Enzyme activities involved in syntrophic acetate oxidation

By comparison of the enzyme systems involved in acetogenesis by the two well–studied acetogens A. woodii and M. thermoacetica with T. phaeum growing syntrophically, it is possible to get a first insight into probable energy-conserving mechanisms. The Wood-Ljungdahl pathway offers no way of energy conservation through substrate level phosphorylation, thus an ion gradient-driven phosphorylation must occur. Though is reasonable to look for membrane-associated enzymes which are likely involved in proton translocation. The data of the proteome analysis have to be treated carefully because it is very likely that not all proteins could be detected. Only 5 - 10 % of the overall proteins could be found in the cell extract. Still we found that the methyl tetrahydrofolate reductase, formate tetrahydrofolate ligase as well as the bifunctional acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) were located at the membrane. Native gels showed that they were

57 found together with a periplasmic [NiFeSe] hydrogenase, nickel-dependent hydrogenase, NAD- and FAD-binding oxidoreductase and a NADH:quinone oxidoreductase in the cytoplasm as well as in the membrane. But none of these proteins or the related enzyme complexes have transmembrane helices, according to several prediction tools. The reason for the missing membrane located proteins might be that due to the weak detergent used. Thus the membrane located enzymes could not be solubilized and hence not be detected via these method.

During acetogenesis with H2/CO2 the methylene tetrahydrofolate reduction was considered the only source for ion translocation, because it is the only exergonic reaction of the Wood-Ljungdahl pathway⁵⁷. It was found out in A. woodii that this reaction is coupled to a sodium translocation catalyzed by a Rnf complex⁴⁷. However during acetate oxidation this reaction is endergonic and the question is how this reaction is powered. It should be mentioned the reaction measured for this enzyme is endergonic at standard condition. However the substrate excess at the beginning should drive the reaction until equilibrium. We observed that the NADH formed during this assay is higher than theoretical based on the free Gibbs energy of this reaction. It is possible that the methylene tetrahydrofolate formed is further oxidized by the methylene tetrahydrofolate dehydrogenase, which means another NAD⁺ is reduced and the product side of the methyl tetrahydrofolate reductase reaction is kept low, which drive this reaction. Since the enzyme is not pure it is difficult to argue what drive this reaction. A similar system to the Rnf complex in A. woodii working in the reverse direction might be another possibility in T. phaeum how this reaction is facilitated. Although an NAD dependency similar to A. woodii could be measured, no Rnf complex could be found in the genome¹⁶⁷. However the NAD/FAD and quinone-related oxidoreductase found in the native gels together with the methylene tetrahydrofolate reductase might indicate a potential electron bifurcation with the CO dehydrogenase or hydrogenases. But none of these gene might be involved in ion translocation due to the absence of transmembrane helices.

The hydrogen partial pressure suggest that no electron bifurcating hydrogenase is present during syntrophic acetate oxidation. The corresponding redox potential is ~ - 320 mV, which is at the same level as the NAD/NADH couple. If an electron bifurcation would occur then electron from NADH and ferredoxin (assuming a redox potential of -420 mV) are used to form hydrogen and a higher redox potential is possible, e.g. – 360 mV which would result in a higher hydrogen partial pressure by at least one order of magnitude (~ 1000 Pa). Such a high partial pressure could be measured during syntrophic ethanol oxidation where an electron bifurcating hydrogenase is

58 expected (Schmidt, A. unpublished). Nonetheless, no activity of a NAD dependent hydrogenase could be found. However recent data with a gentler cell lysis method showed a NADH-dependent hydrogen-evolving hydrogenase activity (Keller, A. unpublished). Thus it is likely that hydrogen is formed with electrons at the redox potential level of NADH.

Similar to hydrogenase, there are genes for several different formate dehydrogenases in the genome, but only one could be detected in the cell-free extract. Additionally parts of the formate hydrogen lyase system could be found during ethanol oxidation, but no activity could be detected for formate dehydrogenases in the native gels. However, enzyme assays proved that such an enzyme is active in both fractions. The activity in the membrane was not shown before⁶⁴. We found out that this activity is present only in freshly prepared cells and the activity is lost after a few hours. The activity in the membrane hint a formate hydrogen lyase system during syntrophic growth. This system directly link the electrons of formate to the formation of hydrogen¹²³ and thus no ferredoxin may be involved during formate oxidation.

Based on these data it is rather difficult to sketch the electron flow of this pathway and locate enzymes involved in energy conservation due to the abundance of different kind of expressed hydrogenases or formate dehydrogenases, as well as NAD related oxidoreductases. More research has to be done to get a clearer view on this topic.

ACKNOWLEDGEMENTS

We thank Antje Wiese for technical assistance and Dr. Andreas Marquardt for mass spectra analysis. We also thank Alexander Schmidt and Dr. Nicolai Müller for scientific support and suggestions.

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Chapter 7 Discussion

Until now, little was known about the energy-conserving mechanism during syntrophic growth on acetate of T. phaeum. This work focused on proving published data and gives an overview over the electron flow and potential energy-conserving enzymes based on proteomic and physiological data. Since certain aspects were already discussed, this section should be considered as an amendment to the previous chapters.

T. phaeum was described as a sulfate-reducing acetogen¹². Genomic data und growth experiments could not confirm a capacity for sulfate reduction. Several important genes were missing. A reason might be the prolonged incubation on acetate and pyruvate. During that time it is likely that those genes were discarded, because they were not needed anymore. This is a common process of bacteria adapting to a new environment¹⁶⁹. The option that T. phaeum was a former sulfate-reducing bacterium might explain the numerous appearances of heterodisulfide reductases (Hdr) genes. Desulfubacterium autotrophicum is a sulfate reducing acetogen able to oxidize acetate with sulfate. In this bacterium hdr genes connect the pool of electrons from the Wood-Ljungdahl pathway to sulfate reduction. The electrons are used to regenerate the disulfide involved in reduction of sulfite to sulfide¹⁷⁰. In methanogens, Hdr is involved in the reduction of methyl-S-CoM to methane. The heterodisulfide CoM-S-S-CoB is formed, which has a redox potential of about -120 mV.

Hydrogen is used for regeneration of the disulfide. In cytochrome-containing methanogens, this exergonic reduction is used to generate a proton motive force. However, such a membrane-associated complex could not be found in other methanogens, which do not have a cytochrome. It is proposed that an electron-bifurcating complex is used to oxidize hydrogen. The electrons are transferred to ferredoxin and the disulfide. The reduced ferredoxin is further used in the first reduction step of CO2 to methane. In cytochrome containing methanogens an Ech hydrogenase is involved in the endergonic reduction of this ferredoxin. The proton motive force which is generated by the Hdr is used do facilitate this reaction¹⁷¹. Disulfides have a rather positive redox potential of around -200 mV¹⁷². Nonetheless, it is difficult to apply these concepts to syntrophic acetate oxidation by T. phaeum. The only reasonable electron donor would be the methyl/methylene couple, which has a similar redox potential. The reduction of the disulfide could proceed in a similar manner as in the methanogen without cytochromes via an electron bifurcation. Proteomic data of T. phaeum suggest that Hdr, FAD and NAD containing oxidoreductase and the bifunctional ACS/CODH form a complex. It is possible that the electrons derived from the oxidation of carbon monoxide are used to

60 facilitate the endergonic oxidation of the thiol with NAD⁺ as electron acceptor. However, enzymatic assays show a NAD⁺ dependent oxidation of methyl-tetrahydrofolate and would disprove this concept. Since this reaction is highly endergonic, it is still questionable whether this reaction occurs in vivo. There might be unidentified mechanisms active which facilitate this reaction in the extract. It is difficult to assign Hdr genes a proper role during syntrophic acetate oxidation, due to the quite positive redox potential of the disulfides compared to the redox potential of the final electron acceptor hydrogen. Especially if NAD⁺ is the electron acceptor for the methyl-tetrahydrofolate oxidation. Hence more energy has to be invested to put the electrons back at the level of hydrogen.

Growth on different substrates in pure and syntrophic culture shows that T. phaeum has difficulties to reach high optical densities and the doubling times are 2-3 times higher than published.

Especially the long lag phase at the beginning of growth prolong the incubation necessary until the culture is outgrown, which was not reported previously. A reason for this growth inhibition might be a poisonous compound produced by fungi or algae due to a contamination of our water distillation machine, which was discovered after the experiments were finished. . After exchanging the machine the culture grew out within 2-3 weeks (A. Keller, personal communication). However doubling time and final OD where similar. Before growth experiments, the distilled water was sterilized by autoclaving, which inactivates all fungi or algae. Nonetheless, the secondary metabolites might remain stable and affect the growth of T. phaeum. Those metabolites could be a sign to induce phage expression and thus to inhibit growth. Another explanation might be the substrate limitation mentioned in chapter 3. Still the low growth yields are a big problem for biochemical experiments, because a lot of cell material is needed for further analysis. The amount might be sufficient for enzyme analysis and proteomics, but for example if a further purification is necessary the amount should be 5 to 10 times higher due to the high protein loss during each purification step. Therefore a different approach is necessary. The recombinant purification of ferredoxin shows that it is possible to purify anaerobic Fe-S containing proteins in E.coli. Although this was done also previously it shows that it is possible to reconstitute the Fe-S cluster even with a modified sequence. This means that the affinity tag does not negatively affect protein folding, while providing a fast purification with high protein yields. Hence it is possible to use this method for other proteins as well. Especially it was shown that other kinds of Fe-S clusters could also be reconstituted the same way. Though all proteins mentioned in this thesis can very likely be purified with this method. Missing Fe-S cluster and potential cofactors can be reconstituted or supplemented to regain activity.

61 The aim of this thesis was to examine possible ways of energy conservation during syntrophic acetate oxidation by comparing the current data with previous predictions of syntrophic acetate oxidation and acetogenesis of other acetogens, like A. woodii. According to the results it is difficult to prove one theory, because there are several possibilities how energy is conserved. Some of them will be discussed. The hydrogen partial pressure of ~ 50 kPa that was measured in cultures of T. phaeum growing syntrophically with acetate suggest that electrons are released at a level equal to NADH (E⁰’

= - 320 mV). This is no problem for the oxidation of methylene-tetrahydrofolate (E⁰’=- - 295 mV¹⁷³), because electrons can be transported directly to NAD⁺. However energy has to be invested for the oxidation of methyl-tetrahydrofolate (E⁰’= −200 mV⁴⁷). Proteomic data of T. phaeum suggest an electron bifurcation complex with the CO dehydrogenase. The low redox potential of CO oxidation (E⁰’= -520 mV²⁴) might be used to facilitate the methyl-tetrahydrofolate oxidation and theoretically the energy is sufficient to reduce NAD⁺. The remaining electrons from the formate oxidation (E⁰’=- 420 mV³⁶) have to be used for energy conservation. The redox potential difference between formate and hydrogen (E≈ -320) might be sufficient to translocate one proton. The formate hydrogen lyase system could be the potential proton translocating enzyme¹⁷⁴, which was found in the genome of T.

phaeum. Inhibition experiments with DCCD indicate that a formate hydrogen lyase system might be expressed. Though enzyme assays showed that methyl-tetrahydrofolate could be oxidized with NAD⁺ without CO addition and proteome analysis could not show a formate hydrogen lyase system. The net ATP gain supports the assumption that only one proton is translocated in this step. If we assume that for the synthesis of one ATP 80 kJ/mole are necessary and 4 protons are used, then the net ATP gain during growth on acetate indicate that only one proton is translocated, because the energy is split between two microorganisms. Since it is not known for T. phaeum how many protons are used for ATP synthesis, those values can differ greatly in vivo. However for the purpose of discussion these assumptions will be used further on.

If we assume that no energy has to be invested into the methyl-tetrahydrofolate oxidation because the reaction is driven by a fast further metabolism keeping the product side low, the electrons from the CO might be used for proton translocation as well. In the genome there is a potential membrane-bound CO dehydrogenase which is similar to that of M. barkeri, which is able to translocate protons¹²⁶. The electrons would be transported via a membrane-bound enzyme system to a hydrogenase and based on the energy excess (∆E = - 200 mV) two protons could be translocated.

Enzyme assays support this concept, but the enzyme could not be found during proteome analysis.

Another proton can be translocated via the formate oxidation mentioned above. The net ATP gain

62 does not support 3 translocated protons. Though we do not know yet how efficient the proton translocations are. Additionally it is possible that formate is not oxidized at all and more likely is used as an electron carrier itself between acetogen and methanogen. Previous experiments detected formate in the medium ¹⁶⁶, while our data did not. The problem is that both experiments rely on an indirect measurement of formate via retention time analysis after HPLC separation. Only the detectors were different. We could detect a peak at the retention time of formate, but the spectra showed an additional peak at 240 nm indicating that this peak does not represent formate. However due to the very low amounts of formate (< 10 µM) it is possible that this peak is artificial and induced by the detector itself (D. Montag, personal communication). Since both methods do not identify formate directly it would be better to use NMR or mass spectrometry after HPLC separation in order to determine the substrate.

These considerations focus on the Wood-Ljungdahl pathway, but recent data suggest a new way of acetate activation. During syntrophic ethanol oxidation ethanol is oxidized to acetaldehyde and then to acetyl-CoA. It was proposed by Alexander Schmidt in our group that a similar pathway maybe used. Thereby acetate might be reduced to acetaldehyde (E⁰’= - 570 mV¹⁷⁵), which is then oxidized to acetyl-CoA (E⁰’ = - 300 mV¹⁷⁵). The electrons for the initial reduction step might come from the CO oxidation, because they are at a similar level and NAD might be a proper electron acceptor for the further oxidation of acetaldehyde. The further oxidation of the methyl group could proceed as discussed above. Preliminary enzyme assays (A. Keller, personal communication) and growth experiments with ethanol as well as genome analysis indicate that such a pathway exists. However, activity of acetate kinase and phosphotransacetylase show that both pathways are possible⁶⁴. The main advantage of this pathway would be that ATP can be gained via substrate level phosphorylation by the formate-tetrahydrofolate ligase, because none has to be invested into acetate activation. The acetate kinase reaction could be used as a valve, if acetaldehyde accumulates in the cell, because it is very toxic. The theoretical Gibbs’ free energy for syntrophic acetate oxidation as well as the net ATP gain measured force that energy has to be reinvested. The reduction of acetate is a likely candidate if it is coupled to the CO dehydrogenase, then protons could be translocated inside the cell as an energy source similar to the ATP synthase and facilitate this reaction. The methyl-tetrahydrofolate oxidation is the other candidate. A process similar to the Rnf complex in A. woodii could be used to invest energy for this reaction.

63 Overall it is very difficult to focus on a potential electron flow scheme. Further analysis is necessary which focus on the purification of the respective enzymes in order to do kinetic studies, co-elution experiments with purified enzyme and cell extract to identify possible enzyme complexes and to identify the native electron acceptor. It is also necessary to do transcriptome analysis to prove which enzyme system is expressed more under certain conditions to show their importance.

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