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Keywords:

  • Sulfur respiration;
  • Disulfide respiration;
  • Polysulfide reductase;
  • Heterodisulfide reductase;
  • Wolinella succinogenes;
  • Pyrodictium abyssi;
  • Methanogenic archaea

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

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 H2 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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

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, sulfite, thiosulfate, organic sulfoxides, elemental sulfur, polysulfide, and organic disulfides) may play important roles [1, 2]. This article will focus on anaerobic respiration with elemental sulfur, with polysulfide, and with organic disulfides. In Section 2the biology of some relevant organisms will be briefly discussed, while Sections 3 and 4will deal with the chemistry of elemental sulfur, polysulfide and organic disulfides. In Sections 5 and 6, a bacterial (Wolinella succinogenes) and an archael system (Pyrodictium) of sulfur respiration will be described in detail. Section 7covers the disulfide respiration involved in catabolism by methanogenic archaea. Sulfur respiration has been reviewed previously in [3–5]and methanogenesis in [6–11].

2Biology of sulfur and disulfide respiration

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

2.1Biology of sulfur respiration

The ability to reduce sulfur using H2 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 fields and hot springs, and shallow and abyssal submarine hydrothermal systems harbor hyperthermophilic archaea and bacteria, which grow optimally above 80°C [55]. Recently, hyperthermophiles have also been discovered in oil-bearing, 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 H2S escaping from the depths. In their hot biotopes, hyperthermophiles form complex ecosystems consisting of a variety of primary producers and decomposers of organic 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 fields, and volcanic hot springs.

Table 1.  Archaeal and bacterial genera harboring members able to reduce elemental sulfur to H2S
 Topt (°C)pHoptElectron donorsReference
Archaea
Crenarchaeota:
Acidianus 70–901.5–2.0H2[12]
Stygiolobus 802.5–3.0H2[13]
Pyrobaculum1026.0H2, peptone, extracts of meat and yeast, bacterial and archaeal cell homogenates[14]
Thermofilum 85–905.0–6.0Peptides[15, 16]
Thermoproteus 85–905.0–6.5H2, peptides, maltose, formate, fumarate, ethanol, malate, methanol, glycogen, starch, amylopectin, formamide[17, 18]
Desulfurococcus 85–906.0–6.4Peptides, starch, pectin, glycogen, yeast extract, casein hydrolysate[19, 20]
Igneococcus 905.5–6.0H2Huber et al., unpublished results
Pyrodictium1055.5–6.0H2[21, 22]
Stetteria 956.0H2[23]
Thermodiscus 885.5H2, yeast extract[17, 24]
Thermosphaera 856.5Yeast extract, peptone[25]
Staphylothermus 926.5Peptone, extracts of meat and yeast[26]
Hyperthermus 95–1077.0Tryptone, peptone[27]
     
Euryarchaeota:
Pyrococcus 96–1006.8–7.0Complex substrates, amino acids, starch, maltose, pyruvate[28, 29]
Thermococcus 75–885.8–9.0Peptides, amino acids, sugars, starch, chitin, pyruvate[30, 31]
Caldococcus 886.4Peptides[32]
Thermoplasma 591.0–2.0Extracts of yeast, meat, and bacteria[33]
Methanopyrus 986.5H2[34]
Methanobacterium 37–657.0H2[34]
Methanothermus 886.5H2[34]
Methanococcus 85–906.0H2, formate[34]
     
Bacteria
Aquifex 856.8H2, sulfur, thiosulfate[35]
Ammonifex 707.5H2[36]
Desulfurobacterium 706.0H2[37]
Desulfuromonas 377.5Acetate, pyruvate, ethanol[38]
Desulfuromusa 356.5–7.0Acetate, propionate[39]
Desulfurella 557.0Acetate[40, 41]
Desulfovibrio 377.2Organic acids, alcohols[42]
Fervidobacterium 65–706.5–7.0Sugars, pyruvate, yeast extract[43, 44]
Geobacter 356.5–7.0Acetate[45]
Pelobacter 376.5–7.0H2, ethanol[46]
Shewanella 306.5–7.0Lactate[47]
Sulfospirillum 376.5–7.5H2, formate[48, 49]
Thermotoga 66–806.5–7.5Sugars, peptone, yeast extract, bacterial and archaeal cell homogenates[50, 51]
Thermosipho 70–756.5–7.5Yeast extract, brain heart infusion, peptone, tryptone[52, 53]
Wolinella 378.5H2, formate[54]

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 H2S from S0 and H2 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 H2S, while methanogenesis is significantly 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.

2.2Biology of disulfide respiration

The ability to use a disulfide 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 disulfides as electron acceptors [3, 60]. The enzymes that catalyze disulfide reduction in D. acetoxidans and P. islandicum have not been investigated. The enzyme responsible for disulfide reduction in D. acetoxidans apparently differs from the enzyme that reacts with polysulfide 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 disulfides [61].

In contrast to these organisms, which use an external disulfide as electron acceptor for respiration, methanogenic archaea generate a disulfide in the final step of methanogenesis. This disulfide is then used as the terminal electron acceptor of the respiratory chain.

3Chemistry of elemental sulfur, polysulfide, and organic disulfides

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

The solubility of elemental sulfur in water at 25°C is very low (5 μg l−1) [62]. The solubility at higher temperatures is not known. Polysulfide is formed by dissolving sulfur flower in an aqueous sulfide solution (Reaction (a)) [63].

  • image((a))

The S8-ring is cleaved by nucleophilic attack of HS. The amount of sulfur that can maximally be dissolved in a sulfide solution at pH 8 and 37°C is nearly equivalent to the sulfide content [63, 64]. Much less polysulfide is formed at pH values below the pK of H2S (Table 2). Tetrasulfide (S2−4) and pentasulfide (S2−5) are the predominant species of polysulfide at pH >6. The pK of proton dissociation of HS4 and HS5 are well below 7. Tetrasulfide and pentasulfide dismutate rapidly according to Reaction (b) [63].

  • image((b))
Table 2.  Proton dissociation constants of compounds involved in polysulfide reduction
ReactionTemperature (°C)pKReference
H2S[RIGHTWARDS ARROW]HS+H+257.0[65]
HS[RIGHTWARDS ARROW]S2−+H+25>17[65]
HS4[RIGHTWARDS ARROW]S2−4+H+206.3[66]
HS5[RIGHTWARDS ARROW]S2−5+H+205.7[66]

As a consequence of the velocity of Reaction (b), it is not known whether S2−4 or S2−5 is the preferred substrate of polysulfide reductase. For the same reason, the product of polysulfide reduction is not known. It is assumed that only one sulfur atom is cleaved from the polysulfide chain during catalysis (Reaction (c)).

  • image
  • image((c))

The redox potential of polysulfide can be estimated from that of elemental sulfur and the equilibrium constant of Reaction (a) assuming that only one species of polysulfide is formed in this reaction. The value given in Table 3 refers to the reduction of S2−4 to HS and is only 15 mV more positive than that of elemental sulfur reduction to HS.

Table 3.  Redox potentials pertinent to sulfur and disulfide respiration
Redox coupleE0′ (mV)Reference
H+/H2−420[67]
HCO3/HCO2−413[67]
S0/HS−275[67]
S2−4/HS−260See text
R-S-S-R/2 R-SH−220[68]
Menaquinone in ethanol −74[69]

Similar to polysulfide reduction, an S-S bond is cleaved in disulfide reduction (reaction (d)).

  • image((d))

The redox potentials of the disulfides 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, disulfides should be the better electron acceptors.

4Polysulfide as a possible intermediate of sulfur respiration

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

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 polysulfide in aqueous solutions of sulfide (Reaction (a)), a product of sulfur respiration, it may be speculated that polysulfide is an intermediate of sulfur respiration in general. To test this hypothesis, Schauder and Müller [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 polysulfide sulfur should be well above 10 μM in the growth medium of sulfur-reducing microorganisms growing in the presence of 1 mM HS+H2S at pH >6. This concentration of polysulfide sulfur (10 μM) is close to the apparent Km measured with polysulfide respiration of W. succinogenes[71](see Table 6). With the assumption that 10 μM polysulfide sulfur is also required for polysulfide respiration to occur in the other bacteria, it follows that polysulfide may be an intermediate of sulfur respiration in most of the known sulfur reducers (see Table 1).

Table 6. Km values for polysulfide in the electron transport from H2 to polysulfide as a function of the amount of Sud protein present [71]
 Sud concentration (μM)Molar ratio (Sud/Psr)Km for polysulfide (μM)
  1. Psr, polysulfide reductase.

Membrane fraction  0 050
Membrane fraction  0.830 7
Cells grown with polysulfide160 1.110
Cells grown with fumarate 15 0.270

The acidophilic archaea grow at temperatures close to 90°C, where polysulfide sulfur concentrations above 10 μM would require a pH >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 polysulfide to allow polysulfide reduction to occur outside of the cytoplasmic membrane. In W. succinogenes, polysulfide reduction occurs in the periplasm, as shown by the orientation of the polysulfide 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 polysulfide reduction by reduced ferredoxin or H2 has been discovered in P. furiosus[73], and therefore it is feasible that polysulfide reduction also occurs in the cytoplasm of the acidophilic archaea. For this to occur, it has to be postulated that elemental sulfur diffuses across the cytoplasmic membrane and forms polysulfide in the cytoplasm according to Reaction (a). Unfortunately, the diffusion 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 25°C (5 μg l−1).

5Sulfur respiration of Wolinella succinogenes

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

The actual electron acceptor of sulfur respiration in W. succinogenes is polysulfide [3, 5, 64, 71]. This anaerobic proteobacterium (?-subgroup) grows by polysulfide respiration with either H2 (Reaction (c)) or formate (Reaction (e)).

  • image
  • image((e))

W. succinogenes has been reported to grow with elemental sulfur as terminal electron acceptor under conditions that were thought not to allow polysulfide formation [74]. In these experiments, the culture medium contained Fe2+ to precipitate as FeS all the sulfide formed by the bacteria, and polysulfide should not be formed from elemental sulfur in the absence of sulfide (Reaction (a)). Recently, a soluble sulfur compound was detected in the Fe2+-containing culture medium at a concentration corresponding to 0.15 mM polysulfide sulfur. The compound was converted to SCN upon the addition of CN 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 sulfide by W. succinogenes.

5.1Bioenergetic data

The ATP gain (ATP/e) of polysulfide 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 polysulfide 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−1 in polysulfide respiration, while that used in fumarate respiration is 127 kJ mol ATP−1. Both values are consistent with the general observation that phosphorylation requires about 100 kJ mol ATP−1 in growing bacteria in most instances [67]. In spite of the large difference between the free energy (or ΔE) available from respiration with polysulfide and from respiration with fumarate, nearly the same electrochemical proton potential across the membrane (Δp) has been measured during the respiration steady state with the two electron acceptors. As a consequence, the H+/e ratio with polysulfide (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 Δp measured with H2 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 H2 (Reaction (c)).

Table 4.  Bioenergetic data of the polysulfide respiration with formate of W. succinogenes
Electron acceptorpHY (g cells/mol formate)ATP/e−ΔE (V)inline image (kJ/mol ATP)Δp (V)H+/e
  1. The data are compared to those of fumarate respiration. The values of pH, Y and ΔE refer to the middle of the exponential growth phase at 37°C. ΔE was calculated from the ΔE0′ given in Table 3 with the given values of pH and equal concentrations of HCO2 and HCO3, polysulfide sulfur and HS, and fumarate and succinate. The numbers in parentheses were estimated as described in the text.

Polysulfide8.43.2 [64](1/6)0.201160.17 [75](1/2)
Fumarate7.97.0 [76]1/3 [77, 78]0.441270.18 [77]1

5.2Electron transport enzymes

The electron transport chain catalyzing polysulfide reduction by H2 or formate consists of polysulfide reductase (Psr) and hydrogenase (Fig. 1) or formate dehydrogenase [80]. The enzymes are integrated in the cytoplasmic membrane with the catalytic subunits exposed to the periplasm [3, 72]. The isolated polysulfide reductase, which consists of the three subunits (PsrA, B, C) predicted from the nucleotide sequence of the polysulfide reductase operon (psrABC) [81], catalyzes polysulfide reduction by BH4 to sulfide, and sulfide oxidation to polysulfide by 2,3-dimethyl-1,4-naphthoquinone (Table 5). The enzyme contains molybdenum and molybdopterin guanine dinucleotide. The amounts of iron and sulfide in the enzyme are consistent with the presence of five 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].

image

Figure 1. Composition of the electron transport chain catalyzing polysulfide respiration with H2 in W. succinogenes. The sulfide 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; MKb, menaquinone bound to polysulfide reductase; PsrA, B, C, polysulfide reductase subunits; HydA, B, C, hydrogenase subunits [84].

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Table 5.  Properties of polysulfide reductase [82, 83]
  1. MGD, molybdopterin guanine dinucleotide; [S], polysulfide; DMN, 2,3-dimethyl-1,4-naphthoquinone; Psr, polysulfide reductase. The turnover numbers and the Km were determined with the enzyme in an anoxic buffer (pH 8.3, 37°C) containing either 50 mM Tris-HCl and 10 mM KBH4 (polysulfide reduction), or 0.2 M triethanolamine (pH 7.9) and 0.2 mM DMN (sulfide oxidation). The menaquinone content given was corrected for the amount of menaquinone associated with the phospholipid present in the preparation (50–200 μmol g protein−1).

Subunits:PsrA (81 kDa)
 PsrB (21 kDa)
 PsrC (34 kDa)
  
Turnover number: 
[S]+BH4[RIGHTWARDS ARROW]HS+BH31700 s−1
Apparent Km50 μM [S]
  
Turnover number: 
HS+DMN+H+[RIGHTWARDS ARROW][S]+DMNH21100 s−1
Apparent Km25 mM HS
  
Contents (mol/mol PsrABC): 
Molybdenum1
MGD1
Iron21
Sulfide22
Menaquinone0.6–1.6
Heme≤0.1
Flavin≤0.1
Other heavy metals≤0.1

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 polysulfide 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 (ΔpsrABC) lacking the psrABC operon does not catalyze polysulfide reduction by H2 or formate when grown with fumarate as terminal electron acceptor, in contrast to the wild-type strain [72]. Surprisingly, the mutant grows with polysulfide. When grown with polysulfide, the mutant forms a membrane-integrated enzyme that replaces polysulfide reductase (Psr). The enzyme formed by the ΔpsrABC mutant grown with polysulfide has not yet been isolated. Its properties appear to differ considerably from those of the polysulfide reductase enzyme. The enzyme does not cross-react with antiserum raised against PsrA. Like polysulfide reductase, the enzyme catalyzes sulfide oxidation by dimethylnaphthoquinone. However, the apparent Km values for sulfide differ drastically. The value measured with polysulfide reductase is 25 mM (Table 5) and that of the enzyme induced in the ΔpsrABC mutant is approximately 1 mM [83]. The enzyme present in the mutant is apparently absent from the wild-type strain, as suggested by this difference in the apparent Km values.

5.3Mechanism of electron transfer from hydrogenase to polysulfide reductase

A mutant of W. succinogenes lacking the hydrogenase structural genes (hydABC) does not grow with H2 and either polysulfide or fumarate [87]. The mutant grown with formate and fumarate does not catalyze the reduction of polysulfide or fumarate by H2, 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 polysulfide 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 diffusion. The mechanism of electron transfer from the dehydrogenases to polysulfide reductase is not known. Electron transfer by menaquinone diffusion appears to be unlikely because the standard redox potential of menaquinone at pH 7 is more than 200 mV more electropositive than that of polysulfide (Table 3).

The experiments illustrated in Fig. 2 suggest that the electron transfer from the dehydrogenases to polysulfide reductase may require diffusion 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 polysulfide (Fig. 2C) and that with fumarate (Fig. 2D) was measured as a function of the amount of phospholipid present in each preparation. The specific 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 polysulfide reductase, fumarate reductase, hydrogenase, and formate dehydrogenase were hardly affected by the dilution of the membrane fraction with phospholipid (data not shown). In contrast, the electron transport activities with polysulfide decreased by 70–80% upon maximal dilution of the membrane fraction with phospholipid, while 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 different effects of membrane dilution can be explained on the basis of the assumption that polysulfide respiration is limited by the diffusion of the electron transport enzymes within the membrane, while fumarate respiration is not. The data of Fig. 2C fit to the Hardt equation, which relates the collision frequency of two protein molecules to their diffusion coefficients (10−8 cm2 s−1) within the membrane and their surface densities [90–92].

image

Figure 2. Electron transport activity with polysulfide (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 μmol/g phospholipid) isolated from the membrane fraction. In A–D, formate (▴) or H2 (•) were applied as electron donor of electron transport. One unit of activity is equivalent to the oxidation of 1 μmol formate or H2 per min at 37°C.

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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 polysulfide or fumarate decreased with the amount of phospholipid fused to the membrane fraction. The effect of membrane dilution on fumarate respiration can be explained on the basis of the relatively high apparent Km of fumarate reductase for menaquinone, which is in the millimolar range [93]. The effect of membrane dilution on polysulfide respiration is more pronounced in the absence of menaquinone (Fig. 2A) than in its presence (Fig. 2C), suggesting that menaquinone is involved also in polysulfide respiration. This view is supported by the finding that the isolated polysulfide reductase contains approximately 1 mol menaquinone per mol enzyme (Table 5). The menaquinone involved in polysulfide reduction is probably bound to polysulfide reductase and dissociates from its binding site upon dilution of the membrane with phospholipid. In contrast, the menaquinone involved in fumarate respiration freely diffuses within the membrane.

The view that the function of menaquinone in the respiration with polysulfide differs 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 K1 instead of menaquinone, fumarate respiration did not decrease upon membrane dilution, and the data were similar to those of Fig. 2D. In contrast, the effect on polysulfide respiration was similar to that upon membrane dilution in the absence of menaquinone (Fig. 2A). Hence, vitamin K1 can replace menaquinone in the pathway of fumarate respiration, but cannot replace menaquinone in the pathway with polysulfide. Vitamin K1 (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 H2[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 polysulfide reduction by H2[94]. This demonstrates that the intact HydC is required for electron transfer from hydrogenase to polysulfide reductase. The result also supports the view that the membrane anchor of polysulfide reductase (PsrC) is involved in the electron transfer from hydrogenase to polysulfide 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 H2 also lack electron transport activity from H2 to polysulfide.

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 polysulfide reductase is the same as that with hydrogenase.

5.4The function of the Sud protein

In the presence of sulfur, fumarate, and nitrate, W. succinogenes grows by polysulfide respiration, while fumarate and nitrate are not reduced [96]. This preference suggests that W. succinogenes is primarily a sulfur (polysulfide) reducer. Its ecological role may be to supply sulfide as a biosynthetic substrate to the methanogens in the rumen of cattle, the habitat of W. succinogenes. The polysulfide concentration in the rumen is estimated to be maximally 6 μM polysulfide sulfur at pH ≤7 and 0.1 mM total sulfide (HS+H2S) [97]. The apparent Km of polysulfide reductase for polysulfide has been measured as 50 μM (Table 5). The same value has been obtained by measuring electron transport from H2 to polysulfide with the membrane fraction of W. succinogenes (Table 6). The apparent Km for polysulfide measured with intact bacteria grown with fumarate is 70 μM. Hence, the Km for polysulfide reduction is about an order of magnitude above the actual polysulfide concentration in the rumen. However, the apparent Km measured with W. succinogenes grown on polysulfide (10 μM) is close to the ruminal polysulfide concentration.

The lower Km for polysulfide measured with cells grown on polysulfide is due to the induction of the soluble periplasmic Sud protein under these conditions [71, 98]. Sud was originally isolated as a sulfide dehydrogenase. The effect of Sud on the Km for polysulfide has been demonstrated using the activity of electron transport from H2 to polysulfide 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 polysulfide concentrations below 0.1 mM. The apparent Km for polysulfide decreases from 50 μM in the absence of Sud to 7 μM in its presence. The stimulating effect of Sud is not observed at higher polysulfide concentrations [71]and is maximal with 0.8 μM Sud dimer added. Higher amounts of Sud do not further increase electron transport activity. Polysulfide-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 polysulfide is more than two orders of magnitude higher than that required for saturation of the electron transport activity of the membrane fraction (0.8 μM). These results suggest that the periplasmic Sud is bound to polysulfide 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 polysulfide sulfur per subunit when incubated in a polysulfide solution [71]. Furthermore, Sud catalyzes sulfur transfer from polysulfide to cyanide according to Reaction (f)

  • image((f))

with a turnover number of about 104 s−1 at 37°C. These results suggest that Sud serves as a polysulfide sulfur transferase from aqueous polysulfide to the active site of polysulfide reductase. Sud appears to raise the affinity of polysulfide reductase for polysulfide. The function of Sud probably is to allow polysulfide respiration to occur at a sufficient speed even at very low polysulfide concentrations.

5.5Mechanism of Δp generation

The mechanism of Δp generation in the polysulfide respiration of W. succinogenes is not known. Two types of mechanism are feasible. Polysulfide reductase may operate as a proton pump during electron transport from H2 or formate to polysulfide. 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 polysulfide reductase in the cytoplasmic membrane during electron transfer is given in Fig. 3. The bound menaquinone (MKb) is assumed to form the hydroquinone anion (MKbH) upon reduction by hydrogenase to account for the H+/e ratio of 1/2 (Table 4). The site of quinone reduction 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 polysulfide reductase. Consistent with the model shown in Fig. 3, the protons required for menaquinone reduction by H2 in the membrane of W. succinogenes have been shown to be taken up from the cytoplasm [99].

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Figure 3. Hypothetical mechanism of Δp generation by electron transport from H2 to polysulfide in W. succinogenes. The electron transfer from hydrogenase (upper part) to polysulfide reductase (lower part) requires collision of the two enzymes within the membrane. Ni, nickel ion; Mo, molybdenum ion linked to molybdopterin guanine dinucleotide; MKb, menaquinone bound to polysulfide reductase.

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6Sulfur respiration in hyperthermophilic archaea

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

6.1Sulfur 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 polysulfide to H2S. An NAD(P)H-dependent sulfide dehydrogenase and a hydrogenase (termed sulfhydrogenase or sulfur:reduced ferredoxin oxidoreductase) couple the oxidation of reduced ferredoxin to the reduction of either protons to H2 or sulfur to H2S [100, 101]. However, the location of these two enzymes in the cytoplasm, plus the finding 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 H2S is proposed to be a mechanism for the disposal of excess reductant generated by fermentation and of toxic H2[28, 51]. As shown for other H2S-producing organisms, sulfur reduction could also lead to the formation of metal sulfides, thus allowing the removal of toxic metals [103].

6.2Sulfur respiration in species of Pyrodictium

In hyperthermophilic sulfur-respiring archaea, the reduction of sulfur to H2S 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 Thermofilum 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 identified as part of a proposed respiratory electron transport chain [105–107]. The hydrogenase has been purified 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 different 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 the electron transfer sequence: hydrogenase[RIGHTWARDS ARROW]quinone[RIGHTWARDS ARROW]cytochrome c. After inactivation of the electron transport activity by UV light, addition of ubiquinones Q-6, Q-10 or purified P. brockii quinone restores activity. Cytochrome c is thought to serve as electron donor to the sulfur reductase, which has not yet been identified [105–107].

The electron transport chain catalyzing sulfur reduction by H2 in P. abyssi isolate TAG11 differs from that of P. brockii with respect to composition and organization of the components [22]. A H2:sulfur oxidoreductase complex, which catalyzes the H2-dependent reduction of sulfur to H2S, has been recently purified 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 purified H2: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 H2:sulfur oxidoreductase or in the membrane fraction of P. abyssi isolate TAG11. Although the respiratory chains of P. brockii and P. abyssi isolate TAG11 differ in electron transport components, the hydrogenases appear to be similar. The H2: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 H2: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 ‘H2-uptake’ hydrogenases, indicating the respiratory role of these enzymes. During purification, the activity of the hydrogenase present in the H2:sulfur oxidoreductase complex, as measured with viologen dyes, increases parallel with H2: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 H2:sulfur oxidoreductase complex, but also a membrane-bound ATPase, likely to function as ATP-synthase, show temperature optima around 100°C [22](R. Dirmeier, unpublished results). Thus far, the electron transport chain of P. brockii and the H2:sulfur oxidoreductase complex from P. abyssi isolate TAG11 are the only described examples of enzymes involved in the membrane-bound sulfur respiration of hyperthermophilic organisms. The stable organization of the different components of the H2:sulfur oxidoreductase complex from P. abyssi isolate TAG11 implies that further investigations will yield a better understanding of sulfur respiration in hyperthermophiles.

7Disulfide respiration in methanogenic archaea

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

Methanogenic archaea derive their metabolic energy from the conversion of a restricted number of substrates to methane. Most methanogens can reduce CO2 to CH4 with H2 as electron donor. A few methanogens can utilize formate, ethanol, or isopropanol as an electron donor for CO2 reduction. Some methanogens can convert methanol, methylamines, and methylmercaptans to CH4 and CO2. Acetate is the only C2-compound utilized by some methanogens as sole energy substrate. It is converted to CH4 and CO2. (For a historical overview on methanogenesis, see [6, 7]; for recent reviews see [8–11].)

In these different 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 (CH3-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 heterodisulfide (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 heterodisulfide 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 heterodisulfide to the thiols H-S-CoM and H-S-CoB, heterodisulfide reductase, is membrane bound and functions as a terminal respiratory reductase. The electron donor for this disulfide 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.

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Figure 4. Structures of coenzyme M (H-S-CoM; 2-mercaptoethanesulfonate), coenzyme B (H-S-CoB; 7-mercaptoheptanoylthreonine phosphate), and the heterodisulfide (CoM-S-S-CoB) of coenzyme M and coenzyme B.

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Figure 5. Scheme of methanogenesis from H2/CO2, methanol, and acetate. As a central intermediate of the various pathways, methyl-coenzyme M (CH3-S-CoM) is formed and is converted to methane and the heterodisulfide 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. H2 and reduced coenzyme F420 (F420H2) are the electron donors for the reduction of CoM-S-S-CoB. The unknown mechanism of electron transfer from the reduced ferredoxin (Fdred) to CoM-S-S-CoB in acetate metabolism is symbolized by a question mark. The role of H2 as an intermediate of this reaction is discussed in the text. CH3-H4MPT, methyl-tetrahydromethanopterin; F420H2, reduced form of coenzyme F420; Fd, ferredoxin; pFd, polyferredoxin.

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Taxonomically, methanogens belong to the archaeal kingdom of euryarchaeota. They are classified in five orders, each of which is as distantly phylogenetically related to the other as the cyanobacteriales to the proteobacteriales. The five orders are Methanobacteriales, Methanococcales, Methanomicrobiales, Methanopyrales, and Methanosarcinales[112]. Of these, only the Methanosarcinales can ferment acetate to CO2 and CH4 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 influence on the composition of the respiratory chains involved in disulfide respiration. Therefore disulfide respiration in Methanosarcina species will be discussed separately from disulfide respiration in organisms belonging to other phylogenetic groups of methanogens.

7.1Disulfide respiration in Methanosarcina species

Since Methanosarcina species can use several growth substrates, these organisms contain different respiratory chains for the reduction of CoM-S-S-CoB. As will be shown below, these respiratory chains are composed of a substrate-specific dehydrogenase, a membrane-bound electron carrier, and heterodisulfide reductase. The following enzymes and electron carriers have been shown to participate in these respiratory chains.

7.1.1Heterodisulfide reductase

Heterodisulfide reductase (Hdr) was purified from the membrane fraction of Methanosarcina barkeri using detergents for solubilization [113–115]. The purified enzyme is composed of two different 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 disulfide reductases, this disulfide reductase does not contain a flavin [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 five transmembrane-spanning helices. Sequence analysis confirmed 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, H2/CO2, or acetate, probes derived from hdrE or hdrD each hybridized to a 2.3-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 heterodisulfide reductase operates in H2/CO2, methanol, and acetate metabolism (A. Künkel and R. Hedderich, unpublished results).

Recently the purification and characterization of heterodisulfide reductase from Methanosarcina thermophila has been reported [116]. The enzyme exhibits properties very similar to those of the M. barkeri enzyme.

7.1.2Hydrogenases

A membrane-bound hydrogenase has been purified from Methanosarcina mazei[117]and Methanosarcina barkeri[118]using detergents for solubilization. The purified enzyme contains Ni, non-heme iron, and acid-labile sulfur. It is composed of two different 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 identified [119]. They are encoded in two separate transcriptional units: vhoGAC (viologen-reactive hydrogenase one) and vhtGAC (viologen-reducing hydrogenase 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 purified 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 purification of the enzymes. The vht operon contains a fourth gene (vhtD), whose gene product is also not present in the purified enzyme. The function of this gene is not known. The finding that the 5′-ends of the vhoG/vhtG genes code for a long signal peptide supports the topology of the hydrogenase shown in Fig. 6.

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Figure 6. Putative scheme of the respiratory chain from H2 to CoM-S-S-CoB and F420H2 to CoM-S-S-CoB in Methanosarcina species. MPox, methanophenazine in the oxidized form; MPred, methanophenazine in the reduced form. For other abbreviations, see Fig. 3.

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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 H2/CO2, methanol, or acetate, while the vhtGAC operon is only expressed during growth on H2/CO2 and methanol but not during growth on acetate. Obviously the two hydrogenases have different 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 H2 to CoM-S-S-CoB, while the VhtGAC hydrogenase could be involved in reactions specific for H2/CO2 and methanol metabolism [8].

7.1.3F420H2 dehydrogenase

The deazaflavin coenzyme F420 (E0′=−360 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 F420 (F420H2) functions as the physiological electron donor for CoM-S-S-CoB reduction in Methanosarcina species. F420H2 dehydrogenase, which catalyzes the oxidation of F420H2, is an integral membrane protein and has been purified from Methanolobus tindarius[122]and Methanosarcina mazei[123]. The enzyme from M. tindarius is composed of five different subunits (45, 40, 22, 18, and 17 kDa) and contains Fe/S centers but no flavin. The enzyme from M. mazei is composed of five 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 purified enzyme catalyzes the reduction of methanophenazine analogues, such as 2-hydroxyphenazine, with a specific activity of 9 U/mg protein and an apparent Km for 2-hydroxyphenazine of 35 μM. 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 F420H2:quinone oxidoreductase has been characterized from the sulfate-reducing archaeon Archaeoglobus fulgidus[126]. The genes encoding this enzyme have been identified 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.

7.1.4Methanophenazine

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 purified F420H2 dehydrogenase [111, 125]. In addition, washed membranes of M. mazei catalyze the reduction of these methanophenazine analogues by H2, suggesting that the methanophenazine functions as electron acceptor of one of the membrane-bound hydrogenases. Furthermore, the membrane-bound heterodisulfide 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.

7.1.5Composition of the different respiratory chains and mechanisms of Δp generation

When Methanosarcina species grow on H2/CO2, the electron donor for the reduction of CoM-S-S-CoB is H2 (Fig. 5). A subcellular system from Methanosarcina mazei consisting of washed inverted vesicles catalyzes the reduction of CoM-S-S-CoB with H2[108, 110].

  • image
  • image((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 CH3OH/H2 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 H2 being oxidized at the extracellular site of the cytoplasmic membrane. A different or additional mechanism for proton translocation must operate. The Δp 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 heterodisulfide reductase (Fig. 6) [111, 124].

During growth on methanol or methylamines, part of the reducing equivalents are transferred to F420 to generate F420H2 (Fig. 5). Washed inverted vesicles of M. mazei catalyze the reduction of CoM-S-S-CoB by F420H2[128].

  • image
  • image((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 F420H2 dehydrogenase, methanophenazine, and heterodisulfide reductase (Fig. 6) [111, 125]. Using washed everted vesicles of M. mazei, it has been shown that both the reduction of 2-hydroxyphenazine with F420H2 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 F420H2 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 Δp 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 enzyme-bound CO and an enzyme-bound methyl group. The methyl group is transferred to coenzyme M via tetrahydromethanopterin (H4MPT). The methyl group of methyl-coenzyme M is subsequently reduced by H-S-CoB to CH4, thereby forming CoM-S-S-CoB. The CO bound to CO dehydrogenase/acetyl-CoA synthase is oxidized to CO2, 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], H2 has been proposed to be an intermediate of this electron transfer reaction. Cell suspensions of M. barkeri catalyze the conversion of external CO to CO2 and H2 when methane formation is inhibited [133, 134]. CO conversion to CO2 and H2 is coupled with the generation of a proton motive force [133, 134]. However, the molecular basis for the generation of H2 is not known. Recently a novel hydrogenase was purified and characterized from acetate-grown cells of M. barkeri, which could catalyze H2 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 different subunits. In Northern blot experiments, the transcript of the ech operon was detected in cells of M. barkeri grown with acetate, methanol, or H2/CO2. The enzyme shares the highest sequence similarity with the CO-induced hydrogenase from Rhodospirillum rubrum[136]and also has significant 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 H2 and CO2. 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].

  • image
  • image((i))

Likewise, in the acetate metabolism of Methanosarcina, bound CO, generated via decarbonylation of acetyl-CoA, might be converted to CO2 and H2, catalyzed by CO dehydrogenase/acetyl-CoA synthase and Ech hydrogenase. If H2 is an intermediate of this electron transport chain, a second membrane-bound hydrogenase (an H2 uptake hydrogenase) must be present in acetate-grown cells that together with heterodisulfide reductase catalyzes CoM-S-S-CoB reduction by H2. This is indeed the case. Acetate-grown cells of Methanosarcina species synthesize the same membrane-bound hydrogenase as H2/CO2-grown cells (VhoGAC in M. mazei) [118, 121]. Thus, the reduction of CoM-S-S-CoB by H2 in acetate metabolism could involve the same electron transport chain as in H2/CO2 metabolism. In summary, an ‘intraspecies’ hydrogen cycling is proposed which includes two different coupling sites for energy conservation: (i) the site for the conversion of bound CO to CO2 and H2 and (ii) the site for the reduction of CoM-S-S-CoB by H2 (Fig. 7).

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Figure 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]. CH3-H4MPT, methyl-tetrahydromethanopterin; MPox, methanophenazine in the oxidized form; MPred, methanophenazine in the reduced form; Hdr, heterodisulfide reductase; Vho, viologen-reactive hydrogenase one.

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Alternatively, Methanosarcina species could contain an electron transport chain that directly channels electrons from CO dehydrogenase/acetyl-CoA synthase via a ferredoxin to heterodisulfide reductase. Such a CO:heterodisulfide oxidoreductase activity has been reconstituted with purified CO dehydrogenase/acetyl-CoA synthase, ferredoxin, washed membranes, and partially purified heterodisulfide reductase [140, 141]. Since this in vitro system still contains the membrane fraction and thus both membrane-bound hydrogenases (VhoGAC and Ech), it cannot be excluded that H2 is an intermediate in this system.

7.2Disulfide respiration in Methanobacteriales, Methanococcales, Methanopyrales, and Methanomicrobiales

Most of the organisms belonging to these phylogenetic groups are restricted to H2/CO2 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 purification, heterodisulfide reductase of this organism was obtained in a tight complex with one of the [Ni-Fe] hydrogenases, the so-called F420-non-reducing hydrogenase [142, 143]. This complex catalyzes the reduction of CoM-S-S-CoB with H2 at significant rates. At alkaline pH, the complex can be dissociated into the two individual enzymes, heterodisulfide reductase and hydrogenase. Heterodisulfide reductase is composed of three different 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 F420-non-reducing hydrogenase is also composed of three different 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 purified from M. thermoautotrophicum as an individual protein [146–148]. It is present in small amounts in the purified H2:heterodisulfide oxidoreductase complex, but a function as electron carrier in this complex has not been clearly shown. After cell lysis, the H2:heterodisulfide 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 different 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 purification. Hence, the major question is how this non-integral membrane protein complex can couple the reduction of CoM-S-S-CoB by H2 with the generation of the proton motive force. At present there is no conclusive answer to this question, and the following findings should be considered.

Coupling of methanogenesis with ADP phosphorylation is not constant. During growth of methanogens on H2/CO2, the growth yield per mol CH4 increases with decreasing H2 concentrations [149, 150]indicating that at low H2 concentrations, energy coupling is tighter than at high H2 concentrations. Hence, at different H2 concentrations, different 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 heterodisulfide reductase [151]. The genome contains, however, two gene clusters that presumably encode two additional hydrogenases, which have not yet been identified 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 significant 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 H2 concentration, one of these hydrogenases interacts with heterodisulfide reductase to couple the reduction of CoM-S-S-CoB by H2 with the formation of a proton motive force. At high H2 concentrations, reduction of CoM-S-S-CoB might not be coupled with energy conservation and might be catalyzed by the soluble H2:heterodisulfide oxidoreductase complex described above. This ‘uncoupling’ might allow a higher flux through the metabolic pathway and could compensate the lower energy yield (see [154]).

There is only limited information about the H2:heterodisulfide oxidoreductase reaction from organisms belonging to the orders Methanococcales, Methanopyrales, and Methanomicrobiales. Heterodisulfide 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 purified enzymes from this organism. Heterodisulfide reductase has been purified from Methanopyros kandleri (R. Hedderich, unpublished results). The enzyme has a subunit composition similar to that of heterodisulfide 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, heterodisulfide 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 finding supports the biochemical data obtained with the H2:heterodisulfide oxidoreductase complex from M. thermoautotrophicum that indicate that heterodisulfide reductase and methylviologen-reducing hydrogenase (Mvh) form a functional complex.

An F420-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 heterodisulfide reductase in vitro.

7.3Other heterodisulfide-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].

  • image((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 different 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 different Fe/S clusters and shows sequence similarity to the subunit HdrD of the M. barkeri heterodisulfide reductase and to the subunits HdrC and HdrB of the M. thermoautotrophicum heterodisulfide 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.

7.4Heterodisulfide reductase – mechanistic considerations

Heterodisulfide reductase from M. barkeri and heterodisulfide reductase from M. thermoautotrophicum differ significantly 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.

image

Figure 8. Schematic alignment of heterodisulfide reductase from M. barkeri (Mb Hdr), heterodisulfide 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 disulfide. The subunits HdrE, HdrA, and TfrA have no sequence similarity and have different functions in the different enzymes.

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Since HdrD, HdrCB, and TfrB are conserved between both heterodisulfide reductases and thiol:fumarate reductase, it is assumed that these polypeptides harbor the catalytic site for the reduction of the disulfide substrate. The heme-containing subunit HdrE of the M. barkeri heterodisulfide 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 disulfide substrate [144], but when the sequence of the M. barkeri heterodisulfide 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 specific function in electron transfer in M. thermoautotrophicum heterodisulfide reductase and does not harbor the catalytic site for the reduction of the disulfide substrate.

The proposed catalytic subunits HdrD and HdrCB do not show any sequence similarity to other characterized disulfide reductases. The M. barkeri enzyme and the proposed catalytic subunits HdrCB of the M. thermoautotrophicum enzyme do not contain a flavin, but only Fe/S centers as prosthetic groups. Hence, a flavin is not the direct electron donor for the reduction of the disulfide. This has important mechanistic consequences. The central problem that needs to be addressed in heterodisulfide reduction is how a one-electron donor, an iron-sulfur cluster, can carry out a concerted reaction involving reductive cleavage of the disulfide substrate. A one-electron-reduced intermediate seems likely to occur in this catalytic cycle. Oxidation of the enzyme with its substrate CoM-S-S-CoB induces an EPR spectrum not common to known iron-sulfur clusters (Gxyz=2.02, 2.00, 1.95; T=<50 K). Redox titrations indicate a midpoint potential of −250 mV for this paramagnetic center (R. Hedderich, S.P.J. Albracht, M.K. Johnson and E. Duin, unpublished results). The nature of this unusual paramagnetic center, which might represent an important intermediate of the catalytic cycle, is currently under investigation. HdrD, HdrCB, and TfrB contain two highly conserved binding motifs for [4Fe-4S] clusters and ten additional conserved cysteine residues [158]. These cysteine residues could ligate an additional iron-sulfur center and could form a redox-active disulfide. The extra iron-sulfur cluster and the redox-active disulfide could form the active site of the enzyme.

There is only one other disulfide reductase known to pose the same mechanistic problem as heterodisulfide reductase, ferredoxin-thioredoxin reductase. This enzyme is found in plants and cyanobacteria, and catalyzes the reduction of thioredoxin with reduced ferredoxin, an iron-sulfur protein, as electron donor. The active site of this enzyme contains a [4Fe-4S] cluster and a redox-active disulfide. Based on spectroscopic data, a thiyl radical stabilized by an iron-sulfur cluster has been postulated as an intermediate of the catalytic cycle for this enzyme [159, 160].

Although ferredoxin:thioredoxin reductase and heterodisulfide reductase do not share any sequence similarity, a similar catalytic mechanism might operate in the enzymes. Their catalytic mechanism clearly differs from that of the enzymes belonging to the family of pyridine nucleotide disulfide oxidoreductases, such as glutathione reductase, NADPH-dependent thioredoxin reductase, and dihydrolipoamide dehydrogenase. In these enzymes, FAD mediates a two-electron/hydride transfer from NAD(P)H to an active site disulfide [161].

7.5Heterodisulfide-reductase-related proteins in non-methanogens

In the DNA and protein databases, there is an emerging group of proteins from non-methanogenic organisms with high sequence similarity to HdrD, HdrCB, and TfrB. This group includes proteins from several bacteria and archaea (Table 7). The genome of A. fulgidus, for example, contains 11 different genes coding for proteins with a high sequence similarity to the proposed catalytic subunit of heterodisulfide reductase [127]. A function has not been assigned to any of these heterodisulfide-reductase-related proteins from non-methanogens. The high sequence similarity to heterodisulfide reductase indicates a role in disulfide reduction or thiol oxidation. It is therefore assumed that these enzymes together with heterodisulfide reductase form a family of disulfide oxidoreductases distinct from the enzymes belonging to the well-characterized family of pyridine nucleotide disulfide oxidoreductases and ferredoxin:thioredoxin reductase.

Table 7.  Gene products with significant sequence similarity to the postulated catalytic subunit HdrD of the M. barkeri heterodisulfide reductase, the postulated catalytic subunits HdrCB of the M. thermoautotrophicum heterodisulfide reductase, and the subunit TfrB of thiol:fumarate reductase
GeneOrganismComment/putative transcriptional unitSequence identity toReference
  1. Note that most of the genes were identified from genome sequencing projects and that the function of these genes and their gene products is unknown.

AF 0506Archaeoglobus fulgidusHdrD (34% from 385 aa)[127]
AF 1773Archaeoglobus fulgidusHdrD (30% from 390 aa)[127]
AF 0755Archaeoglobus fulgidusHomologue of an hdrED fusionHdrD (31% from 380 aa)[127]
AF 1998Archaeoglobus fulgidusHdrD (29% from 305 aa)[127]
AF 0547Archaeoglobus fulgidusPutative operon with a gene encoding a b-type cytochromeHdrD (23% from 431 aa)[127]
AF 0867Archaeoglobus fulgidusHdrD (26% from 220 aa)[127]
AF 0502Archaeoglobus fulgidusPutative operon with genes encoding Fe/S proteins and b-type cytochromesHdrD (32% from 149 aa)[127]
AF 0543Archaeoglobus fulgidusMost similar to hmc6 from D. vulgarisHdrD (20% from 354 aa)[127]
AF 0544Archaeoglobus fulgidusMost similar to hmc6 from D. vulgarisHdrD (20% from 338 aa)[127]
AF 1375Archaeoglobus fulgidushdrABCmvhDGA operonHdrB (30% from 300 aa)[127]
AF 0271Archaeoglobus fulgidusSimilar to C-terminal part of HdrBHdrB (32% from 128 aa)[127]
ywjFBacillus subtilisPutative operon with acdA encoding acyl-CoA dehydrogenaseHdrD (28% from 390 aa)[162]
MTCY 279.05cMycobacterium tuberculosisSimilar to ywjF from B. subtilisHdrD (28% from 288 aa)[163]
hdrDAquifex aeolicusHydrogenase operonHdrD (25% from 409 aa)[164]
hdrBAquifex aeolicushdrABC operonHdrB (30% from 300 aa)[164]
isp2Thiocapsa roseopersicinaHydrogenase operonHdrD (23% from 398 aa)[165]
sdhCSulfolobus acidocaldariusSuccinate dehydrogenase operonHdrB (31% (from 277 aa)[166]
hdrBSynechocystis sp.HdrB (33% from 300 aa)[167]
hmc6Desulfovibrio vulgarishmc operon; encodes a multisubunit membrane complexHdrD (33% from 103 aa)[168]
dsrKChromatium vinosumdsr locus encoding sulfite reductaseHdrD (23% from 437 aa)[169]
glpCEscherichia coliSubunit of anaerobic glycerol-3-phosphate dehydrogenaseHdrD (27% from 251 aa)[170]
glcFEscherichia coliSubunit of glycolate oxidaseHdrD (23% from 370 aa)[171]
ysfDBacillus subtilisSubunit of glycolate oxidaseHdrD (22% from 390 aa)[162]
glcFSynechosystis sp.Subunit of glycolate oxidaseHdrD (22% from 204 aa)[167]

In methanogens, the disulfide used as terminal electron acceptor of the respiratory chain is not an external substrate, but is generated in H2/CO2, methanol, or acetate metabolism (Fig. 5). Likewise, disulfides could be generated in the energy metabolism of other organisms and function as electron acceptor of the respiratory chain. The presence of genes encoding proteins related to heterodisulfide reductase in several non-methanogenic organisms supports this hypothesis. Many of these genes are closely linked to genes encoding integral membrane proteins, such as b-type cytochromes. This further supports the hypothesis of an involvement of these enzymes in respiration.

8Conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

The respiratory systems described differ significantly in the properties of their terminal reductases. Polysulfide reductase, the key enzyme of sulfur respiration in the bacterium W. succinogenes, contains molybdenum bound to a molybdopterin dinucleotide cofactor, and the catalytic subunit is related to enzymes of the family of molybdopterin-dinucleotide-containing oxidoreductases. Heterodisulfide reductase of methanogenic archaea and the H2:sulfur oxidoreductase from the hyperthermophilic archaeon Pyrodictium abyssi isolate TAG11 do not contain such a prosthetic group. Hence, the catalytic mechanism for disulfide or sulfur reduction in these organisms should be different. It is presently not known whether heterodisulfide reductase and sulfur reductase from P. abyssi are related enzymes. The elucidation of the primary structure of sulfur reductase from P. abyssi might provide an answer to this question.

In the respiratory chains described, different electron carriers mediate electron transfer from a dehydrogenase to the terminal reductase. In W. succinogenes a menaquinone tightly bound to polysulfide reductase seems to be the direct acceptor of electrons delivered from hydrogenase or formate dehydrogenase. An unidentified quinone is postulated as electron carrier in P. brockii, and the newly discovered methanophenazine is most likely the physiological electron carrier in methanogens of the order Methanosarcinales. Quinones have not been detected in the membrane fraction of P. abysii. It remains to be shown whether electron transfer in this organism is by direct electron transfer from the hydrogenase to sulfur reductase, which together form a tight complex. Alternatively, an unidentified electron carrier might be involved in this process.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References

This work was supported by grants from the Deutsche Forschungsgemeinschaft to R.H. and K.-O.S. (Schwerpunktprogramm: ‘Neuartige Reaktionen und Katalysemechanismen bei anaeroben Mikroorganismen’) and to A.K. (SFB 472). We thank K. Brune for editing the manuscript.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Biology of sulfur and disulfide respiration
  5. 3Chemistry of elemental sulfur, polysulfide, and organic disulfides
  6. 4Polysulfide as a possible intermediate of sulfur respiration
  7. 5Sulfur respiration of Wolinella succinogenes
  8. 6Sulfur respiration in hyperthermophilic archaea
  9. 7Disulfide respiration in methanogenic archaea
  10. 8Conclusions
  11. Acknowledgements
  12. References
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