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Keywords:

  • Methanosarcina barkeri;
  • Ech hydrogenase;
  • Formylmethanofuran dehydrogenase;
  • Methanogenesis

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

A Methanosarcina barkeri mutant lacking Ech hydrogenase does not catalyze CH4 formation from H2/CO2 since, as was shown previously, the energy-driven reduction of CO2 to formylmethanofuran by H2 is blocked. CH4 formation by this mutant could be restored in the presence of CO or pyruvate. Furthermore, CH4 formation from H2/CO2 plus CO by the Δech mutant was not inhibited by the protonophore TCS. These data show that in vivo the reduction of CO2 to formylmethanofuran can be coupled to the oxidation of CO or pyruvate via a common electron carrier and that the reduction of this electron carrier by H2, catalyzed by Ech hydrogenase, is the energy-driven step in formylmethanofuran-synthesis from CO2, H2 and methanofuran.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The reduction of CO2 to formylmethanofuran (CHO–MFR) is the first step of methanogenesis from H2/CO2[1].

  • image(1)

This reaction is highly endergonic with H2 as electron donor (ΔG°′=+16 kJ mol−1). Under the low hydrogen partial pressures prevailing in the natural habitats of methanogens this reaction becomes even more endergonic (ΔG′=+45 kJ mol−1) [2]. As a consequence the reduction of CO2 to CHO–MFR with H2 requires an additional input of energy to proceed. In cell suspension experiments it was shown that this reduction is driven by reversed electron transport [3–5]. A key enzyme involved in the catalysis of reaction (1) is formylmethanofuran dehydrogenase (FMD) [6,7]. The enzyme catalyzes the reversible dehydrogenation of formylmethanofuran to CO2 and methanofuran. In vitro this enzyme can be assayed with viologen dyes as artificial electron donors or acceptors [2]. FMD from Methanosarcina barkeri is a membrane associated molybdenum iron-sulfur protein composed of six subunits. The direct electron donor of FMD and the hydrogenase participating in reaction (1) were until recently unknown. After the identification of Ech hydrogenase in M. barkeri, it was suggested that this enzyme could play an essential role in this endergonic redox-reaction [8,9]. Ech is a multisubunit membrane-bound [NiFe] hydrogenase with its six subunits showing high sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). To address the in vivo function of Ech hydrogenase, an M. barkeri mutant lacking this enzyme was constructed [10]. The characterisation of the Δech mutant showed that the enzyme is essential for the synthesis of formylmethanofuran from CO2, H2 and methanofuran during methanogenesis from H2/CO2. In vitro Ech hydrogenase catalyzed the reversible reduction of a 2 [4Fe–4S] ferredoxin by H2. The finding that the oxidation of formylmethanofuran to CO2 by the M. barkeri membrane fraction was ferredoxin-dependent strongly indicated that this ferredoxin is also a substrate of FMD. Ferredoxin thus was proposed to function as an electron carrier between Ech hydrogenase and FMD in vivo [10]. Since in the Δech mutant the reduction of CO2 to the level of CO during acetyl-CoA synthesis and the reductive carboxylation of acetyl-CoA to pyruvate, both with H2 as electron donor, were also blocked it was suggested that Ech hydrogenase provides the cell with reduced ferredoxin required for these reactions [10].

Since Ech hydrogenase is tightly membrane-bound via two integral membrane subunits it was suggested that the partial reaction catalyzed by this enzyme is driven by reversed electron transport while the ferredoxin-dependent reduction of CO2 to formylmethanofuran is not. However, direct evidence for this hypothesis is still elusive. In this work, we have used an in vivo system to address this question. Attempts were made to couple formylmethanofuran synthesis with the oxidation of CO to CO2 or with the oxidation of pyruvate to acetyl-CoA and CO2 in the Δech mutant of M. barkeri, which is lacking a functional Ech hydrogenase.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Strains, media, and growth conditions

Methanosarcina barkeri Fusaro (DSM 804) and the Δech mutant of M. barkeri[10] were grown in single cell morphology [11] at 37 °C in high salt (HS) broth medium [12]. 125 mM methanol was added to HS media as carbon and energy source.

2.2Cell suspension experiments

All experiments were performed under strict anaerobic conditions. Cells were collected from late exponential growth phase cultures (ΔOD578=1) by centrifugation at 7000 ×g for 20 min, washed twice in 50 mM Mops (pH 7.0), 2 mM DTT, 400 mM NaCl, 54 mM MgCl2 (=MOPS-HS), and then resuspended in the same buffer. The 1-ml assays were performed at 37 °C in sealed 12-ml anaerobic vials with the following gas mixtures at pressures of 2 × 105 Pa: H2/CO2 (80:20, by vol.), H2/CO2/CO (76:19:5, by vol.), and N2/CO2/CO (76:19:5). Assays were performed in MOPS-HS, supplemented with washed cells (2–3 mg protein ml−1), 50 mM pyruvate where indicated and 16 μM 3,3′,4′,5-tetrachlorosalicylanilide (TCS) where indicated. Assay mixtures were held on ice and started by transfer to 37 °C. Gas phase samples were withdrawn at various time points and assayed for CH4 by gas chromatography. For the determination of total cell protein of whole cells, cells were lysed by sonication and protein content was determined by using the method of Bradford [13]. Cell suspension experiments with [14C] bicarbonate were performed as described above. Assay mixtures contained washed cells (2–3 mg protein/ml) and 0.5 μmol [14C] bicarbonate (1 × 106 Bq). The gas phase was H2/CO2/CO (85:10:5, by vol.).

2.3Analysis of gases

Methane concentrations were determined by gas chromatography via a flame ionisation detector (FID). A calibration curve using known amounts of methane (0.25% and 0.5% in N2) was generated and used to calculate the methane formation of cell suspensions incubated under different conditions. Analysis of [14C] labelled gases was performed by a GC system, allowing the simultaneous analysis and quantification of CH4, CO2 and CO by an FID, and the determination of radioactivity corresponding to CH4, CO2 and CO by a radioactivity gas detector (RAGA type IM 2026/2028, Raytest). For calibration of the radioactivity gas detector [14C] bicarbonate standards were treated under the assay conditions. CO2 was released into the gas phase by the addition of 1 ml of 1 M H2SO4 to the 1-ml assay.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1CO or pyruvate can restore methane formation from H2/CO2 in the Δech mutant of M. barkeri

If, as has been proposed previously [10], formylmethanofuran dehydrogenase, acetyl-CoA-synthase/carbon monoxide dehydrogenase (ACS/CODH) and pyruvate:ferredoxin oxidoreductase (POR) in vivo use the same low potential electron carrier, it should be possible to couple formylmethanofuran synthesis with the oxidation of CO to CO2 or with the oxidation of pyruvate to acetyl-CoA and CO2 in the absence of functional Ech hydrogenase. Since the intact cell provides the most physiological environment, cell suspension experiments with the Δech mutant of M. barkeri were performed. With these cells no CH4 formation from H2/CO2 was observed (less than 2 μmol of CH4 was formed after 24 h in an assay containing 2 mg cell protein). When 5% CO was added to the gas phase, CH4 was formed continuously at rates ranging from 20 to 40 nmol CH4 min−1 (mg cell protein)−1 (Fig. 1(a)). These results were obtained with five independent cell suspension preparations. After 24 h total amounts of 35 to 46 μmol of CH4 were formed (Fig. 1(b)). The total amount of CO present in the reaction mixture was about 50 μmol. Control experiments performed with N2/CO2 plus 5% CO resulted in no detectable CH4 formation within 24 h, showing that H2 could not be replaced by CO in all steps of CO2 reduction to CH4. Since hydrogenases are inhibited by CO, the inhibition of CH4 formation from H2/CO2 by wild-type M. barkeri cells in the presence of 5% CO in the gas phase was tested. At this CO concentration a 50% inhibition of methane formation was observed (data not shown). In the presence of 5% CO methanol-grown cells of wild-type M. barkeri catalyzed CH4 formation from H2/CO2 at a rate of 130 nmol CH4 min−1 (mg protein)−1, in the absence of CO a rate of 260 nmol CH4 min−1 (mg protein)−1 was determined.

image

Figure 1. Methane formation from H2/CO2 plus 5% CO or H2/CO2 plus 50 mM pyruvate by cell suspensions of the M. barkeriΔech mutant. The 1-ml assays containing washed cells (2–3 mg protein ml−1) were performed as described in Materials and Methods. (a) H2/CO2/CO (76:19:5, by vol.) (▾); H2/CO2 (80:20, by vol.) (▴); N2/CO2/CO (76:19:5, by vol.) (▪). (b) Total amount of CH4 formed after 24 h. (c) H2/CO2 (80:20, by vol.), 50 mM pyruvate (•); N2/CO2 (80:20,by vol.), 50 mM pyruvate (▴); H2/CO2 (80:20,by vol.) (▪). (d) Total amount of CH4 formed after 24 h.

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To ascertain that in these experiments the CH4 formed was derived from CO2, the experiments described above were performed with [14C] CO2. The specific radioactivity of the CO2 was 7 ± 0.5 × 106 Bq mol−1. The CH4 formed had a specific activity of 7.9 ± 0.5 × 106 Bq mol−1. In these experiments the specific radioactivity of the remaining CO was <0.1 × 106 Bq mol−1 excluding the formation of [14C] CO from [14C] CO2 via an isotope exchange under the experimental conditions at significant amounts. Hence, the CH4 formed was derived from CO2.

Pyruvate also restored the ability of the mutant to form CH4 from H2/CO2 (Fig. 1(c)). Initial CH4 formation rates were 40 nmol CH4 min−1 (mg protein)−1. After 30 min the reaction slowed down and methane formation rates of 20 nmol CH4 min−1 (mg protein)−1 were observed. From 50 μmol of pyruvate present in the reaction mixture, a maximal amount of 36 μmol CH4 was formed after 24 h (Fig. 1(d)). In assays containing N2/CO2 in the presence of 50 mM pyruvate (no H2 added) low CH4 formation was observed (Fig. 1(c) and (d)). This low amount of CH4 probably is derived from the methyl-group of acetyl-CoA. Acetyl-CoA is generated by oxidative decarboxylation of pyruvate.

3.2Methanogenesis from H2/CO2/CO is not dependent on an energized cytoplasmic membrane

Methanogenesis from H2/CO2 requires an energized cytoplasmic membrane to proceed. When the protonophore TCS was added to these cells, CH4 formation was completely blocked (Fig. 2(a)) [14]. Since M. barkeri has a Na+/H+ antiporter, TCS not only abolishes ΔμH+ but also ΔμNa+[4,15]. As shown above, CO or pyruvate could restore CH4 formation from H2/CO2 in the M. barkeriΔech mutant. To test if methanogenesis in the presence of CO or pyruvate was still dependent on an electrochemical ion gradient, the experiments described above were performed in the presence of TCS. Methanogenesis from H2/CO2 plus CO in the Δech mutant was not inhibited by TCS (Fig. 2(b)). In the experiments with H2/CO2 plus pyruvate, TCS had no influence on the rate of CH4 formation within the first 20 min of the reaction. Approximately 2.5 μmol CH4 were formed in an assay containing 2 mg cell protein. After this initial period the reaction stopped and no further CH4 was formed within 24 h (Fig. 2(c)).

image

Figure 2. Influence of TCS on methane formation from H2/CO2 by cell suspensions of the M. barkeri wild-type (a) and by cell suspensions of the M. barkeriΔech mutant (b) and (c). 1-ml assays containing washed cells (2 mg cell protein ml−1) were performed as described in Materials and Methods. Cell suspensions were preincubated with TCS for 30 min on ice. Control assays without TCS were treated under the same conditions. Reactions were started by increasing the temperature from 0 to 37 °C. (a) H2/CO2 (80:20, by vol.) (▪); H2/CO2 (80:20, by vol.), 16 μM TCS (•). (b) H2/CO2/CO (76:19:5, by vol.) (▾); H2/CO2/CO (76:19:5, by vol.), 16 μM TCS (▴); H2/CO2 (80:20, by vol.) (▪); H2/CO2 (80:20, by vol.), 16 μM TCS (•). (c) H2/CO2 (80:20, by vol.), 50 mM pyruvate (•); H2/CO2 (80:20, by vol.), 50 mM pyruvate, 16 μM TCS (▴); H2/CO2 (80:20, by vol.) (▪); H2/CO2 (80:20, by vol.), 16 μM TCS (▾).

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The results presented show that CH4 formation from H2/CO2 in the Δech mutant could be restored by the addition of CO or pyruvate to cell suspensions. These data indicate that CO- or pyruvate-oxidation is coupled to the reduction of an electron carrier, which can either directly or via additional electron carriers donate electrons to FMD (Fig. 3). Methane formation from H2/CO2 is strictly dependent on an energized membrane [4,14]. Reduction of CO2 to the level of formylmethanofuran was shown to be the energy driven step in this pathway [3–5]. When H2 was replaced by CO as electron donor for the first step of methanogenesis, CH4 formation was no longer dependent on an energized membrane, clearly showing that the partial reaction catalyzed by FMD is not energy-driven. Consequently, reduction of the electron carrier by H2 must be the energy-driven reaction when H2 serves as electron donor for the reduction of CO2 to formylmethanofuran. This reaction is catalyzed by Ech hydrogenase.

image

Figure 3. Reactions providing reducing equivalents for FMD in M. barkeri. Based on in vitro studies an M. barkeri ferredoxin (Fd) is thought to function as a central electron carrier. Reduced ferredoxin required for the reduction of CO2 to formylmethanofuran (CHO–MFR) in wild-type cells is provided by Ech using H2 as electron donor. In the Δech mutant, CO or pyruvate can be used as alternative electron donors for ferredoxin reduction. Only ferredoxin reduction by H2 is driven by reversed electron transport. 1, Ech hydrogenase; 2, acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH); 3, pyruvate:ferredoxin oxidoreductase (POR); 4, formylmethanofuran dehydrogenase (FMD).

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Pyruvate driven methane formation from H2/CO2 stopped after a short initial period when the protonophore TCS was added. A possible explanation could be that pyruvate uptake into the cell is energy-driven, whereas CO is freely diffusible across the membrane.

In vitro studies have shown that an M. barkeri ferredoxin is an efficient electron acceptor/donor of Ech [9] and ACS/CODH [16]. POR of M. barkeri, when assayed in the oxidative direction, was shown to catalyze the reduction of a clostridial ferredoxin [17]. Furthermore, membranes of M. barkeri were found to catalyze H2 production from formylmethanofuran in a strict ferredoxin dependent reaction, indicating that formylmethanofuran dehydrogenase is also electrically connected to this ferredoxin [10]. Based on these in vitro studies it is assumed that the ferredoxin is a central electron carrier in M. barkeri (Fig. 3). It can, however, not be excluded that in vivo additional or alternative electron carriers are operating.

The finding that CO or pyruvate can restore the ability of the Δech mutant to produce methane from H2/CO2 implies that the mutant should be able to grow on H2/CO2 medium supplemented with CO or pyruvate. This was not the case. Neither pyruvate [10] nor CO supplemented media (5% in the gas phase) (Stojanowic and Hedderich, unpublished data) supported growth of the Δech mutant. It has to be considered that both ACS/CODH (responsible for the oxidation of CO to CO2) and POR are only present in anabolic amounts in methanol grown M. barkeri. This could be the reason for the methane formation rates of the Δech mutant from H2/CO2/CO or H2/CO2/pyruvate, which are 6- to 12-fold lower as compared to the methane formation rates from H2/CO2 of wild-type M. barkeri cells grown on methanol. These rates might be too low to support growth of the organism. It also has to be considered that the breakdown of pyruvate in growing cells is very limited and that pyruvate is rapidly incorporated into amino acids as was recently shown with Methanococcus maripaludis[18].

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was supported by the Max-Planck-Gesellschaft, by the Deutsche Forschungsgemeinschaft, and by the Fonds der Chemischen Industrie.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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