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FEMS Microbiology Reviews 87 (1990) 391-396 Published by Elsevier 391 FEMSRE 00191 CO 2 :eduction to acetate in anaerobic bacteria Gabriele Diekert institut flir Mikrobiologie, Universitiit Stuttgart, Stuttgart, F.R.G. Key words: Homoacetogenic bacteria; Acetate formation from CO2; Sodium dependent acetate formation; Methylene-tetraltydrofolate reduction; Energy conservation in homoaeetogens 1. S U M M A R Y The re,ruction of 2 CO 2 to acetate is catalyzed in the energy metabolism of homoacetogenic bacteria, which couple acetate formation to the synthesis of ATP. The carboxyl group of acetate is formed from CO 2 via reduction to a bound carbonyl {[CO]), a reaction that requires the input of metabolic energy when hydrogen is used as the electron donor. The methyl group of acetate is formed via formate and tetrahydrofolate b o u n d C t intermediates including methyl tetrahydrofolate as the intermediates. The methyl group is then 'condensed' with the carbonyl and CoA to acetyl-CoA, which is converted to acetate in the energy metabolism or to cell carbon in the anabolism of the bacteria. The mechanism of ATP synthesis coupled to CO x reduction to acetate is still unclear. The only reaction sufficiently exergonic is the reduction of methylene tetrahydrofolate to methyl tetrahydrofolate. Indirect evidence was presented that this reaction in homoacetogens might be coupled to the electrogenic transport of sodium across the cytoplasmic membrane. The sodium gradient formed via metbylene-THF reduction could be transformed into a proton gradient via a s o d i u m / p r o t o n antiporter. Correspondence to: O. Diekcrt, Institut FOrMikrobiologie,Universifiit Stuttgart, Azenbergstrasse 18, D-7000 Stuttgart 1, F.R.G. ATP would then be synthesized by a proton translocating ATP synthase. 2. I N T R O D U C T I O N Several groups of strictly anaerobic bacteria catalyze the reduction of 2 CO 2 to an acetyl moiety in their metabolism. A m o n g them are the homoacetogenic bacteria, which mediate the CO 2 reduction as a multifunctional pathway operative b o t h in energy metabolism and autotrophic cell carbon synthesis [1-5]. In the catabolism of homoacetogens this pathway can serve as an electron sink for reducing equivalents released in the oxidative degradation of different energy substrates such as sugars (Fig. 1A) or, for instance upon growth of the bacteria on H 2 plus C O 2 or carbon monoxide as the energy sources, for energy conservation coupled to acetate formation from these substrates (Fig. 1B, C). In anabolism, the acetyl CoA formation from C O 2 sepses as a carbon assimilation pathway, which is not only used by homoa¢etogens, b u t also by o t h e r strictly anaerobic autotrophic microorganisms such as sulfidogenic and methanogenic bacteria. For a recent detailed review on this autotrophic CO 2 fixation pathway the reader is referred to [1]. The acetate formation from CO 2 can be reversed in different obligate anaerobes. Some bomoacetogens are able to catalyze acetate oxidation to COg and use the reduction of protons to hyaro- 0168-6445/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties 392 (~ " T "--~ Acetate + CO2 ',-----i----, Py. . . . te--~ ---~Acetate 41HI 2 IHI ICOI? [ CO2 (~) co2 Hexoses" - ~ L formate formyI-THF , CH,-X ~ - ~ . ~ methenyI-THF ~21Hi me th ylene- THF 21HP 3 Hz-~ 61HI CO2 ~ L . CH3-Xx C02~ CH~-THF ICOI / CO..... CH/-× H2 ,-~2 IH) .)... 3 CO2 3CO-~61H) acetate cell carbon Fig. 3. Scheme of acetate and cell carbon synthesis in homoacetogcns. The "framed' reaction is catalyzed by melhytcne tetrahydrofolate reductase. CO / Fig. 1. Simplified scheme of acetate formation from (A) hexoses, (B) H 2 plus CO2, and (C) from CO in homoacctogenic bacteria. gen as the electron sink [6-8] (Fig. 2). Several sulfidogens also degrade acetate oxidatively and use sulfate as the electron acceptor (for a recent review see [9]). Some of the sulfidogens oxidize acetate via the citric acid cycle; others involve the acetyl-CoA pathway (carbon monoxide dehydrogenase pathway) [10,11], which can be considered Acetate oxidation to CO2 (Sullidogenic and acelogenic bacleria) 61HI Acetate / - . CH3-X --, ._~~ AcetyI-CoA - ~ ICOI CO2 COz 2 IHI SO~ + 81HI ~ H2S to be an inversed acetate formation from CO 2 as mediated by homoacetogenic bacteria (Fig. 2). The reduction of CO2 to acetate in homoacetogens involves the formation of a methyl group (a methyl +) and of a carboxyl group, i.e. a CO2 plus 2 reducing equivalents. The methyl group is synthesized via tetrabydrofolate (THF)-bound Cl-intermediates, the carboxyl group via a bound carbon monoxide ([CO]), which is formed from CO2 by the key enzyme in this pathway, namely the carbon monoxide dehydrogenase [12,13]. The methyl and the carboxyl group are then combined together with coenzyme A in a reaction, the mechanism Of which is not yet understood. This latter reaction is probably also catalyzed by the carbon monoxide dehydrogenase [14]. A scheme of acetate and cell carbon formation from CO2 in homoacetogens is given in Fig. 3. (Sulfldogenlc bacter,a) 8 IHI ~ 4 H2 (Acetogen,c bacter|a) Fig. 2. Anaerobic acetate oxidation by homoacetogenic and some sulfidogenic bacteria. 3. ENEROETICAL ASPECTS OF CO2 REDUCTION TO ACETATE IN ACETOGENS Most of the homoacetogenic bacteria are able to grow on H e plus CO2 as the sole energy sources. This implies that CO2 reduction to acetate must be coupled to the synthesis of ATP. One ATP is 393 required for the activation of formate to formyl tetrahydrofolate (formyloTHF). ATP can be formed by a substrate level phosphorylation mechanism in the acetate kinase reaction, which is involved in acetate formation from acetyl-CoA. It must be considered, however, that part of the acetyl-CoA is channelled into cell carbon synthesis rather than into acetate formation. Moreover, the reduction of CO 2 to the bound carbonyl with H 2 as the electron donor according to gous reaction in their energy metabolism, namely the reduction of methylene tetrahydromethanopterin (methylene-THMP) to methyl-THMP: methylene-THMP + 2[HI --, methyl-THMP Evidence was presented that this reaction in methanogens is coupled to the electrogenic transport of sodium across the membrane [19]. Therefore, the role of sodium in the COx reduction to acetate was tested in acetogenic bacteria. CO 2 + H 2 ~ [COl + H20 AG o" = + 2 0 kJ/mol is an endergonic reaction, which is energy driven in homoacetogens [15] and methanogens [16,17]. For these reasons it is still unclear how homoacetogenic bacteria synthesize ATP. The only reaction in acetyl-CoA formation from CO 2 that is sufficiently exergonic to be coupled to energy conservation via a chemiosmotic mechanism is the reduction of methylene tetrahydrofolate to methyl-THF (see 'framed' reaction in Fig. 3): methylene-THF + 2[H] --* methyl-THF With H 2 (or ferredoxin) as the electron donor the AG O' value of this reaction is - 57.3 kJ/mol, with N A D H - 3 9 . 4 kJ/tool. The enzyme has been purified from Clostridium formicoaceticum by Clark and Ljungdahl [18]. It mediates the reduction of methylene-THF with reduced ferredoxin as the electron donor. In the carbon monoxide-utilizing homoacetogen Peptostreptococcus productus the reducing equivalents in this reaction are provided by N A D H (Wohlfarth et al., in press). The enzyme of the latter organism differs considerably from the clostridial enzyme also in other respects. Whereas the clostridial enzyme is an octamer (cf4•4) with a molecular weight of Mr = - 237000 and is oxygen sensitive [18], the P. productus enzyme is not sensitive against oxygen and is an octamer (as) with an M r of ~ 250000 (Wohlfarth et al., in press). From the energetical considerations described above the methylene reduction to the methyl group is most likely the reaction, which is coupled to net ATP formation in homoacetogens growing on H2 plus CO 2. The mechanism of coupling is still unclear. Methanogenic bacteria catalyze an anal(>- 4. THE ROLE OF SODIUM IN ACETATE FORMATION FROM CO2 The influence of sodium on acetate formation from CO 2 was investigated with cell suspensions of P. productus (strain Marburg). It was found that the formation of acetate from H 2 plus CO 2, from formate plus CO2, and frcm CO was significantly stimulated by the addition of sodium. The apparent K m for sodium in acetate formation from CO was near 2 mM. In the absence of added sodium formate excretion was increased [20]. The findings were interpreted to indicate that one of the steps involved in the formation of the methyl group of acetate from formyl-THF was the sodium-dependent reaction. Since formyl-THF formation from formate has never been found to depend on sodium, and in analogy to the methanogenic bacteria (see above), it was concluded that the reduction of methylene-THF to methyl-THF was the sodium dependent step [20]. This was further substantiated by recent findings of G. Gottschalk and collaborators, that in Acetobacterium woodii CO 2 reduction to acetate is coupled to sodium extrusion and that acetate formation from formaldehyde, hydrogen, and carbon monoxide is also dependent on sodium [21]. If the methyl tetrahydrofolate formation from methylene.THF is coupled to the electrogenie transport of sodium across the membranes of homoacetogens, the enzyme must be associated with the membrane. Indirect evidence for a membrane association of the methylene-THF reductase was recently presented for Clostridium thermoauto. trophicum [22]. Evidence for a location of the enzyme in the particulate fraction is also available 394 Na" ( c°2~ ' ~ IHCHOI ICH3OHI H+ H+ Fig. 4. Scheme of energy conservation in homoacetogenic bacteria upon growth on H 2 plus CO . No stoichiometriesof cations translocated per reaction are given. for P. productus (Wohlfarth et al., in press), where up to 60~ of the enzyme can be demonstrated to be present in the membrane. The methylene-THF dehydrogenase served as a cytoplasmic marker enzyme; about o~ ,. ~ of the latter enzyme was located in the ~ytovlasmic fraction after ultracentrifugation Of crude extracts. From the data it was deduced that the methylene-THF reduction is a m e m b r a n e - a s ~ i a t e d process and is coupled to the transport of sodium ions across the membrane. • 5. CONCLUSIONS: H O W IS E N E R G Y CONSERVED IN H O M O A C E T O G E N S ? The mechanism of energy conservation in homoacetogenic bacteria growing on H 2 plus CO2 a~ the sole energy sources is summarized in Fig. 4. i~ should be mentioned that no stoichiometries of monovalent cations transported per reaction are given in the figure. The sodium gradient generated in the methyle.,.,¢-THF reductase reaction is probably i,ot utrectly used for the synthesis of ATP, b u t it is converted to a proton gradient by a s o d i u m / p r o t u n antiporter. The presence of such a n electrogenic antiporter was recently reported for Clostridium thermoaceticum [23]. The electrochemical proton potential then is used for the energy conservation by the action of a proton translocating A T P synthase, which has also been demonstrated for C. thermoaceticum [24]. Part of the proton gradient is consumed for the energy requiting CO 2 reduction to carbon monoxide [15]. Interestingly, from the findings it can b e deduced that homoacetogenic bacteria are able to involve three different primary energy-rich 'intermediates' depending on the C t substrates th.tt they use as the energy source. With H 2 plus CO 2 the energy yielding reaction is the methylene-TEIF reduction, and thus an electrochemical sodium gradient is used for the energy conservation. With carbon monoxide as the substrate a major p~.rt of the energy is derived from the proton gradient generated in the CO oxidation to CO2. Finally, when using methanol as the energy source, homoacetogens synthesize A T P via substrate level phosphorylation in both the acetate kinase reaction and the formyl-THF synthetase reaction; part of the ATP must be required for the generation of a proton gradient for the reduction of CO2 to [CO] and for the formation of a sodium gradient, which most probably drives the endergonic oxidation of methyl tetrahydrofolate to methylene-THF. The latter conclusions, however, remain speculative a n d require further investigations. ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Oodesberg, and by the F e n d s der chemischen Industrie. P FWER~NCES Ill Fuchs, G. (1986) CO2 fixation in acetogenic bacteria: ~'-qriationson a theme. FEMS Microbiol. Rev. 39,181-213. [2] Wood, ,'.~.G., Ragsdule, S.W. and Pezacka, E. (1986) The acetyI-CoA patii~v of autotrophic growth. FEMS Microbiol. Rev. 39, 345-362. [3] Diekert, G., Fuchs, G. and Thauer, R.K. (1985) Properties and function of carbon monoxide dehydrogenase from anaerobic bacteria, in Microbial Gas Metabolism: Mechanistic, Metabolic and Biotechnological Aspcct~ (Peele, R.K. and Dew, C.S., Eds.), pp. 115-130. Society for General Microbiology. [4] Ragsdale, S.W., Wood, H.G., Morton, T.A., Ljungdahl, L.G. and DerVartanian, D.V. (1988) Nickel in CO dehydrogenase, in The Bioinorganic Chemistry of Nickel (Lancaster, J.R., Ed.), pp. 311-332. VCH Publishers, Weinheim. 395 [5] Dieken, G. (1988) Carbon monoxide dehydrogenase of acetogens, in The Bioinorganic Chemistry of Nickel (Lancaster, J.R., Ed.), pp. 299-309. VCH Publishers, Weinheim. [6] Lee, M.J. and Zinder, S.H. (1988) Hydrogen partial pressures in a thermophilic acetate-oxidizing methanogenic coculture. AppL Environ. MicrobioL 54,1457-1461. [7] Lee, MJ. and Zinder, S.H. (1988) Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture. Arch. Microbiol. 150, 513518. [8] Zinder, S.H. and Koch, M. (1984) Non-aceticlastic methanogenesls from acetate: acetate oxidation by a thermophilic syntrophic coculture. Arch. Microbiol. 138, 263272. [9] Widdel, F. (1988) Microbiology and ecology of sulfateand sulfur-reducing bacteria, in Biology of Anaerobic Microorganisms (Zehnder, AJ.B., Ed.), pp. 469-584. John Wiley & Sons Ltd., Chichester. 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Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis. J. Biol. Chem. 260, 3970-3977. [15] Diekert, O., Schrader, E. and Hard~, W. (1986) Encxgetics of CO formation and CO oxidation in cell suspensions of Acetobactermm woodii. Arch. Microbiol. 144, 386-392. [16] Bott, M. and Thauar, R.IC (1987) Proton-motive-forcedriven formation of CO from COz and H2 in methanogenic bacteria. Eur. J. Biochem. 168, 407-412. [17] Bott, M., Eikmanns, B. and Thauer, R.K. (1986) Coupling of carbon monoxide oxidation to CO7. and H 2 with the phosphory|ation of ADP in acetate-grown Methanosarcina barkeri. Eur. J. Biochem. 159, 393-398. [18] Clark, J.E. and Ljungdahl, L.G. (1984) Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum. J. Biol. Chem. 259, 10845-10849. [19] Miiller, V., Winner, C. and Gottschalk, G. (1988) Electron-transport-driven sodium extrusion during methan~ genesis from formaldehyde and molecular hydrogen by Methano~arcina barkeri. Eur. J. Biochem. 178, 519-525. [20] Gcerligs, G., SchSnheit, P. and Diekert, G. (1989) Sodium dependent acetate formation from CO 2 in Peptostreptococcus productus (strain Marburg). FEMS Microbiol. Lett. 57, 253-258. [21] Hugenholtz, J., lvey, D.M. and Ljungdahl, L.G. (1987) Carbon monoxide-driven electron transport in Clostridium thermoautotrophicum membranes. J.Baeteriol. 169, 58455847. [22] Heise, R., Mfiller, V. and Gottschalk, G. (1989) Sodium dependence of acetate formation by the acetogenic bacterium Acetobacterium woodiL J.Bacteriol. 171, 54735478. [23] Terraciano, J.S., Schreurs, WJ.A. and Kashket, E.R. (1987) Membrane H + conductance of Clostri,~um thermo. aceticum anO City,iridium acetobutylicum: evidence for electrogenic N a ÷ / H ÷ antiport in Clostridium thernu~ aceticum. Appl. Environ. Microbiol. 53, 782-786. [24] Mayer. F., lvey. 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