Acetate is quantitatively the most important intermediate in the anaerobic degradation of soluble organic matter. The conversion rate of acetate by methanogenic bacteria is proposed to be the rate limiting step in this degradation The study of acetoclastic methanogens, therefore is of relevance to our understanding of anaerobic processes and their optimal application in treatment of waste water from various sources.
Until now only two genera of methane bacteria have been described which are able to use acetate as sole energy source: Methanosarcina and Methanothrix. Because Methanothrix has a long generation time and low growth yield, most of the research on acetoclastic methanogenesis was done with Methanosarcina until now. The aim of this work was to extend the knowledge of the acetate metabolism in Methanothrix and to compare the results with the knowledge about Methanosarcina.
The study of the acetate metabolism in Methanothrix soehngenii was concentrated around three major subjects:
- Acetate activation to acetyl-coenzyme A by acetyl-coenzyme A synthetase and the energetic consequence of this activation mechanism (Chapters 2-5).
- Cleavage of acetyl-coenzyme A in to a methyl-, carbonyl- and coenzyme A moiety by the enzyme carbon monoxide dehydrogenase and concomitant oxidation of the CO group to CO 2 by the same enzyme (Chapters 6-8).
- Reduction of the methyl group to methane (Chapter 9).
Chapters 2 to 5 deal with several aspects of the acetate degradation and acetate activation in Methanothrix. In chapter 2 the threshold concentrations of acetate utilization and the enzymes responsible for acetate activation of several methanogenic bacteria, including Methanothrix and Methanosarcina, are presented and compared with literature data. The minimum acetate concentrations reached by the acetoclastic Methanosarcina are between 0.2 and 1.2 mM and by Methanothrix between 7 and 70 μM, whereas the hydrogenotrophic methane bacteria, which can use acetate as an additional carbon source achieve acetate concentrations between 0.4 and 0.6 mM. Methanosarcina uses an acetate kinase 1 phosphotransacetylase system to activate acetate with high V max but low affinity. Methanothrix and most hydrogenotrophic methane bacteria have an acetyl-CoA synthetase to activate acetate with relatively high affinity for acetate. The difference in affinity for acetate of Methanosarcina and Methanothrix are consistent with the general model by which Methanosarcina dominates in environments with high acetate concentrations while low acetate concentrations favour Methanothrix. Although the affinity for acetate of the hydrogenotrophic methane bacteria was high, these methanogens were not able to remove acetate to lower concentrations than the acetoclastic methane bacteria. Therefore it is not likely that these hydrogenotrophic methanogens compete strongly for acetate with the acetoclastic methanogens.
In chapter 3 the purification procedure and properties of the acetate activating enzyme of Methanothrix, acetyl-coenzyme A synthetase (ACS), are presented and compared to the acetate activating system of Methanosarcina. ACS activates acetate to acetyl-coenzyme A. ACS is a homodimeric (α 2 ) enzyme with molecular mass of 148 kDa and constitutes up to 4 % of the soluble cell protein. Comparison of the kinetic properties of the ACS from Methanothrix (V max = 55 U/mg, app K m for acetate = 0.86 mM) with the properties of the acetate activating system of Methanosarcina (V max = 660 U/mg, app K m for acetate = 22 mM) confirmed again the hypothesis that Methanothrix dominates in environments with low acetate concentrations while high acetate concentrations favour Methanosarcina.
With varying amounts of ATP weak sigmoidal kinetics were observed for the ACS system of Methanothrix. The Hill-plot gave a slope of 1.58 ± 0.12, suggesting two interacting substrates sites for the ATP. The possible presence of different ATP binding sites was later confirmed by analysis of the deduced amino acid sequence of the ACSa gene.
The energy for the activation of acetate by ACS is provided by the hydrolysis of ATP to AMP and PP i . In Methanothrix the conversion of AMP to ADP and the hydrolysis of PP i to 2 P i are catalyzed by an adenylate kinase and inorganic pyrophosphatase, respectively.
A sum of these reactions leads to the suggestion that for acetate activation in Methanothrix two ATP are needed. Since it is believed that methane formation from acetate can only yield one ATP, it is difficult to envisage how Methanothrix is able to grow at all. One possible site of energy conservation may be coupled to the hydrolysis of pyrophosphate. To investigate the possibility that the energy of the PP i bound could be used to drive endergonic reactions, the pyrophosphatase was isolated from Methanothrix. The properties of the purified inorganic pyrophosphatase are described in Chapter 4. The enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an α2β2 oligomeric structure, giving a molecular mass of 139 ± 7 kDa for the native enzyme. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri- and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg 2+was required for activity. The enzyme was heatstable, insensitive to molecular oxygen, not inhibited by fluoride and constituted upto 0.2 % of the soluble protein. When cells were rigorously disrupted in a Frech Pressure cell, the inorganic pyrophosphatase was found in the soluble cell fraction. However, when gentle lysis procedures were applied up to 5 % of the inorganic pyrophosphatase was associated with the membrane fraction. This membrane association could indicate that hydrolysis of of the pyrophosphate is not solely used to displace the equilibrium of acetate activation. In analogy with the proton translocating inorganic pyrophosphatase of plant vacuoles and phototrophic bacteria, one could envisage a similar proton translocation function for the enzyme in Methanothrix.
Since the energetic aspects formed an intriuging facet of the acetate metabolism in Methanothrix, the interconversion of adenine nucleotides during acetate fermentation in concentrated cell suspensions of Methanothrix soehngenii were investigated and described in chapter 5. Starved cells of Methanothrix contained high levels of AMP (2.2 nmol/mg protein), but had hardly any ADP or ATP. The Energy Charge (EC) of these cells was 0.1. Immediately after the addition of the substrate acetate, the level of ATP started to increase, reaching a maximum of 1.4 nmol/mg protein, corresponding to an EC of 0.7 when half of the acetate had been consumed. Once the acetate was depleted, the ATP concentration decreased to its original level of 0.1 nmol/mg protein, (EC = 0.1). These results showed that although the free energy gain on acetate is very low, Methanothrix is able to conserve some of this free energy in net AT? synthesis.
In chapters 6 to 8 several aspects of the central enzyme in the acetate metabolism of Methanothrix soehngenii, the carbon monoxide dehydrogenase (CDH) are presented.
Chapter 6 decribes the aerobic purification procedure together with several kinetic properties of the CDH. In contrast with the CDH's from most other anaerobic bacteria, the CO oxidizing activity of the purified enzyme of Methanothrix soehngenii was
remarkably stable towards oxygen and it was only slightly inhibited by cyanide. The enzyme constitutes 4 % of the soluble cell protein and showed a high degree of thermostability. Analysis of enzyme kinetic properties revealed a K m of 0.7 mM for CO and of 65 μM for methylviologen. At the optimum pH of 9.0 the V max was 140 μmol of CO oxidized per min per ing protein. Acetyl-coenzyme A / CO exchange activity, 35 nmol.min -1.mg -1of protein, could be detected in anaerobic enzyme preparations, but was absent in aerobic preparations. The enzyme has a tetrameric (ααβ) 2 subunit structure. The Mr of the a subunit is about 89 kDa and of the βsubunit about 20 kDa. The enzyme contained about 12 Fe, 12 acid-labile sulfur and 1 Ni per aB-dimer, which are present in clusters. These iron-sulfur clusters can be studied by Electron Paramagnetic Resonance (EPR) spectroscopy.
In chapter 7 the paramagnetic iron-sulfur centers of purified CDH are described. In EPR spectra of the anaerobically isolated enzyme two major signals were apparent. One with g-values of 2.05, 1.93 and 1.865, and an E m7.5 of -410 mV, which quantified to 0.9 S=1/2 spins per ααβ-dimer. This signal resembles EPR spectra of two dipolarly interacting, ferredoxin-like [4Fe-4S] clusters. Analysis of the deduced amino acid sequence of the CDHa gene confirmed that there is a strech of 64 amino acids, which could be identified as a ferredoxin domain of the archaebacterial type. Taken together the low redox potential and the ferredoxin like sructure of this cluster, it is likely that this center function as electron acceptor in the oxidation of the CO group. The other signal with g-values of 1.997, 1,886 and 1.725, and an E m7.5 of -230 mV, gave 0.1 spin per ααβ-dimer. Until now no structure could be assigned to this unusual signal, although it resembles in some aspects the putative [6Fe-6S] prismane clusters. When the enzyme was incubated with its physiological substrate acetyl-CoA, these two major signals disappeared. Incubation of the enzyme under CO atmosphere resulted in a partial disappearance of the spectral component with g = 1.997, 1.886, 1.725.
In chapter 8 a novel high-spin EPR signal in the oxidized CDH with effective g-values at g = 14.5, 9.6, 5.5, 4.6, 4.2, 3.8 is described. The lines at g = 14.5 and 5.5 were tentatively ascribed to a S = 9/2 system with about 0.3 spins per ααβ-dimer. The other signals were assigned to a S = 5/2 system with 0.1 spins per ααβ-dimer. Both sets of signals disappear upon reduction with E m7.5 = -280 mV. With a very similar reduction potential, E m7.5 = -261 mV, an S = 1/2 signal (0.1 spins per ααβ) appears with the unusual g-tensor 2.005, 1.894, 1.733. Whether these signals belong to the same paramagnetic center exhibiting different spin states is not yet clear. CO dehydrogenase, 69 % enriched in 61Ni, showed the same EPR signals as enzyme preparations isolated from cells grown in media with native Ni. In none of these signals any splitting or broadening from 61Ni could be detected. Also, the characteristic "g = 2.08" EPR signal found in some other CO dehydrogenases, and ascribed to a Ni-Fe-C complex, was never observed under any condition of detection (4 to 100 K) and incubation (ferricyanide, dithionite, CO, coenzyme A, acetyl-CoA).
(Chapter 9 describes the purification and some properties of the methyl-CoM reductase from Methanothrix soehngenii. The enzyme catalyzes the final step in the conversion of acetate: the reduction of methyl-coenzyme Mr to methane. The enzyme had a native molecular mass of 280 kDa in a (ααβγ) 2 subunit structure. The methyl-coenzyme M reductase constituted upto 10 % of the soluble cell protein. The enzyme has K m apparent values of 23 μM and 2 mM for N-7-mercaptoheptanoyl threonine phosphate (HS- HTP) and methyl-coenzyme M, respectively. At the optimum pH of 7.0, 60 nmol of methane were formed per min per ing protein. These properties are comparable to those of methyl-CoM reductase of other methanogenic bacteria, although the specific activity is relatively low.
This thesis clearly showed that the acetate metabolism of the specialist Methanothrix soehngenii has peculiar features. The high energy input in the activation of acetate by an acetyl-CoA synthetase results in a high affinity and a low threshold value, which makes Methanothrix the dominant acetoclastic methanogen in anaerobic ecosystems with low acetate concentrations. The other consequences of the energy input are the low growth yield and long generation time, which causes an outcompetition by Methanosarcina in systems with high acetate concentrations. Although the energy input is quite clear, the energy conservation is not yet well understood. The reduction of the heterodisulfide between coenzyme Mr and mercaptoheptanoyl threonine phosphate is proposed to be the common site for energy conservation in all methanogens. The high activity of heterodisulfide reductase in cell extracts of Methanothrix indicates that this site is also operative in M. soehngenii. Methanothrix, however, needs additional sites of energy conservation to compensate for its high input. One possible site could be the oxidation of CO to CO 2 another site could be formed by the partially membrane associated pyrophosphatase.
The central enzyme in the acetate metabolism of Methanothrix is Carbon monoxide dehydrogenase. This enzyme showed several surprising and novel characteristics: The CO-oxidizing activity appeared to be insensitive towards oxygen, the anaerobically purified enzyme was able, although at low activity, to exchange the carbonyl group of acetyl-CoA with CO and EPR spectroscopy indicated the presence of an unusual iron-sulfur center.
De oxygen-insensitivity is recently observed for the CDH of some sulfate-reducing bacteria which also use a reversed acetyl-CoA pathway for the degradation of acetate. The reason for this insensitivity is not yet clear. The exchange reaction between CO and the carbonylgroup of acetyl-CoA was recently described for the enzyme isolated from Methanosarcina thermophila. This enzyme had also low activity, which indicates that either the right assay conditions are not yet found or that this activity is extremely instable.
Concerning the iron-sulfur centers there are little similarities between the enzymes of the different anaerobic bacteria. One reason why the research to the clusters of CDH of anaerobic bacteria is hampered at the moment is the low spin recovery of the different centers. This makes statements about structure and function difficult. There are good indications that at least one and possibly two ferredoxin-like [4Fe-4S] clusters are present, which play a role in the electron transfer of the CO oxidation. For the presence of a NiFe-C complex in the CDH of Clostridium thermoaceticum and Methanosarcina thermophila, there also good spectroscopic indications. This complex has until now not been observed in the CDH of Methanothrix. The CDH of Methanothrix and also of Clostridium thermoaceticum show in EPR spectroscopy another unusual signal. This signal could be assigned to a putative [6Fe-6S] prismane cluster, which could function in multi- electron transfer or in substrate binding. More clear indications are necessary to identify the structure and function of this cluster.