `colloidal' sulfur has been reported to be reduced with considerable velocities in the presence of enzyme preparations obtained from sulfur-reducing bacteria [4,60]. Since elemental sulfur is readily converted to polysul de in aqueous solutions of sul de (Reaction (a)), a product of sulfur respiration, it may be speculated that polysul de is an intermediate of sulfur respiration in general. To test this hypothesis, Schauder and Muëller [70] measured the maximum amount of sulfur dissolved according to Reaction (a) as a function of ph and temperature. The authors found that the concentration of polysul de sulfur should be well above 10 WM in the growth medium of sulfur-reducing microorganisms growing in the presence of 1 mm HS 3 +H 2 SatpH s 6. This concentration of polysul de sulfur (10 WM) is close to the apparent K m measured with polysul de respiration of W. succinogenes [71] (see Table 6). With the assumption that 10 WM polysul de sulfur is also required for polysul de respiration to occur in the other bacteria, it follows that polysul de may be an intermediate of sulfur respiration in most of the known sulfur reducers (see Table 1). The acidophilic archaea grow at temperatures close to 90³C, where polysul de sulfur concentrations above 10 WM would require a ph s 5 [70]. However, these bacteria have their ph optimum at about 2 (see Table 1). Hence, the environment of these archaea should not contain enough polysul de to allow polysul de reduction to occur outside of the cytoplasmic membrane. In W. succinogenes, polysul- de reduction occurs in the periplasm, as shown by the orientation of the polysul de reductase towards the outside of the cytoplasmic membrane [72]. The orientation of the corresponding enzyme in other sulfur-reducing bacteria is not known. A soluble cytoplasmic enzyme that catalyzes polysul de reduction by reduced ferredoxin or H 2 has been discovered in P. furiosus [73], and therefore it is feasible that polysul de reduction also occurs in the cytoplasm of the acidophilic archaea. For this to occur, it has to be postulated that elemental sulfur di uses across the cytoplasmic membrane and forms polysul- de in the cytoplasm according to Reaction (a). Un-

6 358 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 fortunately, the di usion velocity of elemental sulfur across the cytoplasmic membrane of growing acidophilic archaea is not known. The concentration of elemental sulfur dissolved in the media at the growth temperature of these archaea is probably considerably higher than at 25³C (5 Wg l 31 ). 5. Sulfur respiration of Wolinella succinogenes The actual electron acceptor of sulfur respiration in W. succinogenes is polysul de [3,5,64,71]. This anaerobic proteobacterium (O-subgroup) grows by polysul de respiration with either H 2 (Reaction (c)) or formate (Reaction (e)). HCO 3 2 S23 n H 2 O!HCO 3 3 HS3 S 23 n31 H vg 0 ˆ 330 kj=mol H 2 : e W. succinogenes has been reported to grow with elemental sulfur as terminal electron acceptor under conditions that were thought not to allow polysul de formation [74]. In these experiments, the culture medium contained Fe 2 to precipitate as FeS all the sul de formed by the bacteria, and polysul de should not be formed from elemental sulfur in the absence of sul de (Reaction (a)). Recently, a soluble sulfur compound was detected in the Fe 2 -containing culture medium at a concentration corresponding to 0.15 mm polysul de sulfur. The compound was converted to SCN 3 upon the addition of CN 3 and the Sud protein (see Reaction (f) in Section 5.4), and may serve as the actual substrate in sulfur respiration. Although the nature of the compound is not yet known, the result argues against a direct conversion of elemental sulfur to sul de by W. succinogenes. Table 4 Bioenergetic data of the polysul de respiration with formate of W. succinogenes Electron acceptor ph Y (g cells/mol formate) ATP/e 5.1. Bioenergetic data The ATP gain (ATP/e) of polysul de respiration has not been measured directly. The value given in Table 4 (0.33 mol ATP per mol formate or ATP/ e = 1/6) was estimated from the growth yield (Y) with polysul de using the known ATP gain and the growth yield of fumarate respiration. With this ATP gain, the free energy used for ATP synthesis would be 116 kj mol ATP 31 in polysul de respiration, while that used in fumarate respiration is 127 kj mol ATP 31. Both values are consistent with the general observation that phosphorylation requires about 100 kj mol ATP 31 in growing bacteria in most instances [67]. In spite of the large di erence between the free energy (or ve) available from respiration with polysul de and from respiration with fumarate, nearly the same electrochemical proton potential across the membrane (vp) has been measured during the respiration steady state with the two electron acceptors. As a consequence, the H /e ratio with polysul de (H /e = 1/2) should be maximally half that measured with fumarate (H /e = 1). The two ratios correspond to the ATP/e ratios with an H /ATP ratio of 3, which has been measured with the ATP synthase of W. succinogenes [79]. The values of Y and vp measured with H 2 instead of formate are close to those given in Table 4. Therefore, the remaining data of Table 4 are likely to apply also for respiration with H 2 (Reaction (c)) Electron transport enzymes The electron transport chain catalyzing polysul de reduction by H 2 or formate consists of polysul de reductase (Psr) and hydrogenase (Fig. 1) or formate dehydrogenase [80]. The enzymes are integrated in 3vE (V) vewf ATP=e (kj/mol ATP) Polysul de [64] (1/6) [75] (1/2) Fumarate [76] 1/3 [77,78] [77] 1 The data are compared to those of fumarate respiration. The values of ph, Y and ve refer to the middle of the exponential growth phase at 0 37³C. ve was calculated from the ve 0 given in Table 3 with the given values of ph and equal concentrations of HCO 3 2 and HCO 3 3, polysul de sulfur and HS 3, and fumarate and succinate. The numbers in parentheses were estimated as described in the text. vp (V) H /e

7 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ Fig. 1. Composition of the electron transport chain catalyzing polysul de respiration with H 2 in W. succinogenes. The sul de dehydrogenase (Sud) protein is described in the text (Section 5.4). Mo, molybdenum linked to molybdopterin guanine dinucleotide; Ni, nickel ion; Fe/S, iron-sulfur centers; Cyt b, diheme cytochrome b; MK b, menaquinone bound to polysul de reductase; PsrA, B, C, polysul de reductase subunits; HydA, B, C, hydrogenase subunits [84]. the cytoplasmic membrane with the catalytic subunits exposed to the periplasm [3,72]. The isolated polysul de reductase, which consists of the three subunits (PsrA, B, C) predicted from the nucleotide sequence of the polysul de reductase operon (psrabc) [81], catalyzes polysul de reduction by BH 3 4 to sul de, and sul de oxidation to polysul de by 2,3-dimethyl-1,4-naphthoquinone (Table 5). The enzyme contains molybdenum and molybdopterin guanine dinucleotide. The amounts of iron and sul- de in the enzyme are consistent with the presence of ve tetranuclear iron-sulfur centers. The amino acid sequence derived from the psra nucleotide sequence suggests that the catalytic subunit carries one tetranuclear iron-sulfur center, and PsrB is predicted to carry four iron-sulfur centers [81]. The amino acid sequence of PsrA is similar to that of the catalytic subunits of several molybdo-oxidoreductases, including Escherichia coli formate dehydrogenase and Rhodobacter sphaeroides dimethylsulfoxide reductase [81]. The crystal structures of these single-subunit enzymes are known [85,86]. At the catalytic site of each enzyme, a molybdenum ion is coordinated by two molybdopterin guanine dinucleotide molecules. PsrA is likely the catalytic subunit of polysul de reductase, and likely carries the molybdenum ion coordinated by two molybdopterin guanine dinucleotide molecules, although a lower molybdopterin guanine dinucleotide content has been determined experimentally (Table 5). A mutant (vpsrabc) lacking the psrabc operon does not catalyze polysul de reduction by H 2 or formate when grown with fumarate as terminal electron acceptor, in contrast to the wild-type strain [72]. Surprisingly, the mutant grows with polysul de. When grown with polysul de, the mutant forms a membrane-integrated enzyme that replaces polysul de reductase (Psr). The enzyme formed by the vpsrabc Table 5 Properties of polysul de reductase [82,83] Subunits: PsrA (81 kda) PsrB (21 kda) PsrC (34 kda) Turnover number: [S]+BH 3 4 CHS3 +BH s 31 Apparent K m 50 WM [S] Turnover number: HS 3 +DMN+H C[S]+DMNH s 31 Apparent K m 25 mm HS 3 Contents (mol/mol PsrABC): Molybdenum 1 MGD 1 Iron 21 Sul de 22 Menaquinone 0.6^1.6 Heme 90.1 Flavin 90.1 Other heavy metals 90.1 MGD, molybdopterin guanine dinucleotide; [S], polysul de; DMN, 2,3-dimethyl-1,4-naphthoquinone; Psr, polysul de reductase. The turnover numbers and the K m were determined with the enzyme in an anoxic bu er (ph 8.3, 37³C) containing either 50 mm Tris-HCl and 10 mm KBH 4 (polysul de reduction), or 0.2 M triethanolamine (ph 7.9) and 0.2 mm DMN (sul de oxidation). The menaquinone content given was corrected for the amount of menaquinone associated with the phospholipid present in the preparation (50^200 Wmol g protein 31 ).

8 360 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 mutant grown with polysul de has not yet been isolated. Its properties appear to di er considerably from those of the polysul de reductase enzyme. The enzyme does not cross-react with antiserum raised against PsrA. Like polysul de reductase, the enzyme catalyzes sul de oxidation by dimethylnaphthoquinone. However, the apparent K m values for sul de di er drastically. The value measured with polysul de reductase is 25 mm (Table 5) and that of the enzyme induced in the vpsrabc mutant is approximately 1 mm [83]. The enzyme present in the mutant is apparently absent from the wild-type strain, as suggested by this di erence in the apparent K m values Mechanism of electron transfer from hydrogenase to polysul de reductase Fig. 2. Electron transport activity with polysul de (A and C) or fumarate (B and D) as a function of the phospholipid/membrane protein ratio. The experiments were performed as described previously [90]. In C and D, the liposomes fused to the membrane fraction of W. succinogenes contained menaquinone (20 Wmol/g phospholipid) isolated from the membrane fraction. In A^D, formate (R) orh 2 (b) were applied as electron donor of electron transport. One unit of activity is equivalent to the oxidation of 1 Wmol formate or H 2 per min at 37³C. A mutant of W. succinogenes lacking the hydrogenase structural genes (hydabc) does not grow with H 2 and either polysul de or fumarate [87]. The mutant grown with formate and fumarate does not catalyze the reduction of polysul de or fumarate by H 2, in contrast to the wild-type strain. Growth and electron transport activities are restored upon insertion of hydabc into the genome of the deletion mutant. Hence, the same hydrogenase appears to serve in the electron transport with polysul de and with fumarate. The same holds true for formate dehydrogenase [80]. In fumarate respiration, electron transfer from the dehydrogenases to fumarate reductase is mediated by menaquinone, which is present in the bacterial membrane in more than 10-fold molar excess over the electron transport enzymes [88,89]. Most of the menaquinone is thought to be dissolved in the lipid phase of the membrane and to serve in transferring electrons from the dehydrogenases to fumarate reductase by di usion. The mechanism of electron transfer from the dehydrogenases to polysul de reductase is not known. Electron transfer by menaquinone di usion appears to be unlikely because the standard redox potential of menaquinone at ph 7 is more than 200 mv more electropositive than that of polysul de (Table 3). The experiments illustrated in Fig. 2 suggest that the electron transfer from the dehydrogenases to polysul de reductase may require di usion and collision of the enzymes within the membrane. The membrane fraction of W. succinogenes was fused with increasing amounts of liposomes containing menaquinone, and the electron transport activity with polysul de (Fig. 2C) and that with fumarate (Fig. 2D) was measured as a function of the amount of phospholipid present in each preparation. The speci c activities given are based on the amount of membrane protein and are proportional to the turnover number of the enzymes in electron transport. The activities of polysul de reductase, fumarate reductase, hydrogenase, and formate dehydrogenase were hardly a ected by the dilution of the membrane fraction with phospholipid (data not shown). In contrast, the electron transport activities with polysul- de decreased by 70^80% upon maximal dilution of the membrane fraction with phospholipid, while

9 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ those with fumarate increased slightly with the lower amounts of phospholipid and were similar to the activities of the original membrane fraction with the highest amount of phospholipids. The di erent e ects of membrane dilution can be explained on the basis of the assumption that polysul de respiration is limited by the di usion of the electron transport enzymes within the membrane, while fumarate respiration is not. The data of Fig. 2C t to the Hardt equation, which relates the collision frequency of two protein molecules to their di usion coe cients (10 38 cm 2 s 31 ) within the membrane and their surface densities [90^92]. The experiments shown in Fig. 2A and B were performed like those shown in Fig. 2C and D, except that the liposomes applied did not contain menaquinone. Under these conditions, the electron transport activities with either polysul de or fumarate decreased with the amount of phospholipid fused to the membrane fraction. The e ect of membrane dilution on fumarate respiration can be explained on the basis of the relatively high apparent K m of fumarate reductase for menaquinone, which is in the millimolar range [93]. The e ect of membrane dilution on polysul de respiration is more pronounced in the absence of menaquinone (Fig. 2A) than in its presence (Fig. 2C), suggesting that menaquinone is involved also in polysul de respiration. This view is supported by the nding that the isolated polysul de reductase contains approximately 1 mol menaquinone per mol enzyme (Table 5). The menaquinone involved in polysul de reduction is probably bound to polysul de reductase and dissociates from its binding site upon dilution of the membrane with phospholipid. In contrast, the menaquinone involved in fumarate respiration freely di uses within the membrane. The view that the function of menaquinone in the respiration with polysul de di ers from that with fumarate is supported by the following result [90]. When experiments similar to those shown in Fig. 2C and D were performed with vitamin K 1 instead of menaquinone, fumarate respiration did not decrease upon membrane dilution, and the data were similar to those of Fig. 2D. In contrast, the e ect on polysul de respiration was similar to that upon membrane dilution in the absence of menaquinone (Fig. 2A). Hence, vitamin K 1 can replace menaquinone in the pathway of fumarate respiration, but cannot replace menaquinone in the pathway with polysul de. Vitamin K 1 (four isoprene residues) differs from menaquinone (seven isoprene residues) only in the length of the isoprenoid side chain and its degree of saturation. Hydrogenase catalyzes the reduction of menaquinone by H 2 [84]. The quinone site is located on the diheme cytochrome b subunit of the enzyme (HydC). HydC mutants with one of the heme-b-ligating histidine residues substituted by another amino acid do not catalyze quinone reduction or polysul de reduction by H 2 [94]. This demonstrates that the intact HydC is required for electron transfer from hydrogenase to polysul de reductase. The result also supports the view that the membrane anchor of polysul de reductase (PsrC) is involved in the electron transfer from hydrogenase to polysul de reductase. Bound menaquinone (Table 5) possibly serves as the prosthetic group of PsrC and as the primary acceptor of the electrons delivered by HydC (Fig. 1). This would explain why mutants that do not catalyze quinone reduction by H 2 also lack electron transport activity from H 2 to polysul de. Formate dehydrogenase catalyzes menaquinone reduction by formate [93]. The quinone reactive site of formate dehydrogenase is located on the diheme cytochrome b subunit of the enzyme. The amino acid sequence of this subunit is similar to that of hydrogenase cytochrome b [95]. Especially the four histidine residues coordinating the heme B groups are predicted to be located at similar places on three homologous membrane helices. Therefore, it is likely that the mechanism of electron transfer from formate dehydrogenase to polysul de reductase is the same as that with hydrogenase The function of the Sud protein In the presence of sulfur, fumarate, and nitrate, W. succinogenes grows by polysul de respiration, while fumarate and nitrate are not reduced [96]. This preference suggests that W. succinogenes is primarily a sulfur (polysul de) reducer. Its ecological role may be to supply sul de as a biosynthetic substrate to the methanogens in the rumen of cattle, the habitat of W. succinogenes. The polysul de concentration in the rumen is estimated to be maximally

10 362 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 6 WM polysul de sulfur at ph 9 7 and 0.1 mm total sul de (HS 3 +H 2 S) [97]. The apparent K m of polysul de reductase for polysul de has been measured as 50 WM (Table 5). The same value has been obtained by measuring electron transport from H 2 to polysul de with the membrane fraction of W. succinogenes (Table 6). The apparent K m for polysul de measured with intact bacteria grown with fumarate is 70 WM. Hence, the K m for polysul de reduction is about an order of magnitude above the actual polysul de concentration in the rumen. However, the apparent K m measured with W. succinogenes grown on polysul de (10 WM) is close to the ruminal polysul de concentration. The lower K m for polysul de measured with cells grown on polysul de is due to the induction of the soluble periplasmic Sud protein under these conditions [71,98]. Sud was originally isolated as a sul de dehydrogenase. The e ect of Sud on the K m for polysul de has been demonstrated using the activity of electron transport from H 2 to polysul de catalyzed by the membrane fraction of W. succinogenes (Table 6). The electron transport activity is considerably increased by the presence of the isolated Sud protein at polysul de concentrations below 0.1 mm. The apparent K m for polysul de decreases from 50 WM in the absence of Sud to 7 WM in its presence. The stimulating e ect of Sud is not observed at higher polysul de concentrations [71] and is maximal with 0.8 WM Sud dimer added. Higher amounts of Sud do not further increase electron transport activity. Polysul de-grown W. succinogenes cells contain nearly equimolar amounts of Sud and Psr, whereas the molar ratio is 0.2 in fumarate-grown cells (Table 6). The concentration of Sud in the periplasm of W. succinogenes grown with polysul de is more than two orders of magnitude higher than that required for saturation of the electron transport activity of the membrane fraction (0.8 WM). These results suggest that the periplasmic Sud is bound to polysul de reductase (Fig. 1). Sud consists of two identical subunits (14.3 kda) and does not contain prosthetic groups or heavy metal ions. Sud binds up to 10 mol polysul de sulfur per subunit when incubated in a polysul de solution [71]. Furthermore, Sud catalyzes sulfur transfer from polysul de to cyanide according to Reaction (f) S 23 n CN 3!SCN 3 S 23 n31 f with a turnover number of about 10 4 s 31 at 37³C. These results suggest that Sud serves as a polysul de sulfur transferase from aqueous polysul de to the active site of polysul de reductase. Sud appears to raise the a nity of polysul de reductase for polysul- de. The function of Sud probably is to allow polysul de respiration to occur at a su cient speed even at very low polysul de concentrations Mechanism of vp generation The mechanism of vp generation in the polysul de respiration of W. succinogenes is not known. Two types of mechanism are feasible. Polysul de reductase may operate as a proton pump during electron transport from H 2 or formate to polysul de. Alternatively, the redox reactions of the menaquinone that is probably bound to PsrC may be coupled to proton translocation across the membrane. A schematic view of the stationary complex formed by hydrogenase and polysul de reductase in the cytoplasmic membrane during electron transfer is given in Fig. 3. The bound menaquinone (MK b ) is assumed to form the hydroquinone anion (MK b H 3 ) upon reduction by hydrogenase to account for the H /e ratio of 1/2 (Table 4). The site of quinone reduction Table 6 K m values for polysul de in the electron transport from H 2 to polysul de as a function of the amount of Sud protein present [71] Sud concentration (WM) Molar ratio (Sud/Psr) K m for polysul de (WM) Membrane fraction Membrane fraction Cells grown with polysul de Cells grown with fumarate Psr, polysul de reductase.

11 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ Fig. 3. Hypothetical mechanism of vp generation by electron transport from H 2 to polysul de in W. succinogenes. The electron transfer from hydrogenase (upper part) to polysul de reductase (lower part) requires collision of the two enzymes within the membrane. Ni, nickel ion; Mo, molybdenum ion linked to molybdopterin guanine dinucleotide; MK b, menaquinone bound to polysul de reductase. is envisaged to be located in a lipophilic environment. This would require the existence of proton paths for proton uptake during quinone reduction and for proton release during quinol oxidation. It is assumed that the former path is provided by hydrogenase and the latter by polysul de reductase. Consistent with the model shown in Fig. 3, the protons required for menaquinone reduction by H 2 in the membrane of W. succinogenes have been shown to be taken up from the cytoplasm [99]. 6. Sulfur respiration in hyperthermophilic archaea 6.1. Sulfur reduction in fermentative hyperthermophiles Several heterotrophic sulfur reducers, such as Pyrococcus furiosus, exhibit a fermentative type of metabolism in which sulfur acts as an additional electron sink. From this organism, two enzymes have been isolated that catalyze the reduction of sulfur or polysul de to H 2 S. An NAD(P)H-dependent sul- de dehydrogenase and a hydrogenase (termed sulfhydrogenase or sulfur:reduced ferredoxin oxidoreductase) couple the oxidation of reduced ferredoxin to the reduction of either protons to H 2 or sulfur to H 2 S [100,101]. However, the location of these two enzymes in the cytoplasm, plus the nding that products of maltose fermentation are virtually identical during growth with or without sulfur, argue against a conventional membrane-bound respiratory type of metabolism in the presence of sulfur [102]. In these sulfur-reducing heterotrophs, the reduction of sulfur to H 2 S is proposed to be a mechanism for the disposal of excess reductant generated by fermentation and of toxic H 2 [28,51]. As shown for other H 2 S-producing organisms, sulfur reduction could also lead to the formation of metal sul des, thus allowing the removal of toxic metals [103] Sulfur respiration in species of Pyrodictium In hyperthermophilic sulfur-respiring archaea, the reduction of sulfur to H 2 S is catalyzed by membrane-bound respiratory chains. Lithotrophs such as Pyrodictium brockii and Stygiolobus azoricus use molecular hydrogen as electron donor for this reaction [21,13], while organotrophic organisms such as Thermodiscus maritimus and Thermo lum pendens use peptides or carbohydrates [16,104]. Evidently, the lithotrophic sulfur-respiring archaea must couple electron transport to sulfur with phosphorylation of ADP. In the membranes of P. brockii, a hydrogenase, a quinone, and a cytochrome c have been identi ed as part of a proposed respiratory electron transport chain [105^107]. The hydrogenase has been puri ed and found to be of the Ni/Fe-type and to consist of two subunits (66^68 kda and 45 kda) [106]. TLC analysis of the quinone has shown migration characteristics similar to that of ubiquinone-6 (Q-6), but NMR analysis has revealed evidence for a quinone di erent from all quinones compared. Cytochrome c (13^14 kda) is the only cytochrome detected in the membranes of P. brockii. Inhibition experiments with the quinone analogue HQNO have suggested

12 364 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 the electron transfer sequence: hydrogenasecquinoneccytochrome c. After inactivation of the electron transport activity by UV light, addition of ubiquinones Q-6, Q-10 or puri ed P. brockii quinone restores activity. Cytochrome c is thought to serve as electron donor to the sulfur reductase, which has not yet been identi ed [105^107]. The electron transport chain catalyzing sulfur reduction by H 2 in P. abyssi isolate TAG11 di ers from that of P. brockii with respect to composition and organization of the components [22]. A H 2 :sulfur oxidoreductase complex, which catalyzes the H 2 -dependent reduction of sulfur to H 2 S, has been recently puri ed from the membranes of P. abyssi isolate TAG11. The catalytic properties of the enzyme complex suggest that it represents the entire respiratory chain of the organism, with hydrogenase, electron transport components, and sulfur reductase arranged in one stable multi-enzyme complex. The puri ed H 2 :sulfur oxidoreductase consists of at least nine subunits, two of which are b-type cytochromes, and one a cytochrome c. The cytochrome c (30 kda) is approximately twice as large as that of P. brockii. It should be pointed out that among hyperthermophiles, c-type cytochromes have been detected only in species of Pyrodictium [22,105]. No quinone has been detected in the H 2 :sulfur oxidoreductase or in the membrane fraction of P. abyssi isolate TAG11. Although the respiratory chains of P. brockii and P. abyssi isolate TAG11 di er in electron transport components, the hydrogenases appear to be similar. The H 2 :sulfur oxidoreductase consists of two subunits (66 and 45 kda), similar in size to the P. brockii hydrogenase subunits. The N-terminal amino acid sequence of the 66-kDa subunit is similar to the N-terminal sequence of the catalytic subunits of Ni/Fe-hydrogenases. The content of 1.6 mol nickel/mol H 2 :sulfur oxidoreductase suggests that its hydrogenase, like the P. brockii enzyme, is of the Ni/ Fe-type. Both hydrogenases are insensitive to oxygen and function as `H 2 -uptake' hydrogenases, indicating the respiratory role of these enzymes. During puri cation, the activity of the hydrogenase present in the H 2 :sulfur oxidoreductase complex, as measured with viologen dyes, increases parallel with H 2 :sulfur oxidoreductase activity. Present data suggest that energy conservation via respiration in hyperthermophiles appears to be similar to that of mesophiles. A membrane-bound respiratory chain generates a chemiosmotic potential, which is utilized by a membrane-bound ATP synthase to form ATP. Yet, due to their extreme habitats, hyperthermophiles have adapted their system to high temperatures. For example, in P. abyssi isolate TAG11, not only the H 2 :sulfur oxidoreductase complex, but also a membrane-bound ATPase, likely to function as ATP-synthase, show temperature optima around 100³C [22] (R. Dirmeier, unpublished results). Thus far, the electron transport chain of P. brockii and the H 2 :sulfur oxidoreductase complex from P. abyssi isolate TAG11 are the only described examples of enzymes involved in the membranebound sulfur respiration of hyperthermophilic organisms. The stable organization of the di erent components of the H 2 :sulfur oxidoreductase complex from P. abyssi isolate TAG11 implies that further investigations will yield a better understanding of sulfur respiration in hyperthermophiles. 7. Disul de respiration in methanogenic archaea Methanogenic archaea derive their metabolic energy from the conversion of a restricted number of substrates to methane. Most methanogens can reduce CO 2 to CH 4 with H 2 as electron donor. A few methanogens can utilize formate, ethanol, or isopropanol as an electron donor for CO 2 reduction. Some methanogens can convert methanol, methylamines, and methylmercaptans to CH 4 and CO 2. Acetate is the only C 2 -compound utilized by some Fig. 4. Structures of coenzyme M (H-S-CoM; 2-mercaptoethanesulfonate), coenzyme B (H-S-CoB; 7-mercaptoheptanoylthreonine phosphate), and the heterodisul de (CoM-S-S-CoB) of coenzyme M and coenzyme B.

13 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ methanogens as sole energy substrate. It is converted to CH 4 and CO 2. (For a historical overview on methanogenesis, see [6,7]; for recent reviews see [8^ 11].) In these di erent pathways of energy metabolism, two unique thiol-containing coenzymes play a central role: coenzyme M (H-S-CoM; 2-mercaptoethanesulfonate) and coenzyme B (H-S-CoB; 7-mercaptoheptanoylthreonine phosphate) (Fig. 4). Coenzyme M is converted to its methylthioether (CH 3 -S-CoM), which is the central intermediate of methanogenesis (Fig. 5) (see [6,7]). This methylthioether reacts with coenzyme B to yield methane and the heterodisul de (CoM-S-S-CoB) of the two methanogenic thiol-containing coenzymes. This reaction is catalyzed by the soluble methyl-coenzyme M reductase (for a review see [11]). The heterodisul de thus generated plays a central role in energy conservation; the reduction of CoM-S-S-CoB is coupled with the generation of a proton motive force [108^111]. Hence, CoM-S-S- CoB is the terminal electron acceptor of a respiratory chain in these organisms. The enzyme reducing the heterodisul de to the thiols H-S-CoM and H-S- CoB, heterodisul de reductase, is membrane bound and functions as a terminal respiratory reductase. The electron donor for this disul de respiration varies with the growth substrate. In the following, the composition of the various respiratory chains involved in the reduction of CoM-S-S-CoB will be discussed in more detail. Taxonomically, methanogens belong to the archaeal kingdom of euryarchaeota. They are classi ed in ve orders, each of which is as distantly phylogenetically related to the other as the cyanobacteriales to the proteobacteriales. The ve orders are Methanobacteriales, Methanococcales, Methanomicrobiales, Methanopyrales, and Methanosarcinales [112]. Of these, only the Methanosarcinales can ferment acetate to CO 2 and CH 4 and can grow on methanol, methylamines, and methylthiols as sole energy source. In addition, the Methanosarcinales contain cytochromes, whereas cytochromes have not been found in organisms belonging to the other orders of methanogens (see [8]). The ability to use a variety of substrates and the presence of cytochromes have an important in uence on the composition of the respiratory chains involved in disul de respiration. Therefore disul de respiration in Methanosarcina species will be discussed separately from disul de respiration in organisms belonging to other phylogenetic groups of methanogens Disul de respiration in Methanosarcina species Since Methanosarcina species can use several growth substrates, these organisms contain di erent respiratory chains for the reduction of CoM-S-S- CoB. As will be shown below, these respiratory chains are composed of a substrate-speci c dehydrogenase, a membrane-bound electron carrier, and heterodisul de reductase. The following enzymes and electron carriers have been shown to participate in these respiratory chains Heterodisul de reductase Heterodisul de reductase (Hdr) was puri ed from the membrane fraction of Methanosarcina barkeri using detergents for solubilization [113^115]. The puri ed enzyme is composed of two di erent subunits, a 23-kDa polypeptide designated HdrE and a 46- kda polypeptide designated HdrD. The enzyme contains approximately 0.6 mol heme b/mol enzyme and about 20 mol non-heme iron and acid-labile sulfur/ mol enzyme. In contrast to most other disul de reductases, this disul de reductase does not contain a avin [115]. The 23-kDa polypeptide shows peroxidase activity, which indicates that this polypeptide contains heme. Spectroscopic studies have shown it to be a heme b [113,114]. The genes encoding the two subunits HdrD and HdrE form the transcription unit hdred. From the deduced amino acid sequence, it can be predicted that HdrE is an integral membrane protein with ve transmembrane-spanning helices. Sequence analysis con rmed that HdrE is a b-type cytochrome as it shows sequence similarity to other b-type cytochromes [115]. Analysis of the deduced amino acid sequence of HdrD indicates that it is a hydrophilic polypeptide that contains two classical binding motifs for [4Fe-4S] clusters close to its N-terminus. The C-terminal domain of the polypeptide contains several cysteine residues, which could ligate an additional iron-sulfur cluster, as will be discussed below. In Northern blot experiments with RNA isolated from cells grown on methanol, H 2 /CO 2, or acetate, probes derived from hdre or hdrd each hybridized to a 2.3-

14 366 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 Fig. 5. Scheme of methanogenesis from H 2 /CO 2, methanol, and acetate. As a central intermediate of the various pathways, methyl-coenzyme M (CH 3 -S-CoM) is formed and is converted to methane and the heterodisul de of coenzyme M and coenzyme B (CoM-S-S-CoB). CoM-S-S-CoB thus generated functions as the terminal electron acceptor of the various respiratory chains. H 2 and reduced coenzyme F 420 (F 420 H 2 ) are the electron donors for the reduction of CoM-S-S-CoB. The unknown mechanism of electron transfer from the reduced ferredoxin (Fd red ) to CoM-S-S-CoB in acetate metabolism is symbolized by a question mark. The role of H 2 as an intermediate of this reaction is discussed in the text. CH 3 -H 4 MPT, methyl-tetrahydromethanopterin; F 420 H 2, reduced form of coenzyme F 420 ; Fd, ferredoxin; pfd, polyferredoxin. kb mrna, indicating that this operon is expressed during growth on each of these substrates [115]. In Southern blot hybridizations with total DNA from M. barkeri, only one copy of hdre and hdrd could be detected. This indicates that the same heterodisul- de reductase operates in H 2 /CO 2, methanol, and acetate metabolism (A. Kuënkel and R. Hedderich, unpublished results). Recently the puri cation and characterization of heterodisul de reductase from Methanosarcina thermophila has been reported [116]. The enzyme exhibits properties very similar to those of the M. barkeri enzyme Hydrogenases A membrane-bound hydrogenase has been puri- ed from Methanosarcina mazei [117] and Methanosarcina barkeri [118] using detergents for solubilization. The puri ed enzyme contains Ni, non-heme iron, and acid-labile sulfur. It is composed of two di erent subunits with apparent molecular masses of 60 kda and 40 kda. In the genome of M. mazei, the structural genes for two closely related membrane-bound hydrogenases have been identi ed [119]. They are encoded in two separate transcriptional units: vhogac (viologen-reactive hydrogenase one) and vhtgac (viologen-reducing hydrogenase

15 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ two). The genes vhoa and vhta each encode a 60- kda subunit, which harbors the binding motifs for the Ni-Fe active site. The genes vhog and vhtg each encode a 40-kDa subunit, which contains 10 conserved cysteine residues. Similar conserved residues have been shown to ligate three iron-sulfur clusters in the Desulfovibrio gigas hydrogenase [120]. Each transcriptional unit contains one additional gene, vhoc or vhtc, whose gene product is not present in the puri ed enzyme. These genes encode b-type cytochromes. It is assumed that the hydrophobic VhoC and VhtC subunits were separated from their two hydrophilic subunits during puri cation of the enzymes. The vht operon contains a fourth gene (vhtd), whose gene product is also not present in the puri ed enzyme. The function of this gene is not known. The nding that the 5P-ends of the vhog/vhtg genes code for a long signal peptide supports the topology of the hydrogenase shown in Fig. 6. Northern blot experiments have shown that the expression of the genes encoding the two membrane-bound hydrogenases in M. mazei is substrate-dependent [121]. The vhogac operon is expressed during growth on H 2 /CO 2, methanol, or acetate, while the vhtgac operon is only expressed during growth on H 2 /CO 2 and methanol but not during growth on acetate. Obviously the two hydrogenases have di erent functions in the metabolism of Methanosarcina mazei. The amino acid sequences of the homologous structural subunits (VhoG/VhtG and VhoA/VhtA) are almost identical. The C-termini of VhoC and VhtC are not homologous. This might indicate that the two hydrogenases interact with different electron acceptors via this subunit. Since the VhoGAC hydrogenase is synthesized during growth on all substrates, it could be part of the respiratory chain from H 2 to CoM-S-S-CoB, while the VhtGAC hydrogenase could be involved in reactions speci c for H 2 /CO 2 and methanol metabolism [8] F 420 H 2 dehydrogenase The deaza avin coenzyme F 420 (E 0 0 = 3360 mv) is a two-electron redox carrier in methanogenic archaea. It functions as the physiological electron acceptor or donor of several oxidoreductases of the pathways of energy metabolism [6]. The reduced form of coenzyme F 420 (F 420 H 2 ) functions as the physiological electron donor for CoM-S-S-CoB reduction in Methanosarcina species. F 420 H 2 dehydrogenase, which catalyzes the oxidation of F 420 H 2,is an integral membrane protein and has been puri ed from Methanolobus tindarius [122] and Methanosarcina mazei [123]. The enzyme from M. tindarius is composed of ve di erent subunits (45, 40, 22, 18, and 17 kda) and contains Fe/S centers but no avin. The enzyme from M. mazei is composed of ve different subunits (40, 37, 22, 20, and 16 kda) and contains approximately 7 mol non-heme iron and 7 mol acid-labile sulfur. In addition, the enzyme contains FAD as prosthetic group. The puri ed enzyme catalyzes the reduction of methanophenazine analogues, such as 2-hydroxyphenazine, with a speci c activity of 9 U/mg protein and an apparent K m for 2-hydroxyphenazine of 35 WM. In vivo the lipophilic methanophenazine present in the membrane is assumed to be the physiological electron acceptor of this enzyme [111,124,125] as will be discussed below. An F 420 H 2 :quinone oxidoreductase has been characterized from the sulfate-reducing archaeon Archaeoglobus fulgidus [126]. The genes encoding this enzyme have been identi ed in the totally sequenced genome of A. fulgidus [127]. The subunits of this enzyme show high sequence similarity to subunits of energy-conserving NADH:quinone oxidoreductase Methanophenazine Methanogenic archaea do not contain quinones. Recently a compound was isolated from membranes of M. mazei that could have a function in the respiratory chains of Methanosarcina species similar to that of quinones in the respiratory chains of other organisms [111]. This novel electron carrier is called methanophenazine and is a 2-hydroxyphenazine derivative connected to a polyisoprenoid side chain via an ether bridge. Since this component is almost insoluble in water, water-soluble analogues of methanophenazine, such as 2-hydroxyphenazine and 2-bromophenazine, have been used for in vitro enzyme assays. These water-soluble analogues have been shown to function as electron acceptors of the puri- ed F 420 H 2 dehydrogenase [111,125]. In addition, washed membranes of M. mazei catalyze the reduction of these methanophenazine analogues by H 2, suggesting that the methanophenazine functions as

16 368 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 electron acceptor of one of the membrane-bound hydrogenases. Furthermore, the membrane-bound heterodisul de reductase uses reduced 2-hydroxyphenazine as an electron donor for the reduction of CoM-S-S-CoB [111,125]. From these data, it is reasonable to assume that methanophenazine plays an important role in membrane-bound electron transport in vivo Composition of the di erent respiratory chains and mechanisms of vp generation When Methanosarcina species grow on H 2 /CO 2, the electron donor for the reduction of CoM-S-S- CoB is H 2 (Fig. 5). A subcellular system from Methanosarcina mazei consisting of washed inverted vesicles catalyzes the reduction of CoM-S-S-CoB with H 2 [108,110]. Fig. 6. Putative scheme of the respiratory chain from H 2 to CoM-S-S-CoB and F 420 H 2 to CoM-S-S-CoB in Methanosarcina species. MP ox, methanophenazine in the oxidized form; MP red, methanophenazine in the reduced form. For other abbreviations, see Fig. 3. H 2 CoM-S-S-CoB! H-S-CoM H-S-CoB vg 0 ˆ 340 kj=mol: g This reaction is coupled with proton translocation across the cytoplasmic membrane into the lumen of the inverted vesicles. Two H are translocated per molecule of CoM-S-S-CoB reduced in this in vitro system. Results of experiments with intact cells and CH 3 OH/H 2 as substrate indicate a stoichiometry of 3^4 H translocated per CoM-S-S-CoB reduced. The discrepancy can be explained by the fact that only about 50% of the vesicles in the in vitro system are intact and thus couple CoM-S-S-CoB reduction with H translocation. A stoichiometry of 3^4 H translocated per CoM-S-S-CoB reduced indicates that the proton motive force is not generated solely by transmembrane electron transport, with H 2 being oxidized at the extracellular site of the cytoplasmic membrane. A di erent or additional mechanism for proton translocation must operate. The vp generated drives the phosphorylation of ADP with inorganic phosphate. From the present data, it is assumed that the respiratory chain is composed of one of the membrane-bound hydrogenases ^ most probably VhoGAC ^methanophenazine, and heterodisul de reductase (Fig. 6) [111,124]. During growth on methanol or methylamines, part of the reducing equivalents are transferred to F 420 to generate F 420 H 2 (Fig. 5). Washed inverted vesicles of M. mazei catalyze the reduction of CoM-S-S-CoB by F 420 H 2 [128]. F 420 H 2 CoM-S-S-CoB! F 420 H-S-CoM H-S-CoB vg 0 ˆ 329 kj=mol: h The reaction is coupled with proton translocation across the cytoplasmic membrane with a stoichiometry of 2 H translocated [109]. Recent data indicate that this respiratory chain is composed of F 420 H 2 dehydrogenase, methanophenazine, and heterodisul- de reductase (Fig. 6) [111,125]. Using washed everted vesicles of M. mazei, it has been shown

17 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ that both the reduction of 2-hydroxyphenazine with F 420 H 2 and the reoxidation of reduced hydroxyphenazine by CoM-S-S-CoB are coupled to proton translocation across the cytoplasmic membrane [124]. The mechanism of proton translocation is unknown. Since oxidation of F 420 H 2 and reduction of CoM-S-S-CoB both occur on the cytoplasmic side, transmembrane electron transport without proton translocation can be excluded as the mechanism of vp generation. Protons are translocated either by a redox-driven proton pump or by the redox reactions of methanophenazine (Fig. 6). During growth on acetate, cleavage of the acetate molecule is catalyzed by CO dehydrogenase/acetyl CoA synthase. This reaction generates enzymebound CO and an enzyme-bound methyl group. The methyl group is transferred to coenzyme M via tetrahydromethanopterin (H 4 MPT). The methyl group of methyl-coenzyme M is subsequently reduced by H-S-CoB to CH 4, thereby forming CoM- S-S-CoB. The CO bound to CO dehydrogenase/acetyl-coa synthase is oxidized to CO 2, and the reducing equivalents are used for the reduction of CoM-S- S-CoB. A ferredoxin has been shown to be the direct electron acceptor of CO dehydrogenase/acetyl CoA synthase (Fig. 5) (for a recent review see [9]). It is not yet known how the electrons are transferred from the ferredoxin to CoM-S-S-CoB. Based on studies with whole cells [129,130] and cell extracts [131,132], H 2 has been proposed to be an intermediate of this electron transfer reaction. Cell suspensions of M. barkeri catalyze the conversion of external CO to CO 2 and H 2 when methane formation is inhibited [133,134]. CO conversion to CO 2 and H 2 is coupled with the generation of a proton motive force [133,134]. However, the molecular basis for the generation of H 2 is not known. Recently a novel hydrogenase was puri- ed and characterized from acetate-grown cells of M. barkeri, which could catalyze H 2 formation via this metabolic pathway [135]. The hydrogenase was designated Ech (E for E. coli, c for the third (c) hydrogenase, and h for hydrogenase) because its properties are similar to those of the E. coli hydrogenase 3. The M. barkeri enzyme is an integral membrane protein composed of six di erent subunits. In Northern blot experiments, the transcript of the ech operon was detected in cells of M. barkeri grown with acetate, methanol, or H 2 /CO 2. The enzyme shares the highest sequence similarity with the COinduced hydrogenase from Rhodospirillum rubrum [136] and also has signi cant sequence similarity to the Escherichia coli hydrogenases 3 and 4 [137,138]. R. rubrum can grow in the dark on CO as sole energy source, forming H 2 and CO 2. This reaction is coupled with the formation of a proton motive force in this organism. Since the CO dehydrogenase in R. rubrum is a soluble enzyme, the membrane-bound CO-induced hydrogenase is most likely the site of energy conservation [136]. CO H 2 O! CO 2 H 2 vg 0 ˆ 320 kj=mol: Likewise, in the acetate metabolism of Methanosarcina, bound CO, generated via decarbonylation of acetyl-coa, might be converted to CO 2 and H 2, catalyzed by CO dehydrogenase/acetyl-coa synthase and Ech hydrogenase. If H 2 is an intermediate of this electron transport chain, a second membranebound hydrogenase (an H 2 uptake hydrogenase) must be present in acetate-grown cells that together with heterodisul de reductase catalyzes CoM-S-S- CoB reduction by H 2. This is indeed the case. Acetate-grown cells of Methanosarcina species synthesize the same membrane-bound hydrogenase as H 2 /CO 2 - grown cells (VhoGAC in M. mazei) [118,121]. Thus, the reduction of CoM-S-S-CoB by H 2 in acetate metabolism could involve the same electron transport chain as in H 2 /CO 2 metabolism. In summary, an `intraspecies' hydrogen cycling is proposed which includes two di erent coupling sites for energy conservation: (i) the site for the conversion of bound CO to CO 2 and H 2 and (ii) the site for the reduction of CoM-S-S-CoB by H 2 (Fig. 7). Alternatively, Methanosarcina species could contain an electron transport chain that directly channels electrons from CO dehydrogenase/acetyl-coa synthase via a ferredoxin to heterodisul de reductase. Such a CO:heterodisul de oxidoreductase activity has been reconstituted with puri ed CO dehydrogenase/acetyl-coa synthase, ferredoxin, washed membranes, and partially puri ed heterodisul de reductase [140,141]. Since this in vitro system still contains the membrane fraction and thus both mem- i

18 370 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381 Fig. 7. Scheme of methanogenesis from acetate in Methanosarcina species. Recent data indicate that the 2 [4Fe-4S] ferredoxin (Fd) from M. barkeri mediates electron transfer between CO dehydrogenase/acetyl-coa synthase and Ech hydrogenase [139]. CH 3 -H 4 MPT, methyltetrahydromethanopterin; MP ox, methanophenazine in the oxidized form; MP red, methanophenazine in the reduced form; Hdr, heterodisul de reductase; Vho, viologen-reactive hydrogenase one. brane-bound hydrogenases (VhoGAC and Ech), it cannot be excluded that H 2 is an intermediate in this system Disul de respiration in Methanobacteriales, Methanococcales, Methanopyrales, and Methanomicrobiales Most of the organisms belonging to these phylogenetic groups are restricted to H 2 /CO 2 as energy substrates. These organisms do not contain cytochromes and thus b-type cytochromes can be excluded as membrane anchors and electron carriers of membrane-bound dehydrogenases and reductases. Reduction of CoM-S-S-CoB has been investigated mainly with Methanobacterium thermoautotrophicum, which belongs to the order Methanobacteriales. Upon puri cation, heterodisul de reductase of this organism was obtained in a tight complex with one of the [Ni-Fe] hydrogenases, the so-called F 420 -nonreducing hydrogenase [142,143]. This complex catalyzes the reduction of CoM-S-S-CoB with H 2 at signi cant rates. At alkaline ph, the complex can be dissociated into the two individual enzymes, heterodisul de reductase and hydrogenase. Heterodisul de reductase is composed of three di erent subunits ^ HdrA, -B, and -C ^ encoded by the two separate transcriptional units hdra and hdrcb. The enzyme contains FAD and iron-sulfur clusters. HdrA contains an FAD binding motif and four binding motifs for [4Fe-4S] clusters. HdrC contains two binding motifs for [4Fe-4S] clusters [144]. The F 420 -non-reducing hydrogenase is also composed of three di erent subunits: a hydrogenase large subunit containing the binuclear Ni-Fe active site, a hydrogenase small subunit containing three iron-sulfur clusters, and an additional small subunit with unknown function. The operon encoding the three subunits of this hydrogenase (mvhdga, methylviologen-reducing hydrogenase) contains an additional open reading frame (mvhb), which encodes a polyferredoxin [145]. The polyferredoxin has been puri ed from M. thermoautotrophicum as an individual protein [146^148]. It is present in small amounts in the puri ed H 2 :heterodisul de oxidoreductase complex, but a function as electron carrier in this

19 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ complex has not been clearly shown. After cell lysis, the H 2 :heterodisul de oxidoreductase complex is present in the soluble fraction of M. thermoautotrophicum. The complex contains only hydrophilic polypeptides, as indicated by the deduced amino acid sequence of the proteins. The three transcriptional units encoding the di erent subunits of the complex do not contain additional open reading frames encoding hydrophobic proteins, which in theory could have been separated from the catalytic subunits during the puri cation. Hence, the major question is how this non-integral membrane protein complex can couple the reduction of CoM-S-S-CoB by H 2 with the generation of the proton motive force. At present there is no conclusive answer to this question, and the following ndings should be considered. Coupling of methanogenesis with ADP phosphorylation is not constant. During growth of methanogens on H 2 /CO 2, the growth yield per mol CH 4 increases with decreasing H 2 concentrations [149,150] indicating that at low H 2 concentrations, energy coupling is tighter than at high H 2 concentrations. Hence, at di erent H 2 concentrations, di erent electron transport chains could be involved in the reduction of CoM-S-S-CoB. The genome of M. thermoautotrophicum contains no additional gene cluster encoding a second heterodisul de reductase [151]. The genome contains, however, two gene clusters that presumably encode two additional hydrogenases, which have not yet been identi ed at the protein level [135,151]. The genes encoding the large and small subunits of these postulated hydrogenases are closely linked to genes encoding iron-sulfur proteins and integral membrane proteins. Both the hydrophilic and the hydrophobic subunits of these putative hydrogenases show signi cant sequence similarity to subunits of the energy-conserving NADH:quinone oxidoreductase from various organisms [152]. Similar gene clusters are present in the genome of M. jannaschii [153]. These putative enzymes are interesting candidates for proton pumps. It may be speculated that under certain physiological conditions, such as low H 2 concentration, one of these hydrogenases interacts with heterodisul de reductase to couple the reduction of CoM-S-S-CoB by H 2 with the formation of a proton motive force. At high H 2 concentrations, reduction of CoM-S-S-CoB might not be coupled with energy conservation and might be catalyzed by the soluble H 2 :heterodisul de oxidoreductase complex described above. This `uncoupling' might allow a higher ux through the metabolic pathway and could compensate the lower energy yield (see [154]). There is only limited information about the H 2 :heterodisul de oxidoreductase reaction from organisms belonging to the orders Methanococcales, Methanopyrales, and Methanomicrobiales. Heterodisul de reductase activity has been detected in organisms belonging to these phylogenetic groups [155]. As in M. thermoautotrophicum, most of the activity is located in the soluble fraction. The genome of Methanococcus jannaschii contains two copies of hdrcb and one copy of hdra [153]. No data have been obtained with puri ed enzymes from this organism. Heterodisul de reductase has been puri ed from Methanopyros kandleri (R. Hedderich, unpublished results). The enzyme has a subunit composition similar to that of heterodisul de reductase from M. thermoautotrophicum. The N-terminal amino acid sequence of the 35-kDa subunit is highly similar to that of HdrB from M. thermoautotrophicum. The gene encoding the subunit HdrA has been cloned and sequenced, and the deduced amino acid sequence shares high sequence similarity with HdrA from M. thermoautotrophicum [115]. Hence, heterodisul de reductase in this organism seems to be quite similar to the enzyme from M. thermoautotrophicum. It is interesting to note that the sulfate-reducing archaeon A. fulgidus contains homologues of the genes hdra, hdrb, hdrc, mvhd, mvhg, and mvha in a putative transcriptional unit hdracbmvhdga (genes AF1377^AF1372) [127]. This nding supports the biochemical data obtained with the H 2 :heterodisul- de oxidoreductase complex from M. thermoautotrophicum that indicate that heterodisul de reductase and methylviologen-reducing hydrogenase (Mvh) form a functional complex. An F 420 -non-reducing hydrogenase, similar to the M. thermoautotrophicum enzyme, is also present in the Methanococcales. The enzyme from Methanococcus voltae has been characterized in detail [156]. The enzyme from M. voltae does not form a tight complex with heterodisul de reductase in vitro.

20 372 R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^ Other heterodisul de-generating reactions Methyl-coenzyme M reduction with coenzyme B is not the only reaction in which CoM-S-S-CoB is generated. Most methanogens contain a soluble fumarate reductase, which catalyzes the reduction of fumarate with H-S-CoM and H-S-CoB to succinate and CoM-S-S-CoB [157,158]. Fumarate H-S-CoM H-S-CoB! succinate CoM-S-S-CoB: j This reaction is part of a biosynthetic pathway for the biosynthesis of 2-oxoglutarate. Since this anabolic reaction generates CoM-S-S-CoB, it also has a link to energy conservation. The reaction is catalyzed by thiol:fumarate reductase (Tfr). The enzyme is composed of two di erent subunits, TfrA and TfrB [157,158]. TfrA contains FAD and has high sequence similarity to the catalytic subunit of fumarate reductases and succinate dehydrogenases. TfrB contains three binding motifs for di erent Fe/S clusters and shows sequence similarity to the subunit HdrD of the M. barkeri heterodisul de reductase and to the subunits HdrC and HdrB of the M. thermoautotrophicum heterodisul de reductase. It is reasonable to assume that the subunit TfrA harbors the catalytic site for fumarate reduction and TfrB the catalytic site for thiol oxidation Heterodisul de reductase ^ mechanistic considerations Heterodisul de reductase from M. barkeri and heterodisul de reductase from M. thermoautotrophicum di er signi cantly in their subunit composition and cofactor content. However, a sequence comparison of the enzymes indicates that they have homologous subunits. Subunit HdrD of the M. barkeri enzyme is a homologue of a fusion protein consisting of the M. thermoautotrophicum HdrC and HdrB subunits [115]. The N-terminal part of HdrD, which contains two binding motifs for [4Fe-4S] clusters, is similar to HdrC, while the C-terminal part of HdrD is similar to HdrB. The subunit TfrB of thiol:fumarate reductase is highly similar to HdrD and HdrCB (Fig. 8) [158]. The b-type cytochrome HdrE of M. barkeri is not present in M. thermoautotrophicum. Instead, the M. thermoautotrophicum enzyme contains the FAD-containing subunit HdrA. Since HdrD, HdrCB, and TfrB are conserved between both heterodisul de reductases and thiol:fumarate reductase, it is assumed that these polypeptides harbor the catalytic site for the reduction of the disul de substrate. The heme-containing subunit HdrE of the M. barkeri heterodisul de reductase is clearly involved in electron transfer. The function of the subunit HdrA of the M. thermoautotrophicum enzyme is not known. Until recently it was thought to harbor the catalytic site for the reduction of the disul de substrate [144], but when the sequence of the M. barkeri heterodisul de reductase became available, it was obvious that subunit HdrA or a related protein is not part of this enzyme. Therefore, it is assumed that HdrA has a speci c function in electron transfer in M. thermoautotrophicum heterodisul de reductase and does not harbor the catalytic site for the reduction of the disul de substrate. The proposed catalytic subunits HdrD and HdrCB do not show any sequence similarity to other characterized disul de reductases. The M. barkeri enzyme and the proposed catalytic subunits HdrCB Fig. 8. Schematic alignment of heterodisul de reductase from M. barkeri (Mb Hdr), heterodisul de reductase from M. thermoautotrophicum (Mt Hdr), and thiol:fumarate reductase from M. thermoautotrophicum (Mt Tfr). The subunits HdrD, HdrCB, and TfrB, which show a high degree of sequence similarity, are shown in blue. In addition to the 8 cysteine residues that ligate the two [4Fe-4S] clusters, these subunits contain 10 conserved cysteine residues (10 C) which might ligate an additional Fe/S cluster and might form a redox-active disul de. The subunits HdrE, HdrA, and TfrA have no sequence similarity and have di erent functions in the di erent enzymes.