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CO in methanogenesis

CO in methanogenesis Ann Microbiol (2010) 60:1–12 DOI 10.1007/s13213-009-0008-5 REVIEW ARTICLE James G. Ferry Received: 22 September 2009 /Accepted: 9 November 2009 /Published online: 2 February 2010 Springer-Verlag and the University of Milan 2010 Abstract Although CO is present in methanogenic envi- also deposited in diverse O -free habitats where anaerobic ronments, an understanding of CO metabolism by metha- microbial food chains decompose the organic matter to CH nogens has lagged behind other methanogenic substrates and CO in a process called biomethanation. A portion of the and investigations of CO metabolism in non-methanogenic CH is converted back to CO by anaerobic methylotrophs 4 2 species. This review features studies on the metabolism of and the remainder escapes into aerobic zones where it is CO by methanogens from 1931 to the present. The path- oxidized to CO by O -requiring methylotrophs, thereby 2 2 ways for CO metabolism of freshwater versus marine completing the global carbon cycle. species are contrasted and the ecological implications The biomethanation of organic matter occurs in diverse discussed. The biochemistry and role of CO dehydroge- habitats such as freshwater sediments, rice paddies, sewage nase/acetyl-CoA synthase in the pathway for conversion of digesters, the rumen, the lower intestinal tract of monogas- acetate to methane and biosynthesis of cell carbon is pre- tric animals, landfills, hydrothermal vents, coastal marine sented. Finally, a proposal for the role of CO and primitive sediments, and the subsurface (Liu and Whitman 2008). A forms of the CO dehydrogenase/acetyl-CoA synthase in the minimum of three interacting metabolic groups of anae- origin and early evolution of life is discussed. robes comprise a consortium converting complex organic matter to CO and CH (Fig. 2). The fermentative group I 2 4 . . . Keywords Carbon monoxide Methanogenesis Acetate anaerobes decompose complex organic matter to acetate, . . Acetyl-CoA synthase Ecology Evolution higher volatile fatty acids, H , and CO . The H -producing 2 2 2 acetogenic group II anaerobes decompose the higher vola- tile fatty acids to acetate, H , and CO . The group III and 2 2 Introduction IV methanogens convert the metabolic products of the first two groups to CH by two major pathways. At least two- The decomposition of complex organic matter in diverse thirds of the CH produced in nature derives from the anaerobic environments is an essential link in the global conversion of the methyl group of acetate by the Group III carbon cycle (Fig. 1) producing approximately 1 billion methanogens. The Group IV methanogens produce approx- metric tons of methane each year. In the cycle, CO is fixed imately one-third by reducing CO with electrons supplied 2 2 into complex organic matter by photosynthesis that is from the oxidation of H or formate. Thus, methanogens decomposed primarily by O -requiring aerobic microorgan- are dependent on the first two groups to supply substrates isms in oxygenated habitats with release of CO back into for their growth. The production of H by Groups I and II 2 2 the atmosphere. However, a portion of the organic matter is is thermodynamically unfavorable, requiring the CO - reducing methanogenic Group IV to maintain low concen- trations of H . A variety of other simple compounds serve J. G. Ferry (*) as minor substrates for methanogenesis. Ethanol and Department of Biochemistry and Molecular Biology, secondary alcohols are used as electron donors for the The Pennsylvania State University, reduction of CO to CH . The methyl groups of methanol, 2 4 University Park, PA 16802, USA methylamines, and methylated sulfides are also dismutated e-mail: jgf3@psu.edu 2 Ann Microbiol (2010) 60:1–12 CH (Fischer et al. 1931). However, the direct conversion CO of CO by methanogens (Eq. 1) could not be concluded (A) since both H and acetate appeared and disappeared (F) 2 (Fischer et al. 1932) consistent with non-methanogens as (B) (E) organic the primary anaerobes metabolizing CO to methanogenic CH 4 substrates (Eqs. 2 and 3). matter 4CO þ 2H O ! 3CO þ CH ð1Þ 2 2 4 (C) (D) H + CO 2 2 acetate 4CO þ 4H O ! 4H þ 4CO ð2aÞ 2 2 2 formate Fig. 1 The global carbon cycle. a Fixation of CO into organic matter, b aerobic decomposition of organic matter to CO , c anaerobic 4H þ CO ! 2H O þ CH ð2bÞ 2 2 2 4 decomposition of organic matter to fermentative end products, d anaerobic conversion of fermentative end products to CH , e anaerobic oxidation of CH to CO , f aerobic oxidation of CH to CO 4 2 4 2 4CO þ 2H O ! CH COOH þ 2CO ð3aÞ 2 3 2 to CO and CH . Although CO is present in methanogenic 2 4 environments (Conrad and Seiler 1980), an understanding CH COOH ! CO þ CH ð3bÞ 3 2 4 of CO metabolism by methanogens has lagged behind other substrates. This review chronicles studies on the metabo- In 1933, it was reported (Stephenson and Stickland lism of CO by methanogens from 1931 to the present. For 1933) that a pure culture was able to convert CO to CH by a more comprehensive general understanding of metha- first converting CO to H (Eq. 2ab) that, 14 years later in nogens, the reader is referred to several recent review 1947, was confirmed with resting cell suspensions of a articles describing the ecology, physiology, and biochem- pure culture (Kluyver and Schnellen 1947). Another gap in istry of methanogenesis (Ferry 2008; Ferry and Lessner time had passed before studies on CO metabolism by 2008; Liu and Whitman 2008; Oelgeschlager and Rother methanogens resumed in 1977 and 1984 when it was 2008; Spanheimer and Muller 2008; Thauer et al. 2008). shown that several species remove CO from the gas phase when growing with CO plus H and, for the first time, that 2 2 CO as an energy source two freshwater methanogens are able to grow with CO as the sole energy source, albeit under conditions unable to The first recorded report portending the ability of metha- sustain proliferation in the native environment (Daniels nogens to metabolize CO appeared in 1931 when it was et al. 1977). Only recently, a marine species was shown to demonstrated that sewage sludge converts CO to CO and grow with CO under conditions suitable for proliferation in the native environment via a novel pathway for meth- anogenesis consistent with CO-dependent growth in the native environment (Lessner et al. 2006). Freshwater environments Methanothermobacter thermoautotrophicus (formerly, Methanobacterium thermoautotrophicum strain ΔH) was shown to grow with CO as the sole energy source dis- proportionating CO according to Eq. 1, although the growth rate was only 1% of that with H /CO (Daniels et al. 1977). 2 2 Cell-free extracts showed CO dehydrogenase activity with coenzyme F as the electron acceptor. The activity was reversibly inactivated by cyanide and O consistent with an active-site metal center; however, the enzyme was not purified and studied in greater detail. Methanosarcina barkeri strain MS isolated in 1966 Fig. 2 A freshwater anaerobic microbial food chain. By permission (Ferry 2008) (Bryant and Boone 1987) can be adapted to grow in an Ann Microbiol (2010) 60:1–12 3 atmosphere of 50% CO as the only carbon and energy dizes H and reduces ferredoxin in reaction 1 (Fig. 3)that source (O'Brien et al. 1984). Net H formation is observed supplies electrons for reaction 2. The EchA-F complex is when the headspace CO is greater than 20%. Below this necessary, since the reduction of CO with H (reactions 1 2 2 concentration of CO, H is consumed and the rate of CH plus 2) is thermodynamically unfavorable under standard 2 4 production increases substantially with an increased growth conditions of one atmosphere H (ΔG°′=+16 kJ/mole) and rate approaching a doubling time of 65 h. Methanosarcina considerably more so with the much lower concentrations barkeri strain MS also produces CH and CO with the of H in the environment. This thermodynamic barrier is 4 2 2 growth substrates methanol plus a headspace of 50% CO overcome by reverse electron transport to ferredoxin driven (O'Brien et al. 1984). However, it was reported that H by the electrochemical proton gradient and catalyzed by the accumulated and methanol was not metabolized until the EchA-F hydrogenase complex. However, when cultured CO decreased to below 30% in the headspace, at which with CO, it is presumed that ferredoxin is the electron point H and methanol was rapidly consumed proportional acceptor of the CO dehydrogenase and the oxidation of CO to an increase in the rate of CH formation and growth. coupled to reduction of CO (reaction 2) becomes thermo- 4 2 Based on these characteristics, it was concluded that dynamically favorable (ΔG°′ =−4 kJ/mole) (Ferry and methanogenesis in the freshwater species M. barkeri strain House 2006) obviating the requirement for EchA-F hydrog- MS is inhibited by concentrations of CO greater than ap- enase (Stojanowic and Hedderich 2004). proximately 50% in the headspace, consistent with results obtained with M. thermoautotrophicum (Daniels et al. 1977). Marine environments The authors speculate that CO is metabolized according to Eqs. 2a and b, and the inhibition results from inhibition of Recent investigations of Methanosarcina acetivorans dem- hydrogenase catalyzing the oxidation of H (O'Brien et al. onstrate that this marine isolate displays robust growth with 1984). Although the pathway for reduction of CO to CH CO as the sole carbon and energy source (Lessner et al. 2 4 with H was not investigated, it is presumed to follow the 2006;Rother et al. 2007). The generation time (∼20 h) is less well-studied pathway determined for freshwater methano- than that reported for other methanogens, and growth occurs gens, as shown in Fig. 3 for Methanosarcina species, with with CO concentrations greater than 1 atmosphere. Although one possible exception. With H as the electron donor, the a few methanogens have been shown to utilize CO for EchA-F (Escherichia coli-type hydrogenase) complex oxi- growth, the experiments have routinely utilized concentra- tions above 50% in the atmosphere which is several-fold greater than is likely encountered in the environment. The CO CO CO CO CO CO 2 2 2 2 2 2 lowest concentrations that support growth have not been Fd Fd Fd Fd Fd Fd MF MF MF MF MF MF re re re re re red d d d d d 1 1 + + + + + + reported for any methanogen, nor has a measurement of CO 2H 2H 2H 2H 2H 2H Ec Ec Ec Ec Ec Ech h h h h hA A A A A A- - - - - -F F F F F F 2 2 Fd Fd Fd Fd Fd Fd ox ox ox ox ox ox concentrations in the native habitats. Thus, a role for CO as CHO CHO CHO CHO CHO CHO M M M M M MF F F F F F H H H H H H 2 2 2 2 2 2 the sole carbon and energy source in the native habitats of 3 3 THS THS THS THS THS THSP P P P P Pt t t t t t MF MF MF MF MF MF methanogens is still in question. Nonetheless, M. acetivorans CHO CHO CHO CHO CHO CHO THS THS THS THS THS THSP P P P P Pt t t t t t was isolated from sediments rich in decaying kelp appended + + + + + + H H H H H H ADP ADP ADP AT AT ATP- P- P- 4 4 with flotation bladders containing up to 10% CO as a + + + H H H 12 12 as as ase e e H H H H H H O O O O O O 2 2 2 2 2 2 potential source of CO for growth of this marine isolate AT AT ATP P P + + + CH CH CH CH CH CH THSP THSP THSP THSP THSP THSPt t t t t t (Abbott and Hollenberg 1976; Sowers et al. 1984). F F F F F F H H H H H H 420 420 420 420 420 420 2 2 2 2 2 2 6 6 5 5 A biochemical and quantitative proteomic analysis of H H H H H H 2 2 2 2 2 2 F F F F F F 420 420 420 420 420 420 methanol- and acetate-grown versus CO-grown M. aceti- CH CH CH CH CH CH THS THS THS THS THS THSP P P P P Pt t t t t t 2 2 2 2 2 2 vorans (Lessner et al. 2006) has provided, for the first time, F F F F F F H H H H H H 420 420 420 420 420 420 2 2 2 2 2 2 7 7 8 8 evidence of a pathway for CO-dependent methanogenesis H H H H H H 2 2 2 2 2 2 F F F F F F 42 42 42 42 42 420 0 0 0 0 0 9 9 supporting growth (Fig. 4) that has been confirmed in part CH CH CH CH CH CH THS THS THS THS THS THSP P P P P Pt t t t t t 3 3 3 3 3 3 by a qualitative proteomic analysis (Rother et al. 2007). In + + + + + + HS HS HS HS HS HSCo Co Co Co Co CoM M M M M M M M M M M Mt t t t t trA rA rA rA rA rA-H -H -H -H -H -H 2N 2N 2N 2N 2N 2Na a a a a a the pathway, 3 CO are oxidized to 3 CO that provides CH CH CH CH CH CH SC SC SC SC SC SCoM oM oM oM oM oM 3 3 3 3 3 3 electrons for the subsequent reduction of 1 CO to form a HS HS HS HS HS HSCo Co Co Co Co CoB B B B B B T T T T T TH H H H H HSPt SPt SPt SPt SPt SPt VhoAC VhoAC VhoAC VhoAC VhoAC VhoACG G G G G G + + + + + + 10 10 11 11 methyl group attached to the cofactor tetrahydrosarcinap- 4H 4H 4H 4H 4H 4H Hd Hd Hd Hd Hd Hdr r r r r rD D D D D DE E E E E E CoM CoM CoM CoM CoM CoMS S S S S S S S S S S SC C C C C Co o o o o oB B B B B B terin (THSPt) (reactions 1–6) similar to the pathway of fresh- H H H H H H CH CH CH CH CH CH 2 2 2 2 2 2 4 4 4 4 4 4 water methanogens (Fig. 3) except the electron donor. The methyl group of methyl-THSPt is transferred to coenzyme M (HS-CoM), although the route may be different from Fig. 3 The CO reduction pathway of freshwater Methanosarcina freshwater strains. The methyltransferase that transfers the species utilizing H . Fd Ferredoxin, THSPt tetrahydrosarcinapterin, HS-CoB coenzyme B, HS-CoM coenzyme M, MF methanofuran methyl group from methyl-THSPt to HS-CoM (MtrA-H) in 4 Ann Microbiol (2010) 60:1–12 Fig. 4 Pathway for conversion of CO to acetate and methane 2 C 2 C 2 C 2 CO O O O 2 2 2 2 1a 1a 1a 1a by the marine isolate 2 F 2 F 2 F 2 Fd d d d – re re re red d d d 7C 7C 7C 7CO O O O 7C 7C 7C 7CO O O O + 1 + 1 + 1 + 14 4 4 4e e e e 2 2 2 2 Methanosarcina acetivorans. 2 2 2 2 2 F 2 F 2 F 2 Fd d d d 1b 1b 1b 1b Fd Ferredoxin, THSPt ox ox ox ox 4e 4e 4e 4e + 2F + 2F + 2F + 2Fd d d d 2F 2F 2F 2Fd d d d r r r r o o o o 2 C 2 C 2 C 2 CH H H HO O O O M M M MF F F F tetrahydrosarcinapterin, 1c 1c 1c 1c HS-CoB coenzyme B, HS-CoM – 10e 10e 10e 10e + 5F + 5F + 5F + 5F 5F 5F 5F 5F H H H H 3 3 3 3 42 42 42 420 0 0 0 42 42 42 420 0 0 0 2 2 2 2 coenzyme M, MF methanofuran 2 C 2 C 2 C 2 CH H H HO O O O T T T TH H H HSPt SPt SPt SPt + + + + H H H H 4 4 4 4 CH CH CH CH CO CO CO COO O O OH H H H 3 3 3 3 H H H H O O O O 2 2 2 2 AT AT AT ATP P P P + + 2 C 2 C 2 C 2 CH H H H T T T TH H H HSP SP SP SPtttt 13 13 13 13 AD AD AD ADP P P P 2 F 2 F 2 F 2 F H H H H AD AD AD ADP P P P 42 42 42 420 0 0 0 2 2 2 2 -2 -2 -2 -2 + + + + ATP ATP ATP ATP- - - - CH CH CH CH CO CO CO COP P P PO O O O 5 5 5 5 H H H H 3 3 3 3 4 4 4 4 as as as ase e e e 2 F 2 F 2 F 2 F AT AT AT ATP P P P 42 42 42 420 0 0 0 12 12 12 12 2 C 2 C 2 C 2 CH H H H T T T TH H H HSPt SPt SPt SPt 2 2 2 2 CH CH CH CH CO CO CO COS S S SC C C Co o o oA A A A 3 3 3 3 2 F 2 F 2 F 2 F H H H H 42 42 42 420 0 0 0 2 2 2 2 6 6 6 6 11 11 11 11 2 F 2 F 2 F 2 F 42 42 42 420 0 0 0 CO CO CO CO Co Co Co CoA A A A 2 C 2 C 2 C 2 CH H H H T T T TH H H HSPt SPt SPt SPt 3 3 3 3 7 7 7 7 + + + + HS HS HS HSCo Co Co CoM M M M 8 8 8 8 Mt Mt Mt Mtr r r rA A A A- - - -H H H H 2N 2N 2N 2Na a a a CH CH CH CH SC SC SC SCoM oM oM oM HS HS HS HSCo Co Co CoB B B B 3 3 3 3 F F F F 42 42 42 420 0 0 0 T T T TH H H HSPt SPt SPt SPt Fp Fp Fp Fpo o o oA A A A- - - -O O O O + + + + H H H H 10 10 10 10 Hd Hd Hd HdrDE rDE rDE rDE 9 9 9 9 F F F F H H H H 42 42 42 420 0 0 0 2 2 2 2 C C C Co o o oM M M MS S S S SC SC SC SCoB oB oB oB CH CH CH CH 4 4 4 4 all known methanogenic pathways is down-regulated in are required to accommodate variations in the energy avail- CO-grown M. acetivorans versus methanol- and acetate- able to pump sodium. Thus, at low environmental CO con- grown cells (Lessner et al. 2006) suggesting a reduced centrations, the available energy is low and the transfer from involvement of MtrA-H in the pathway (reaction 8, Fig. 4). methyl-THSPt to HS-CoM would necessarily shift more The MtrA-H complex is membrane-bound and couples the towards the sodium-independent pathway. With increased methyl transfer reaction to translocation of sodium from the CO levels and greater available energy, methyl transfer cytoplasm across the membrane to the periplasm, forming a would shift to the sodium-pumping MtrA-H complex to opti- gradient (Gottschalk and Thauer 2001). Indeed, the sodium mize the thermodynamic efficiency. Under laboratory con- requirement for methanogenesis is thought to originate from ditions where cells are routinely cultured with greater than this enzyme, and the sodium gradient is postulated to drive 0.5 atmosphere of CO, the postulated soluble sodium- energy-requiring reactions (Gottschalk and Thauer 2001). independent route may be dispensable. The mechanism for Three homologs annotated as putative corrinoid-containing providing electrons for reductive demethylation of CH -S- proteins are up-regulated in CO-grown versus methanol- and CoM to CH is a prominent characteristic of the proposed acetate-grown M. acetivorans consistent with a role during pathway for conversion of CO to CH in M. acetivorans that growth with CO (Rother et al. 2007). Corrinoid cofactors further distinguishes it from the well-characterized CO bind the methyl group in a variety of methyltransferases from reduction pathway of freshwater methanogens. In freshwater methanogens including the MtrA-H complex (Gottschalk species of Methanosarcina, HS-CoB donates electrons for and Thauer 2001); thus, it has been postulated (Lessner et al. the reductive demethylation of CH -S-CoM to CH cata- 3 4 2006) that the up-regulated corrinoid proteins participate in lyzed by methylreductase (reaction 10, Fig. 3). The hetero- transfer of the methyl group from methyl-THSPt to HS-CoM disulfide CoM-S-S-CoB is also a product of reaction 10 that via a soluble sodium-independent pathway (reaction 7, is reduced to the active sulfhydryl forms of the cofactors Fig. 4) distinct from MtrA-H. In fact, one of the homologs catalyzed by the VhoACG/HdrDE complex (reaction 11, overproduced in Escherichia coli (MA4384) catalyzes trans- Fig. 3). H is oxidized by the VhoACG hydrogenase with fer of the methyl group from methyl-THSPt to HS-CoM transfer of electrons to the heterodisulfide reductase HdrDE (unpublished) supporting the proposal. The proposal does that is coupled to formation of an electrochemical potential not rule out that both pathways may function simultaneously. that drives ATP synthesis (reaction 12, Fig. 3). On the other Indeed, it is conceivable that both sodium-dependent MtrA- hand, the proteomic and biochemical evidence indicates that H and the postulated soluble sodium-independent pathway HdrDE and the FpoA-O complex (F H dehydrogenase) 420 2 Ann Microbiol (2010) 60:1–12 5 participate in the transfer of electrons to the heterodisulfide acetyl-CoA is further converted to acetate catalyzed by in M. acetivorans (reaction 10, Fig. 4). HdrDE and the phosphotransacetylase and acetate kinase (reactions 12 and FpoA-O complex also function in the pathway of methanol 13) with the synthesis of ATP. Thus, it appears that ATP is conversion to methane in Methanosarcina species (Ferry synthesized via both substrate level and chemiosmotic and Kastead 2007), including M. acetivorans (Li et al. 2007), mechanisms. The CdhA-E complexes from freshwater wherein methanophenazine mediates electron transfer be- Methanosarcina species have CO dehydrogenase activity tween FpoA-O and HdrDE. Methanophenazine is a quinone- with ferredoxin as the electron acceptor (Terlesky and Ferry like compound that translocates protons from the cytoplasm 1988b), as does the M. acetivorans enzyme (unpublished to the periplasm forming an electrochemical potential that results) consistent with the CdhA-E also functioning in drives ATP synthesis. Additionally, FpoA-O pumps protons steps 1a and 1b (Fig. 4). Quantitative RTPCR analysis contributing to the electrochemical potential in methanol- indicates modest up-regulation of two genes annotated as grown Methanosarcina species. Thus, it is postulated encoding CooS in CO-grown versus acetate-grown cells (Lessner et al. 2006) that the FpoA-O/HdrDE complex (Lessner et al. 2006). CooS is a CO dehydrogenase first contributes to the electrochemical potential that drives ATP described in Rhodospirillum rubrum (32) that also reduces synthesis in CO-grown M. acetivorans (Fig. 4). The ferredoxin; thus, the putative CooS in M. acetivorans is mechanism for the formation of formate is not known. also a candidate for catalyzing reactions 1a and 1b. Pheno- The products acetate and formate are yet another feature typic analyses of mutant strains of M. acetivorans deleted of the pathway in M. acetivorans that contrasts with M. of the cooS genes suggest they contribute to but are not barkeri (Rother and Metcalf 2004; Lessner et al. 2006; required for CO-dependent growth (Rother et al. 2007). Oelgeschlager and Rother 2009). Quantitative proteomic There are no known enzymes in M. acetivorans responsible analyses (Lessner et al. 2006) suggest a pathway in which for reactions leading from CO to coenzyme F . CO dehydrogenase/acetyl-CoA synthase (CdhA-E) cata- Notably, H is not an intermediate in the pathway for lyzes synthesis of acetyl-CoA from the methyl group of CO conversion to CH in M. acetivorans (Lessner et al. methyl-THSPt, CO, and CoA-SH (reaction 11, Fig. 5). The 2006) in contrast to freshwater M. barkeri (Fig. 3). Indeed, cell membr cell membra ane ne AT ATP P Co CoA A- -S SH H Ac Ack k Pta Pta -2 -2 CH CH COO¯ COO¯ MA MA3 36 60 06 6 CH CH COPO COPO M MA A3607 3607 CH CH CO COS SC CoA oA 3 3 4 4 3 3 3 3 Fd Fd THM THMP PT T o o ADP ADP -2 -2 HPO HPO 4 4 CoM- CoM-S SH H HCO HCO ¯ ¯ 3 3 Cd Cdh h Ca Cam m Mt Mtr r M MA A1 10 01 11- 1-16 16 C Co oA-SH A-SH CH CH -THM -THMPT PT 3 3 M MA A2536 2536 M MA A3860-65 3860-65 + + M MA A02 0269-76 69-76 Na Na CO CO CH CH -S- -S-Co CoM M 3 3 2 2 Ma Ma-Rn -Rnf f Co CoB B- -S SH H Fd Fd THM THMP PT T r r + + + + Na Na /H /H M MA A0 0658-65 658-65 Fd Fd o o M MA A454 4546-47 6-47 Mc Mcr r M MA A45 4550 50 + + MP MPH MP MPH H H 2 2 CH CH 4 4 CoM- CoM-S-S- S-S-Co CoB B M MA A0687-88 0687-88 + + - -2 2 AD ADP + P + H HP PO O H H AT ATP P 4 4 CoM-SH + CoM-SH + CoB-S CoB-SH H Hdr Hdr- -DE DE + + Na Na M MA A4152-60 4152-60 At Atp p Mr Mrp p M MA A456 4566-72 6-72 + + H H Fig. 5 Pathway for the conversion of acetate to methane by anhydrase, Ma-Rnf M. acetivorans Rnf, MP methanophenazine, Hdr- Methanosarcina acetivorans. Ack Acetate kinase, Pta phosphotransa- DE heterodisulfide reductase, Mrp multiple resistance/pH regulation + + + cetylase, CoA-SH coenzyme A, THMPT tetrahydromethanopterin, Fd Na /H antiporter, Atp H -transporting ATP synthase. Carbon transfer reduced ferredoxin, Fd oxidized ferredoxin, Cdh CO dehydrogenase/ reactions are catalyzed by the enzymes shown in blue. Electron acetyl-CoA synthase, CoM-SH coenzyme M, Mtr methyl-THMPT: transfer reactions are catalyzed by enzymes shown in green.By CoM-SH methyltransferase, CoB-SH coenzyme B, Cam carbonic permission (Li et al. 2006) 6 Ann Microbiol (2010) 60:1–12 M. acetivorans does not encode a functional EchA-F Raybuck et al. 1991). Although a two-subunit CO dehy- hydrogenase (Galagan et al. 2002). Thus, the observation drogenase has been purified and characterized from an that M. acetivorans is more tolerant of high concentrations acetate-utilizing species from the genus Methanosaeta of CO compared with M. barkeri is consistent with the (formerly Methanothrix) (Boone and Kamagata 1998) that postulated CO inhibition of hydrogenase in M. barkeri catalyzes the exchange of CO with the carbonyl group of (O'Brien et al. 1984). The question then arises why M. acetyl-CoA (Jetten et al. 1989, 1991a, b; Eggen et al. 1991), acetivorans evolved a pathway for utilization of CO inde- the majority of mechanistic investigations have been with pendent of H . One possibility is that, in marine environ- the five-subunit CdhA-E complexes from the acetate- ments, sulfate-reducing microbes outcompete methanogens utilizing species Methanosarcina thermophila and M. for H (Zinder 1993), presenting the possibility that if H barkeri described here. 2 2 were an intermediate it could be lost to sulfate reducers. The subunits of the CdhABCDE complex from Meth- Another possibility is that Methanosarcina species are at a anosarcina species are correspondingly designated αεβγδ disadvantage utilizing H in the marine environment where and encoded in operons arranged in the order cdhABCDE the concentrations are kept low by sulfate-reducers and, (Maupin-Furlow and Ferry 1996a, b; Grahame et al. 2005). therefore, have not evolved hydrogenases. Indeed, it is Use of a plasmid-mediated lacZ fusion reporter system has noted that Methanosarcina species have considerably revealed that the operon encoding the complex of M. higher threshold concentrations for H than do obligate thermophila (Grahame et al. 2005) is 54-fold down- CO -reducing species (Thauer et al. 2008). regulated in M. acetivorans grown on methanol compared The formation of methanethiol and dimethylsulfide to acetate, consistent with a role for the complex in the during growth of M. acetivorans on CO was recently re- pathway for utilization of acetate (Apolinario et al. 2005). ported (Moran et al. 2008). The authors speculate that The results confirm an earlier report on the regulation of methyl-groups generated in the CO-dependent reduction cdhA in M. thermophila examined by northern blotting of CO are transferred to sulfide present in the growth (Sowers et al. 1993). Unlike the genome of M. thermophila medium and the process could be coupled to energy con- that harbors only one operon encoding CdhA-E (Grahame servation. However, the rate of dimethylsulfide formation et al. 2005), the genomes of M. acetivorans (Galagan et al. from CO is only 1–2% of that of CH formation suggesting 2002)and Methanosarcina mazei (Deppenmeier et al. the process is not of major consequence in the energy 2002) contain duplicate cdh operons with greater than yielding metabolism during growth on CO (Oelgeschlager 95% identity, raising the question as to whether both are and Rother 2009). transcribed during growth on acetate. A proteomic analysis of M. acetivorans indicates that one complex is expressed at least 16-fold over the other (Li et al. 2006, 2007), CO dehydrogenase/Acetyl-COA synthase consistent with the predominance of a single Cdh complex purified from acetate-grown M. mazei strain Go1 (formerly Conversion of acetate to CH Methanosarcina frisia) (Maestrojuan et al. 1992), although the organism encodes duplicate cdh operons with high The five-subunit CdhA-E complex is central to pathways identity (Eggen et al. 1996). However, in apparent contrast for conversion of acetate to methane (Kohler and Zehnder to this result, global transcriptional profiling of M. mazei 1984; Krzycki and Zeikus 1984; Nelson and Ferry 1984; Go1 suggests both cdh operons are transcribed at approx- Krzycki et al. 1985; Bott et al. 1986; Terlesky et al. 1986; imately equal levels (Hovey et al. 2005). Bott and Thauer 1987; Zinder and Anguish 1992; Gokhale The complex from Methanosarcina species is resolvable et al. 1993; Kemner and Zeikus 1994) as illustrated in into three components (Abbanat and Ferry 1991; Grahame Fig. 5 for M. acetivorans (Li et al. 2006). The complex and Demoll 1996; Kocsis et al. 1999). The CdhAE com- functions in the pathway to cleave the C-C and C-S bonds ponent contains the α and ε subunits, the CdhDE compo- of acetyl-CoA yielding a carbonyl group that is oxidized nent contains the γ and δ subunits, and the CdhC to CO and a methyl group that is transferred to THSPt component contains the β subunit. The CdhAE component (Terlesky et al. 1987; Fischer and Thauer 1989; Abbanat has CO dehydrogenase activity with ferredoxin as the and Ferry 1990; Raybuck et al. 1991; Grahame 1991, 1993; electron acceptor (Terlesky and Ferry 1988a, b; Fischer Grahame and Demoll 1995; Grahame et al. 1996; Bhaskar and Thauer 1990). The crystal structure (Gong et al. 2008) et al. 1998). In subsequent steps of the pathway, electrons of the M. barkeri CdhAE (Fig. 6)shows a α ε 2 2 derived from the oxidation of the carbonyl group are configuration with the α subunit harboring a Ni-Fe-S C transferred to the methyl group producing CH . The com- cluster and four Fe S clusters (B, D, E, and F in Fig. 6) 4 4 4 plex also catalyzes the synthesis of acetyl-CoA from a consistent with earlier EPR spectroscopic investigations methyl donor, CO and CoA-SH (Abbanat and Ferry 1990; (Krzycki et al. 1989). The C, B, and D clusters are posi- Ann Microbiol (2010) 60:1–12 7 Fig. 6 The Methanosarcina barkeri α ε CdhAE component.a Side as spheres, with iron atoms in purple, nickel atoms in blue, and the 2 2 view shown as ribbons with the α-subunits colored in cyan and green remaining atoms in CPK. b Side view of the metal clusters. By and the ε-subunits in tan and orange. Metal cluster atoms are shown permission (Gong et al. 2008) tioned similarly in the crystal structure of the homolog electron acceptor for the α ε component (Grahame and 2 2 from Moorella thermoacetica (Ragsdale 2007), an acetate- Stadtman 1987) suggesting a potential role for the ε-subunit producing species of the Bacteria domain. However, the E in FAD-mediated CO oxidation during growth on CO. and F clusters are unique to CdhAE and proposed to The CdhDE component transfers the methyl group of function in electron transport from the active site C cluster acetyl-CoA to THSPt and contains an iron-sulfur center and to ferredoxin. The C cluster (Fig. 7) resembles the M. corrinoid cofactors that transfer the methyl group during thermoacetica structure comprised of a pseudocubane catalysis (Grahame 1991, 1993; Jablonski et al. 1993; NiFe S cluster bridged to an exogeneous iron atom. Ad- Maupin-Furlow and Ferry 1996a). Analyses of the CdhD 3 4 ditional electron density provides support for coupling and CdhE subunits overproduced independently in E. coli between CO bound to the nickel and H O/OH bound to the indicates that the iron-sulfur center is located in the CdhE exogenous iron in the critical C = O bond-forming step subunit and that both subunits bind a corrinoid cofactor. leading to CO (Fig. 7). The structure also identifies a gas Which of the two subunits interact with THSPt has not been channel extending from the C cluster to the surface of the determined. A structure has not been reported for either protein in a direction presumed to interact with the CdhC subunit; however, EPR analyses of the intact CdhDE com- component. Structural and sequence alignments indicate ponent (Jablonski et al. 1993) indicate that the corrinoids that the ε-subunit is a member of the DHS-like NAD/FAD- are maintained in the base-off state with a E ′ of -486 mV 2+/1+ 2+ binding domain clan. Indeed, the structure reveals a cavity for the Co couple that facilitates reduction of Co by able to accommodate an FAD or NAD cofactor located approximately 12 kcal/mol relative to base-on cobamides. 1+ between the interface of the α- and ε-subunits and near the Reduction to the Co redox state is a requirement for DFe -S cluster suggesting a potential role in electron methylation of corrinoid cofactors. The EPR analysis also 4 4 2+/1+ transfer. The authors of the structure note that FAD is an identified a [4Fe-4S] cluster with an E ' of -502 mV, Fig. 7 Proposed coupling of the CO and H O species in the C cluster of the Methanosarcina barkeri CdhAE component 2 8 Ann Microbiol (2010) 60:1–12 redox dependence of acetyl-CoA synthesis by the CdhC component shows one-electron Nernst behavior and that two electrons are required for reductive activation of the active site Ni in the process of forming the enzyme-acetyl intermediate (Gencic and Grahame 2008). Autotrophy Many methanogens grow autotrophically with CO as sole source of carbon (Ferry and Kastead 2007). For example, Fig. 8 Structure of the A cluster from Moorella thermoacetica.By M. thermoautotrophicum is able to grow with H and CO 2 2 permission (Ragsdale 2007) in a simple mineral salts medium (Zeikus and Wolfe 1972; Schonheit et al. 1980) synthesizing acetyl-CoA (Stupperich 2+/1+ approximately isopotential with the Co couple suggest- and Fuchs 1983; Stupperich et al. 1983; Ruhlemann et al. 2+ ing the cluster is likely involved in reducing Co . 1985), the starting point for synthesis of all cellular com- The CdhC component contains an A cluster that is the ponents (Fuchs and Stupperich 1980). Acetyl-CoA is proposed site of acetyl-CoA cleavage or synthesis (Gra- synthesized via methyl-tetrahydromethanopterin (methyl- hame and Demoll 1996; Murakami and Ragsdale 2000; THMPt) providing the methyl group and CO the carboxyl Gencic and Grahame 2003; Funk et al. 2004). Although a group (Lange and Fuchs 1985, 1987). The CO is generated structure is not available, a variety of spectroscopic studies from CO and H in an energy-dependent reaction (Conrad 2 2 suggest the A cluster is comprised of an Fe S center and Thauer 1983; Eikmanns et al. 1985) and methyl- 4 4 bridged to a binuclear Ni-Ni site (Gu et al. 2003; Funk et al. THMPt via steps in the pathway for CO reduction to 2004) similar in structure (Fig. 8) to that proposed for the methane (Lange and Fuchs 1987) similar to that shown in homolog from M. thermoacetica (Ragsdale 2007). In Fig. 3 (reactions 2–8). The THMPt in M. thermoautotro- addition to CO oxidation, the CdhAE component is phicum is an analog of THSPt found in Methanosarcina required for acetyl-CoA cleavage or synthesis (Murakami species. Autotrophic methanogens such as M. thermoauto- and Ragsdale 2000). The authors propose a mechanism trophicum have CO dehydrogenase activity whereas het- for acetyl-CoA synthesis or cleavage involving an intra- erotrophic methanogens lack CO dehydrogenase and molecular electron transfer reaction between the C cluster require acetate as the carbon source that is converted to in the CdhAE component and cluster A in the CdhC acetyl-CoA (Bott et al. 1985). Indeed, the genome of M. component for each catalytic cycle thereby maintaining Ni thermoautotrophicum contains a gene cluster encoding in cluster A in the catalytically active Ni(I) redox state. The subunits with high identity to subunits of the CdhA-E ab CO CO CO CO CO CO CO CO CO CO + + + + CH CH CH CH SH SH SH SH + + + + R R R R- - - -SH SH SH SH 2 2 2 2 2 2 3 3 3 3 3 C 3 C 3 C 3 C 3 C 3 CO O O O O O A A A A 2 C 2 C 2 C 2 C 2 C 2 CO O O O O O 2 2 2 2 2 2 CH CH CH CH CO CO CO COS S S S- - - -R R R R 3 3 3 3 CH CH CH CH CH CH -THM -THM -THM -THM -THM -THMP P P P P PT T T T T T 3 3 3 3 3 3 Fe Fe Fe FeS S S S + H + H + H + H CO CO CO CO CO CO 2 2 2 2 2 2 2 2 P P P P iiii B B B B R- R- R- R- R- R-S S S S S SH H H H H H B B B B R-S R-S R-S R-SH H H H R-S R-S R-S R-SH H H H CH CH CH CH CH CH COS COS COS COS COS COS-R -R -R -R -R -R 3 3 3 3 3 3 -2 -2 -2 -2 A A A A CH CH CH CH CO CO CO COP P P PO O O O 3 3 3 3 4 4 4 4 P P P P P P iiiiii C C C C AD AD AD ADP P P P R-S R-S R-S R-S R-S R-SH H H H H H C C C C AT AT AT ATP P P P -2 -2 -2 -2 -2 -2 CH CH CH CH CH CH CO CO CO CO CO COP P P P P PO O O O O O 3 3 3 3 3 3 4 4 4 4 4 4 FeS FeS FeS FeS + H + H + H + H S S S S 2 2 2 2 AD AD AD AD AD ADP P P P P P D D D D AT AT AT AT AT ATP P P P P P CH CH CH CH CO CO CO COOH OH OH OH 3 3 3 3 R- R- R- R-S S S SH H H H C C C C C CH H H H H H COOH COOH COOH COOH COOH COOH Fig. 9 The proposed cyclic energy-conserving pathway that func- phate. a Solid lines indicate the cyclic pathway (steps A–C). The tioned in the primitive (a) and chemoautotrophic cell independent of broken arrow indicates the priming reaction that is not part of the surface-catalyzed reactions (b) (Ferry and House 2006). The stippled cyclic pathway. b THMPT Tetrahydromethanopterin. By permission areas represent the lipid membranes. Pi represents inorganic phos- (Ferry and House 2006) Ann Microbiol (2010) 60:1–12 9 complex of acetate-utilizing Methanosarcina species (The thioesters from CO and a methyl group derived from co- Comprehensive Microbial Resource. J. Craig Venter Institute, evolution of enzymes leading to the reduction of CO to the http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi). Fur- methyl level that is the extant Wood-Ljungdahl pathway thermore, a CO dehydrogenase was purified from the (Fig. 9b). Thus, it is proposed that the Wood-Ljungdahl autotrophic methanogen Methanococcus vannielii that con- pathway first evolved as an energy-yielding pathway that tains nickel and is composed of subunits with molecular was adapted for biosynthesis of cell carbon, and evolution weights similar to the CdhAE component (DeMoll et al. of the first autotroph, once the pre-biotic organic soup was 1987). These results are consistent with synthesis of acetyl- exhausted for incorporation into cell material (Ferry and CoA in autotrophic methanogens catalyzed by a CO House 2006). It is further proposed that the pathway shown dehydrogenase with properties similar to the CdhA-E in Fig. 9b was the foundation for evolution of methanogenic complex of acetate-utilizing species. pathways. Evolution Conclusions It is proposed that Earth’s atmosphere at the time of the origin of life contained significant amounts of CO (Holland Methanogens play an essential role in the global carbon 1984; Kasting 1990; Kharecha et al. 2005) and that the cycle by serving as terminal organisms in anaerobic micro- CdhA-E complex of methanogens and homologs in the bial food chains that convert complex organic matter to Bacteria domain evolved early during the origin and evo- CH and CO . They utilize simple molecules for growth 4 2 lution of primitive life forms (Lindahl and Chang 2001). In that include acetate and several one-carbon compounds. the chemoautotrophic origin of life (Russell et al. 1988; Although first reported in 1931, comparatively little is Wachtershauser 1988; Russell and Hall 1997), the primary known today of the ecology, physiology, and biochemistry pre-biotic initiation reaction for carbon fixation was the of CO utilization by methanogens. Only a few species surface-catalyzed synthesis of an acetate thioester from are reported to metabolize CO, and it is not yet known CO and H S driven by a geochemical energy source. A unequivocally if CO is a viable energy source for prominent feature of the chemoautotrophic theory is the methanogens in diverse environments. However, recent evolution of biological CO fixation pathways of which investigations of M. acetivorans suggest this methanogen one is a primitive form of the extant Wood–Ljungdahl utilizes CO as a growth substrate in nature converting CO, pathway (Wachtershauser 1997; Pereto et al. 1999; Martin abundant in the native habitat, to CH and acetate via an and Russell 2003; Russell and Martin 2004) in which unusual pathway. CO is also an important intermediate 2CO molecules are reduced to acetyl-CoA implying early during growth of methanogens on acetate and assimilation evolution of primitive forms of enzymes with catalytic of CO for cell carbon. Clearly, more research is warranted capabilities of the CdhA-E complex of methanogens and to determine the extent of CO utilization by methanogens homologs in the Bacteria domain. A role for a primitive in native environments. form of CdhC has been proposed in a modification of the Acknowledgements Research in the laboratory of J.G.F has been chemoautotrophic theory (Ferry and House 2006). In the supported by the NIH, DOE, NSF, and NASA. modified theory, the primitive CdhC catalyzes C-S bond formation (reaction A in Fig. 9a) regenerating the CH COSR thioester from acetate and HS-R that is the energy source References in the first energy-yielding metabolic cycle. Reaction A in Fig. 9a is driven by the conversion of FeS and H Sto pyrite Abbanat DR, Ferry JG (1990) Synthesis of acetyl-CoA by the and H a previously proposed energy source in the carbon monoxide dehydrogenase complex from acetate-grown chemoautotrophic theory (Wachtershauser 1988). The theory Methanosarcina thermophila. 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Biochem J 27:1517–1527 Zeikus JG, Wolfe RS (1972) Methanobacterium thermoautotrophi- Stojanowic A, Hedderich R (2004) CO reduction to the level of cum sp. n., an anaerobic, autotrophic, extreme thermophile. J formylmethanofuran in Methanosarcina barkeri is non-energy Bacteriol 109:707–713 driven when CO is the electron donor. FEMS Microbiol Lett Zinder S (1993) Physiological ecology of methanogens. In: Ferry JG (ed) 235:163–167 Methanogenesis. Chapman and Hall, New York, NY, pp 128–206 Stupperich E, Fuchs G (1983) Autotrophic acetyl coenzyme A Zinder SH, Anguish T (1992) Carbon monoxide, hydrogen, and synthesis in vitro from two CO in Methanobacterium. FEBS formate metabolism during methanogenesis from acetate by Lett 156:345–348 thermophilic cultures of Methanosarcina and Methanothrix Stupperich E, Hammel KE, Fuchs G, Thauer RK (1983) Carbon strains. Appl Environ Microbiol 58:3323–3329 monoxide fixation into the carboxyl group of acetyl coenzyme http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

CO in methanogenesis

Annals of Microbiology , Volume 60 (1) – Feb 2, 2010

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Copyright © 2010 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-009-0008-5
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Abstract

Ann Microbiol (2010) 60:1–12 DOI 10.1007/s13213-009-0008-5 REVIEW ARTICLE James G. Ferry Received: 22 September 2009 /Accepted: 9 November 2009 /Published online: 2 February 2010 Springer-Verlag and the University of Milan 2010 Abstract Although CO is present in methanogenic envi- also deposited in diverse O -free habitats where anaerobic ronments, an understanding of CO metabolism by metha- microbial food chains decompose the organic matter to CH nogens has lagged behind other methanogenic substrates and CO in a process called biomethanation. A portion of the and investigations of CO metabolism in non-methanogenic CH is converted back to CO by anaerobic methylotrophs 4 2 species. This review features studies on the metabolism of and the remainder escapes into aerobic zones where it is CO by methanogens from 1931 to the present. The path- oxidized to CO by O -requiring methylotrophs, thereby 2 2 ways for CO metabolism of freshwater versus marine completing the global carbon cycle. species are contrasted and the ecological implications The biomethanation of organic matter occurs in diverse discussed. The biochemistry and role of CO dehydroge- habitats such as freshwater sediments, rice paddies, sewage nase/acetyl-CoA synthase in the pathway for conversion of digesters, the rumen, the lower intestinal tract of monogas- acetate to methane and biosynthesis of cell carbon is pre- tric animals, landfills, hydrothermal vents, coastal marine sented. Finally, a proposal for the role of CO and primitive sediments, and the subsurface (Liu and Whitman 2008). A forms of the CO dehydrogenase/acetyl-CoA synthase in the minimum of three interacting metabolic groups of anae- origin and early evolution of life is discussed. robes comprise a consortium converting complex organic matter to CO and CH (Fig. 2). The fermentative group I 2 4 . . . Keywords Carbon monoxide Methanogenesis Acetate anaerobes decompose complex organic matter to acetate, . . Acetyl-CoA synthase Ecology Evolution higher volatile fatty acids, H , and CO . The H -producing 2 2 2 acetogenic group II anaerobes decompose the higher vola- tile fatty acids to acetate, H , and CO . The group III and 2 2 Introduction IV methanogens convert the metabolic products of the first two groups to CH by two major pathways. At least two- The decomposition of complex organic matter in diverse thirds of the CH produced in nature derives from the anaerobic environments is an essential link in the global conversion of the methyl group of acetate by the Group III carbon cycle (Fig. 1) producing approximately 1 billion methanogens. The Group IV methanogens produce approx- metric tons of methane each year. In the cycle, CO is fixed imately one-third by reducing CO with electrons supplied 2 2 into complex organic matter by photosynthesis that is from the oxidation of H or formate. Thus, methanogens decomposed primarily by O -requiring aerobic microorgan- are dependent on the first two groups to supply substrates isms in oxygenated habitats with release of CO back into for their growth. The production of H by Groups I and II 2 2 the atmosphere. However, a portion of the organic matter is is thermodynamically unfavorable, requiring the CO - reducing methanogenic Group IV to maintain low concen- trations of H . A variety of other simple compounds serve J. G. Ferry (*) as minor substrates for methanogenesis. Ethanol and Department of Biochemistry and Molecular Biology, secondary alcohols are used as electron donors for the The Pennsylvania State University, reduction of CO to CH . The methyl groups of methanol, 2 4 University Park, PA 16802, USA methylamines, and methylated sulfides are also dismutated e-mail: jgf3@psu.edu 2 Ann Microbiol (2010) 60:1–12 CH (Fischer et al. 1931). However, the direct conversion CO of CO by methanogens (Eq. 1) could not be concluded (A) since both H and acetate appeared and disappeared (F) 2 (Fischer et al. 1932) consistent with non-methanogens as (B) (E) organic the primary anaerobes metabolizing CO to methanogenic CH 4 substrates (Eqs. 2 and 3). matter 4CO þ 2H O ! 3CO þ CH ð1Þ 2 2 4 (C) (D) H + CO 2 2 acetate 4CO þ 4H O ! 4H þ 4CO ð2aÞ 2 2 2 formate Fig. 1 The global carbon cycle. a Fixation of CO into organic matter, b aerobic decomposition of organic matter to CO , c anaerobic 4H þ CO ! 2H O þ CH ð2bÞ 2 2 2 4 decomposition of organic matter to fermentative end products, d anaerobic conversion of fermentative end products to CH , e anaerobic oxidation of CH to CO , f aerobic oxidation of CH to CO 4 2 4 2 4CO þ 2H O ! CH COOH þ 2CO ð3aÞ 2 3 2 to CO and CH . Although CO is present in methanogenic 2 4 environments (Conrad and Seiler 1980), an understanding CH COOH ! CO þ CH ð3bÞ 3 2 4 of CO metabolism by methanogens has lagged behind other substrates. This review chronicles studies on the metabo- In 1933, it was reported (Stephenson and Stickland lism of CO by methanogens from 1931 to the present. For 1933) that a pure culture was able to convert CO to CH by a more comprehensive general understanding of metha- first converting CO to H (Eq. 2ab) that, 14 years later in nogens, the reader is referred to several recent review 1947, was confirmed with resting cell suspensions of a articles describing the ecology, physiology, and biochem- pure culture (Kluyver and Schnellen 1947). Another gap in istry of methanogenesis (Ferry 2008; Ferry and Lessner time had passed before studies on CO metabolism by 2008; Liu and Whitman 2008; Oelgeschlager and Rother methanogens resumed in 1977 and 1984 when it was 2008; Spanheimer and Muller 2008; Thauer et al. 2008). shown that several species remove CO from the gas phase when growing with CO plus H and, for the first time, that 2 2 CO as an energy source two freshwater methanogens are able to grow with CO as the sole energy source, albeit under conditions unable to The first recorded report portending the ability of metha- sustain proliferation in the native environment (Daniels nogens to metabolize CO appeared in 1931 when it was et al. 1977). Only recently, a marine species was shown to demonstrated that sewage sludge converts CO to CO and grow with CO under conditions suitable for proliferation in the native environment via a novel pathway for meth- anogenesis consistent with CO-dependent growth in the native environment (Lessner et al. 2006). Freshwater environments Methanothermobacter thermoautotrophicus (formerly, Methanobacterium thermoautotrophicum strain ΔH) was shown to grow with CO as the sole energy source dis- proportionating CO according to Eq. 1, although the growth rate was only 1% of that with H /CO (Daniels et al. 1977). 2 2 Cell-free extracts showed CO dehydrogenase activity with coenzyme F as the electron acceptor. The activity was reversibly inactivated by cyanide and O consistent with an active-site metal center; however, the enzyme was not purified and studied in greater detail. Methanosarcina barkeri strain MS isolated in 1966 Fig. 2 A freshwater anaerobic microbial food chain. By permission (Ferry 2008) (Bryant and Boone 1987) can be adapted to grow in an Ann Microbiol (2010) 60:1–12 3 atmosphere of 50% CO as the only carbon and energy dizes H and reduces ferredoxin in reaction 1 (Fig. 3)that source (O'Brien et al. 1984). Net H formation is observed supplies electrons for reaction 2. The EchA-F complex is when the headspace CO is greater than 20%. Below this necessary, since the reduction of CO with H (reactions 1 2 2 concentration of CO, H is consumed and the rate of CH plus 2) is thermodynamically unfavorable under standard 2 4 production increases substantially with an increased growth conditions of one atmosphere H (ΔG°′=+16 kJ/mole) and rate approaching a doubling time of 65 h. Methanosarcina considerably more so with the much lower concentrations barkeri strain MS also produces CH and CO with the of H in the environment. This thermodynamic barrier is 4 2 2 growth substrates methanol plus a headspace of 50% CO overcome by reverse electron transport to ferredoxin driven (O'Brien et al. 1984). However, it was reported that H by the electrochemical proton gradient and catalyzed by the accumulated and methanol was not metabolized until the EchA-F hydrogenase complex. However, when cultured CO decreased to below 30% in the headspace, at which with CO, it is presumed that ferredoxin is the electron point H and methanol was rapidly consumed proportional acceptor of the CO dehydrogenase and the oxidation of CO to an increase in the rate of CH formation and growth. coupled to reduction of CO (reaction 2) becomes thermo- 4 2 Based on these characteristics, it was concluded that dynamically favorable (ΔG°′ =−4 kJ/mole) (Ferry and methanogenesis in the freshwater species M. barkeri strain House 2006) obviating the requirement for EchA-F hydrog- MS is inhibited by concentrations of CO greater than ap- enase (Stojanowic and Hedderich 2004). proximately 50% in the headspace, consistent with results obtained with M. thermoautotrophicum (Daniels et al. 1977). Marine environments The authors speculate that CO is metabolized according to Eqs. 2a and b, and the inhibition results from inhibition of Recent investigations of Methanosarcina acetivorans dem- hydrogenase catalyzing the oxidation of H (O'Brien et al. onstrate that this marine isolate displays robust growth with 1984). Although the pathway for reduction of CO to CH CO as the sole carbon and energy source (Lessner et al. 2 4 with H was not investigated, it is presumed to follow the 2006;Rother et al. 2007). The generation time (∼20 h) is less well-studied pathway determined for freshwater methano- than that reported for other methanogens, and growth occurs gens, as shown in Fig. 3 for Methanosarcina species, with with CO concentrations greater than 1 atmosphere. Although one possible exception. With H as the electron donor, the a few methanogens have been shown to utilize CO for EchA-F (Escherichia coli-type hydrogenase) complex oxi- growth, the experiments have routinely utilized concentra- tions above 50% in the atmosphere which is several-fold greater than is likely encountered in the environment. The CO CO CO CO CO CO 2 2 2 2 2 2 lowest concentrations that support growth have not been Fd Fd Fd Fd Fd Fd MF MF MF MF MF MF re re re re re red d d d d d 1 1 + + + + + + reported for any methanogen, nor has a measurement of CO 2H 2H 2H 2H 2H 2H Ec Ec Ec Ec Ec Ech h h h h hA A A A A A- - - - - -F F F F F F 2 2 Fd Fd Fd Fd Fd Fd ox ox ox ox ox ox concentrations in the native habitats. Thus, a role for CO as CHO CHO CHO CHO CHO CHO M M M M M MF F F F F F H H H H H H 2 2 2 2 2 2 the sole carbon and energy source in the native habitats of 3 3 THS THS THS THS THS THSP P P P P Pt t t t t t MF MF MF MF MF MF methanogens is still in question. Nonetheless, M. acetivorans CHO CHO CHO CHO CHO CHO THS THS THS THS THS THSP P P P P Pt t t t t t was isolated from sediments rich in decaying kelp appended + + + + + + H H H H H H ADP ADP ADP AT AT ATP- P- P- 4 4 with flotation bladders containing up to 10% CO as a + + + H H H 12 12 as as ase e e H H H H H H O O O O O O 2 2 2 2 2 2 potential source of CO for growth of this marine isolate AT AT ATP P P + + + CH CH CH CH CH CH THSP THSP THSP THSP THSP THSPt t t t t t (Abbott and Hollenberg 1976; Sowers et al. 1984). F F F F F F H H H H H H 420 420 420 420 420 420 2 2 2 2 2 2 6 6 5 5 A biochemical and quantitative proteomic analysis of H H H H H H 2 2 2 2 2 2 F F F F F F 420 420 420 420 420 420 methanol- and acetate-grown versus CO-grown M. aceti- CH CH CH CH CH CH THS THS THS THS THS THSP P P P P Pt t t t t t 2 2 2 2 2 2 vorans (Lessner et al. 2006) has provided, for the first time, F F F F F F H H H H H H 420 420 420 420 420 420 2 2 2 2 2 2 7 7 8 8 evidence of a pathway for CO-dependent methanogenesis H H H H H H 2 2 2 2 2 2 F F F F F F 42 42 42 42 42 420 0 0 0 0 0 9 9 supporting growth (Fig. 4) that has been confirmed in part CH CH CH CH CH CH THS THS THS THS THS THSP P P P P Pt t t t t t 3 3 3 3 3 3 by a qualitative proteomic analysis (Rother et al. 2007). In + + + + + + HS HS HS HS HS HSCo Co Co Co Co CoM M M M M M M M M M M Mt t t t t trA rA rA rA rA rA-H -H -H -H -H -H 2N 2N 2N 2N 2N 2Na a a a a a the pathway, 3 CO are oxidized to 3 CO that provides CH CH CH CH CH CH SC SC SC SC SC SCoM oM oM oM oM oM 3 3 3 3 3 3 electrons for the subsequent reduction of 1 CO to form a HS HS HS HS HS HSCo Co Co Co Co CoB B B B B B T T T T T TH H H H H HSPt SPt SPt SPt SPt SPt VhoAC VhoAC VhoAC VhoAC VhoAC VhoACG G G G G G + + + + + + 10 10 11 11 methyl group attached to the cofactor tetrahydrosarcinap- 4H 4H 4H 4H 4H 4H Hd Hd Hd Hd Hd Hdr r r r r rD D D D D DE E E E E E CoM CoM CoM CoM CoM CoMS S S S S S S S S S S SC C C C C Co o o o o oB B B B B B terin (THSPt) (reactions 1–6) similar to the pathway of fresh- H H H H H H CH CH CH CH CH CH 2 2 2 2 2 2 4 4 4 4 4 4 water methanogens (Fig. 3) except the electron donor. The methyl group of methyl-THSPt is transferred to coenzyme M (HS-CoM), although the route may be different from Fig. 3 The CO reduction pathway of freshwater Methanosarcina freshwater strains. The methyltransferase that transfers the species utilizing H . Fd Ferredoxin, THSPt tetrahydrosarcinapterin, HS-CoB coenzyme B, HS-CoM coenzyme M, MF methanofuran methyl group from methyl-THSPt to HS-CoM (MtrA-H) in 4 Ann Microbiol (2010) 60:1–12 Fig. 4 Pathway for conversion of CO to acetate and methane 2 C 2 C 2 C 2 CO O O O 2 2 2 2 1a 1a 1a 1a by the marine isolate 2 F 2 F 2 F 2 Fd d d d – re re re red d d d 7C 7C 7C 7CO O O O 7C 7C 7C 7CO O O O + 1 + 1 + 1 + 14 4 4 4e e e e 2 2 2 2 Methanosarcina acetivorans. 2 2 2 2 2 F 2 F 2 F 2 Fd d d d 1b 1b 1b 1b Fd Ferredoxin, THSPt ox ox ox ox 4e 4e 4e 4e + 2F + 2F + 2F + 2Fd d d d 2F 2F 2F 2Fd d d d r r r r o o o o 2 C 2 C 2 C 2 CH H H HO O O O M M M MF F F F tetrahydrosarcinapterin, 1c 1c 1c 1c HS-CoB coenzyme B, HS-CoM – 10e 10e 10e 10e + 5F + 5F + 5F + 5F 5F 5F 5F 5F H H H H 3 3 3 3 42 42 42 420 0 0 0 42 42 42 420 0 0 0 2 2 2 2 coenzyme M, MF methanofuran 2 C 2 C 2 C 2 CH H H HO O O O T T T TH H H HSPt SPt SPt SPt + + + + H H H H 4 4 4 4 CH CH CH CH CO CO CO COO O O OH H H H 3 3 3 3 H H H H O O O O 2 2 2 2 AT AT AT ATP P P P + + 2 C 2 C 2 C 2 CH H H H T T T TH H H HSP SP SP SPtttt 13 13 13 13 AD AD AD ADP P P P 2 F 2 F 2 F 2 F H H H H AD AD AD ADP P P P 42 42 42 420 0 0 0 2 2 2 2 -2 -2 -2 -2 + + + + ATP ATP ATP ATP- - - - CH CH CH CH CO CO CO COP P P PO O O O 5 5 5 5 H H H H 3 3 3 3 4 4 4 4 as as as ase e e e 2 F 2 F 2 F 2 F AT AT AT ATP P P P 42 42 42 420 0 0 0 12 12 12 12 2 C 2 C 2 C 2 CH H H H T T T TH H H HSPt SPt SPt SPt 2 2 2 2 CH CH CH CH CO CO CO COS S S SC C C Co o o oA A A A 3 3 3 3 2 F 2 F 2 F 2 F H H H H 42 42 42 420 0 0 0 2 2 2 2 6 6 6 6 11 11 11 11 2 F 2 F 2 F 2 F 42 42 42 420 0 0 0 CO CO CO CO Co Co Co CoA A A A 2 C 2 C 2 C 2 CH H H H T T T TH H H HSPt SPt SPt SPt 3 3 3 3 7 7 7 7 + + + + HS HS HS HSCo Co Co CoM M M M 8 8 8 8 Mt Mt Mt Mtr r r rA A A A- - - -H H H H 2N 2N 2N 2Na a a a CH CH CH CH SC SC SC SCoM oM oM oM HS HS HS HSCo Co Co CoB B B B 3 3 3 3 F F F F 42 42 42 420 0 0 0 T T T TH H H HSPt SPt SPt SPt Fp Fp Fp Fpo o o oA A A A- - - -O O O O + + + + H H H H 10 10 10 10 Hd Hd Hd HdrDE rDE rDE rDE 9 9 9 9 F F F F H H H H 42 42 42 420 0 0 0 2 2 2 2 C C C Co o o oM M M MS S S S SC SC SC SCoB oB oB oB CH CH CH CH 4 4 4 4 all known methanogenic pathways is down-regulated in are required to accommodate variations in the energy avail- CO-grown M. acetivorans versus methanol- and acetate- able to pump sodium. Thus, at low environmental CO con- grown cells (Lessner et al. 2006) suggesting a reduced centrations, the available energy is low and the transfer from involvement of MtrA-H in the pathway (reaction 8, Fig. 4). methyl-THSPt to HS-CoM would necessarily shift more The MtrA-H complex is membrane-bound and couples the towards the sodium-independent pathway. With increased methyl transfer reaction to translocation of sodium from the CO levels and greater available energy, methyl transfer cytoplasm across the membrane to the periplasm, forming a would shift to the sodium-pumping MtrA-H complex to opti- gradient (Gottschalk and Thauer 2001). Indeed, the sodium mize the thermodynamic efficiency. Under laboratory con- requirement for methanogenesis is thought to originate from ditions where cells are routinely cultured with greater than this enzyme, and the sodium gradient is postulated to drive 0.5 atmosphere of CO, the postulated soluble sodium- energy-requiring reactions (Gottschalk and Thauer 2001). independent route may be dispensable. The mechanism for Three homologs annotated as putative corrinoid-containing providing electrons for reductive demethylation of CH -S- proteins are up-regulated in CO-grown versus methanol- and CoM to CH is a prominent characteristic of the proposed acetate-grown M. acetivorans consistent with a role during pathway for conversion of CO to CH in M. acetivorans that growth with CO (Rother et al. 2007). Corrinoid cofactors further distinguishes it from the well-characterized CO bind the methyl group in a variety of methyltransferases from reduction pathway of freshwater methanogens. In freshwater methanogens including the MtrA-H complex (Gottschalk species of Methanosarcina, HS-CoB donates electrons for and Thauer 2001); thus, it has been postulated (Lessner et al. the reductive demethylation of CH -S-CoM to CH cata- 3 4 2006) that the up-regulated corrinoid proteins participate in lyzed by methylreductase (reaction 10, Fig. 3). The hetero- transfer of the methyl group from methyl-THSPt to HS-CoM disulfide CoM-S-S-CoB is also a product of reaction 10 that via a soluble sodium-independent pathway (reaction 7, is reduced to the active sulfhydryl forms of the cofactors Fig. 4) distinct from MtrA-H. In fact, one of the homologs catalyzed by the VhoACG/HdrDE complex (reaction 11, overproduced in Escherichia coli (MA4384) catalyzes trans- Fig. 3). H is oxidized by the VhoACG hydrogenase with fer of the methyl group from methyl-THSPt to HS-CoM transfer of electrons to the heterodisulfide reductase HdrDE (unpublished) supporting the proposal. The proposal does that is coupled to formation of an electrochemical potential not rule out that both pathways may function simultaneously. that drives ATP synthesis (reaction 12, Fig. 3). On the other Indeed, it is conceivable that both sodium-dependent MtrA- hand, the proteomic and biochemical evidence indicates that H and the postulated soluble sodium-independent pathway HdrDE and the FpoA-O complex (F H dehydrogenase) 420 2 Ann Microbiol (2010) 60:1–12 5 participate in the transfer of electrons to the heterodisulfide acetyl-CoA is further converted to acetate catalyzed by in M. acetivorans (reaction 10, Fig. 4). HdrDE and the phosphotransacetylase and acetate kinase (reactions 12 and FpoA-O complex also function in the pathway of methanol 13) with the synthesis of ATP. Thus, it appears that ATP is conversion to methane in Methanosarcina species (Ferry synthesized via both substrate level and chemiosmotic and Kastead 2007), including M. acetivorans (Li et al. 2007), mechanisms. The CdhA-E complexes from freshwater wherein methanophenazine mediates electron transfer be- Methanosarcina species have CO dehydrogenase activity tween FpoA-O and HdrDE. Methanophenazine is a quinone- with ferredoxin as the electron acceptor (Terlesky and Ferry like compound that translocates protons from the cytoplasm 1988b), as does the M. acetivorans enzyme (unpublished to the periplasm forming an electrochemical potential that results) consistent with the CdhA-E also functioning in drives ATP synthesis. Additionally, FpoA-O pumps protons steps 1a and 1b (Fig. 4). Quantitative RTPCR analysis contributing to the electrochemical potential in methanol- indicates modest up-regulation of two genes annotated as grown Methanosarcina species. Thus, it is postulated encoding CooS in CO-grown versus acetate-grown cells (Lessner et al. 2006) that the FpoA-O/HdrDE complex (Lessner et al. 2006). CooS is a CO dehydrogenase first contributes to the electrochemical potential that drives ATP described in Rhodospirillum rubrum (32) that also reduces synthesis in CO-grown M. acetivorans (Fig. 4). The ferredoxin; thus, the putative CooS in M. acetivorans is mechanism for the formation of formate is not known. also a candidate for catalyzing reactions 1a and 1b. Pheno- The products acetate and formate are yet another feature typic analyses of mutant strains of M. acetivorans deleted of the pathway in M. acetivorans that contrasts with M. of the cooS genes suggest they contribute to but are not barkeri (Rother and Metcalf 2004; Lessner et al. 2006; required for CO-dependent growth (Rother et al. 2007). Oelgeschlager and Rother 2009). Quantitative proteomic There are no known enzymes in M. acetivorans responsible analyses (Lessner et al. 2006) suggest a pathway in which for reactions leading from CO to coenzyme F . CO dehydrogenase/acetyl-CoA synthase (CdhA-E) cata- Notably, H is not an intermediate in the pathway for lyzes synthesis of acetyl-CoA from the methyl group of CO conversion to CH in M. acetivorans (Lessner et al. methyl-THSPt, CO, and CoA-SH (reaction 11, Fig. 5). The 2006) in contrast to freshwater M. barkeri (Fig. 3). Indeed, cell membr cell membra ane ne AT ATP P Co CoA A- -S SH H Ac Ack k Pta Pta -2 -2 CH CH COO¯ COO¯ MA MA3 36 60 06 6 CH CH COPO COPO M MA A3607 3607 CH CH CO COS SC CoA oA 3 3 4 4 3 3 3 3 Fd Fd THM THMP PT T o o ADP ADP -2 -2 HPO HPO 4 4 CoM- CoM-S SH H HCO HCO ¯ ¯ 3 3 Cd Cdh h Ca Cam m Mt Mtr r M MA A1 10 01 11- 1-16 16 C Co oA-SH A-SH CH CH -THM -THMPT PT 3 3 M MA A2536 2536 M MA A3860-65 3860-65 + + M MA A02 0269-76 69-76 Na Na CO CO CH CH -S- -S-Co CoM M 3 3 2 2 Ma Ma-Rn -Rnf f Co CoB B- -S SH H Fd Fd THM THMP PT T r r + + + + Na Na /H /H M MA A0 0658-65 658-65 Fd Fd o o M MA A454 4546-47 6-47 Mc Mcr r M MA A45 4550 50 + + MP MPH MP MPH H H 2 2 CH CH 4 4 CoM- CoM-S-S- S-S-Co CoB B M MA A0687-88 0687-88 + + - -2 2 AD ADP + P + H HP PO O H H AT ATP P 4 4 CoM-SH + CoM-SH + CoB-S CoB-SH H Hdr Hdr- -DE DE + + Na Na M MA A4152-60 4152-60 At Atp p Mr Mrp p M MA A456 4566-72 6-72 + + H H Fig. 5 Pathway for the conversion of acetate to methane by anhydrase, Ma-Rnf M. acetivorans Rnf, MP methanophenazine, Hdr- Methanosarcina acetivorans. Ack Acetate kinase, Pta phosphotransa- DE heterodisulfide reductase, Mrp multiple resistance/pH regulation + + + cetylase, CoA-SH coenzyme A, THMPT tetrahydromethanopterin, Fd Na /H antiporter, Atp H -transporting ATP synthase. Carbon transfer reduced ferredoxin, Fd oxidized ferredoxin, Cdh CO dehydrogenase/ reactions are catalyzed by the enzymes shown in blue. Electron acetyl-CoA synthase, CoM-SH coenzyme M, Mtr methyl-THMPT: transfer reactions are catalyzed by enzymes shown in green.By CoM-SH methyltransferase, CoB-SH coenzyme B, Cam carbonic permission (Li et al. 2006) 6 Ann Microbiol (2010) 60:1–12 M. acetivorans does not encode a functional EchA-F Raybuck et al. 1991). Although a two-subunit CO dehy- hydrogenase (Galagan et al. 2002). Thus, the observation drogenase has been purified and characterized from an that M. acetivorans is more tolerant of high concentrations acetate-utilizing species from the genus Methanosaeta of CO compared with M. barkeri is consistent with the (formerly Methanothrix) (Boone and Kamagata 1998) that postulated CO inhibition of hydrogenase in M. barkeri catalyzes the exchange of CO with the carbonyl group of (O'Brien et al. 1984). The question then arises why M. acetyl-CoA (Jetten et al. 1989, 1991a, b; Eggen et al. 1991), acetivorans evolved a pathway for utilization of CO inde- the majority of mechanistic investigations have been with pendent of H . One possibility is that, in marine environ- the five-subunit CdhA-E complexes from the acetate- ments, sulfate-reducing microbes outcompete methanogens utilizing species Methanosarcina thermophila and M. for H (Zinder 1993), presenting the possibility that if H barkeri described here. 2 2 were an intermediate it could be lost to sulfate reducers. The subunits of the CdhABCDE complex from Meth- Another possibility is that Methanosarcina species are at a anosarcina species are correspondingly designated αεβγδ disadvantage utilizing H in the marine environment where and encoded in operons arranged in the order cdhABCDE the concentrations are kept low by sulfate-reducers and, (Maupin-Furlow and Ferry 1996a, b; Grahame et al. 2005). therefore, have not evolved hydrogenases. Indeed, it is Use of a plasmid-mediated lacZ fusion reporter system has noted that Methanosarcina species have considerably revealed that the operon encoding the complex of M. higher threshold concentrations for H than do obligate thermophila (Grahame et al. 2005) is 54-fold down- CO -reducing species (Thauer et al. 2008). regulated in M. acetivorans grown on methanol compared The formation of methanethiol and dimethylsulfide to acetate, consistent with a role for the complex in the during growth of M. acetivorans on CO was recently re- pathway for utilization of acetate (Apolinario et al. 2005). ported (Moran et al. 2008). The authors speculate that The results confirm an earlier report on the regulation of methyl-groups generated in the CO-dependent reduction cdhA in M. thermophila examined by northern blotting of CO are transferred to sulfide present in the growth (Sowers et al. 1993). Unlike the genome of M. thermophila medium and the process could be coupled to energy con- that harbors only one operon encoding CdhA-E (Grahame servation. However, the rate of dimethylsulfide formation et al. 2005), the genomes of M. acetivorans (Galagan et al. from CO is only 1–2% of that of CH formation suggesting 2002)and Methanosarcina mazei (Deppenmeier et al. the process is not of major consequence in the energy 2002) contain duplicate cdh operons with greater than yielding metabolism during growth on CO (Oelgeschlager 95% identity, raising the question as to whether both are and Rother 2009). transcribed during growth on acetate. A proteomic analysis of M. acetivorans indicates that one complex is expressed at least 16-fold over the other (Li et al. 2006, 2007), CO dehydrogenase/Acetyl-COA synthase consistent with the predominance of a single Cdh complex purified from acetate-grown M. mazei strain Go1 (formerly Conversion of acetate to CH Methanosarcina frisia) (Maestrojuan et al. 1992), although the organism encodes duplicate cdh operons with high The five-subunit CdhA-E complex is central to pathways identity (Eggen et al. 1996). However, in apparent contrast for conversion of acetate to methane (Kohler and Zehnder to this result, global transcriptional profiling of M. mazei 1984; Krzycki and Zeikus 1984; Nelson and Ferry 1984; Go1 suggests both cdh operons are transcribed at approx- Krzycki et al. 1985; Bott et al. 1986; Terlesky et al. 1986; imately equal levels (Hovey et al. 2005). Bott and Thauer 1987; Zinder and Anguish 1992; Gokhale The complex from Methanosarcina species is resolvable et al. 1993; Kemner and Zeikus 1994) as illustrated in into three components (Abbanat and Ferry 1991; Grahame Fig. 5 for M. acetivorans (Li et al. 2006). The complex and Demoll 1996; Kocsis et al. 1999). The CdhAE com- functions in the pathway to cleave the C-C and C-S bonds ponent contains the α and ε subunits, the CdhDE compo- of acetyl-CoA yielding a carbonyl group that is oxidized nent contains the γ and δ subunits, and the CdhC to CO and a methyl group that is transferred to THSPt component contains the β subunit. The CdhAE component (Terlesky et al. 1987; Fischer and Thauer 1989; Abbanat has CO dehydrogenase activity with ferredoxin as the and Ferry 1990; Raybuck et al. 1991; Grahame 1991, 1993; electron acceptor (Terlesky and Ferry 1988a, b; Fischer Grahame and Demoll 1995; Grahame et al. 1996; Bhaskar and Thauer 1990). The crystal structure (Gong et al. 2008) et al. 1998). In subsequent steps of the pathway, electrons of the M. barkeri CdhAE (Fig. 6)shows a α ε 2 2 derived from the oxidation of the carbonyl group are configuration with the α subunit harboring a Ni-Fe-S C transferred to the methyl group producing CH . The com- cluster and four Fe S clusters (B, D, E, and F in Fig. 6) 4 4 4 plex also catalyzes the synthesis of acetyl-CoA from a consistent with earlier EPR spectroscopic investigations methyl donor, CO and CoA-SH (Abbanat and Ferry 1990; (Krzycki et al. 1989). The C, B, and D clusters are posi- Ann Microbiol (2010) 60:1–12 7 Fig. 6 The Methanosarcina barkeri α ε CdhAE component.a Side as spheres, with iron atoms in purple, nickel atoms in blue, and the 2 2 view shown as ribbons with the α-subunits colored in cyan and green remaining atoms in CPK. b Side view of the metal clusters. By and the ε-subunits in tan and orange. Metal cluster atoms are shown permission (Gong et al. 2008) tioned similarly in the crystal structure of the homolog electron acceptor for the α ε component (Grahame and 2 2 from Moorella thermoacetica (Ragsdale 2007), an acetate- Stadtman 1987) suggesting a potential role for the ε-subunit producing species of the Bacteria domain. However, the E in FAD-mediated CO oxidation during growth on CO. and F clusters are unique to CdhAE and proposed to The CdhDE component transfers the methyl group of function in electron transport from the active site C cluster acetyl-CoA to THSPt and contains an iron-sulfur center and to ferredoxin. The C cluster (Fig. 7) resembles the M. corrinoid cofactors that transfer the methyl group during thermoacetica structure comprised of a pseudocubane catalysis (Grahame 1991, 1993; Jablonski et al. 1993; NiFe S cluster bridged to an exogeneous iron atom. Ad- Maupin-Furlow and Ferry 1996a). Analyses of the CdhD 3 4 ditional electron density provides support for coupling and CdhE subunits overproduced independently in E. coli between CO bound to the nickel and H O/OH bound to the indicates that the iron-sulfur center is located in the CdhE exogenous iron in the critical C = O bond-forming step subunit and that both subunits bind a corrinoid cofactor. leading to CO (Fig. 7). The structure also identifies a gas Which of the two subunits interact with THSPt has not been channel extending from the C cluster to the surface of the determined. A structure has not been reported for either protein in a direction presumed to interact with the CdhC subunit; however, EPR analyses of the intact CdhDE com- component. Structural and sequence alignments indicate ponent (Jablonski et al. 1993) indicate that the corrinoids that the ε-subunit is a member of the DHS-like NAD/FAD- are maintained in the base-off state with a E ′ of -486 mV 2+/1+ 2+ binding domain clan. Indeed, the structure reveals a cavity for the Co couple that facilitates reduction of Co by able to accommodate an FAD or NAD cofactor located approximately 12 kcal/mol relative to base-on cobamides. 1+ between the interface of the α- and ε-subunits and near the Reduction to the Co redox state is a requirement for DFe -S cluster suggesting a potential role in electron methylation of corrinoid cofactors. The EPR analysis also 4 4 2+/1+ transfer. The authors of the structure note that FAD is an identified a [4Fe-4S] cluster with an E ' of -502 mV, Fig. 7 Proposed coupling of the CO and H O species in the C cluster of the Methanosarcina barkeri CdhAE component 2 8 Ann Microbiol (2010) 60:1–12 redox dependence of acetyl-CoA synthesis by the CdhC component shows one-electron Nernst behavior and that two electrons are required for reductive activation of the active site Ni in the process of forming the enzyme-acetyl intermediate (Gencic and Grahame 2008). Autotrophy Many methanogens grow autotrophically with CO as sole source of carbon (Ferry and Kastead 2007). For example, Fig. 8 Structure of the A cluster from Moorella thermoacetica.By M. thermoautotrophicum is able to grow with H and CO 2 2 permission (Ragsdale 2007) in a simple mineral salts medium (Zeikus and Wolfe 1972; Schonheit et al. 1980) synthesizing acetyl-CoA (Stupperich 2+/1+ approximately isopotential with the Co couple suggest- and Fuchs 1983; Stupperich et al. 1983; Ruhlemann et al. 2+ ing the cluster is likely involved in reducing Co . 1985), the starting point for synthesis of all cellular com- The CdhC component contains an A cluster that is the ponents (Fuchs and Stupperich 1980). Acetyl-CoA is proposed site of acetyl-CoA cleavage or synthesis (Gra- synthesized via methyl-tetrahydromethanopterin (methyl- hame and Demoll 1996; Murakami and Ragsdale 2000; THMPt) providing the methyl group and CO the carboxyl Gencic and Grahame 2003; Funk et al. 2004). Although a group (Lange and Fuchs 1985, 1987). The CO is generated structure is not available, a variety of spectroscopic studies from CO and H in an energy-dependent reaction (Conrad 2 2 suggest the A cluster is comprised of an Fe S center and Thauer 1983; Eikmanns et al. 1985) and methyl- 4 4 bridged to a binuclear Ni-Ni site (Gu et al. 2003; Funk et al. THMPt via steps in the pathway for CO reduction to 2004) similar in structure (Fig. 8) to that proposed for the methane (Lange and Fuchs 1987) similar to that shown in homolog from M. thermoacetica (Ragsdale 2007). In Fig. 3 (reactions 2–8). The THMPt in M. thermoautotro- addition to CO oxidation, the CdhAE component is phicum is an analog of THSPt found in Methanosarcina required for acetyl-CoA cleavage or synthesis (Murakami species. Autotrophic methanogens such as M. thermoauto- and Ragsdale 2000). The authors propose a mechanism trophicum have CO dehydrogenase activity whereas het- for acetyl-CoA synthesis or cleavage involving an intra- erotrophic methanogens lack CO dehydrogenase and molecular electron transfer reaction between the C cluster require acetate as the carbon source that is converted to in the CdhAE component and cluster A in the CdhC acetyl-CoA (Bott et al. 1985). Indeed, the genome of M. component for each catalytic cycle thereby maintaining Ni thermoautotrophicum contains a gene cluster encoding in cluster A in the catalytically active Ni(I) redox state. The subunits with high identity to subunits of the CdhA-E ab CO CO CO CO CO CO CO CO CO CO + + + + CH CH CH CH SH SH SH SH + + + + R R R R- - - -SH SH SH SH 2 2 2 2 2 2 3 3 3 3 3 C 3 C 3 C 3 C 3 C 3 CO O O O O O A A A A 2 C 2 C 2 C 2 C 2 C 2 CO O O O O O 2 2 2 2 2 2 CH CH CH CH CO CO CO COS S S S- - - -R R R R 3 3 3 3 CH CH CH CH CH CH -THM -THM -THM -THM -THM -THMP P P P P PT T T T T T 3 3 3 3 3 3 Fe Fe Fe FeS S S S + H + H + H + H CO CO CO CO CO CO 2 2 2 2 2 2 2 2 P P P P iiii B B B B R- R- R- R- R- R-S S S S S SH H H H H H B B B B R-S R-S R-S R-SH H H H R-S R-S R-S R-SH H H H CH CH CH CH CH CH COS COS COS COS COS COS-R -R -R -R -R -R 3 3 3 3 3 3 -2 -2 -2 -2 A A A A CH CH CH CH CO CO CO COP P P PO O O O 3 3 3 3 4 4 4 4 P P P P P P iiiiii C C C C AD AD AD ADP P P P R-S R-S R-S R-S R-S R-SH H H H H H C C C C AT AT AT ATP P P P -2 -2 -2 -2 -2 -2 CH CH CH CH CH CH CO CO CO CO CO COP P P P P PO O O O O O 3 3 3 3 3 3 4 4 4 4 4 4 FeS FeS FeS FeS + H + H + H + H S S S S 2 2 2 2 AD AD AD AD AD ADP P P P P P D D D D AT AT AT AT AT ATP P P P P P CH CH CH CH CO CO CO COOH OH OH OH 3 3 3 3 R- R- R- R-S S S SH H H H C C C C C CH H H H H H COOH COOH COOH COOH COOH COOH Fig. 9 The proposed cyclic energy-conserving pathway that func- phate. a Solid lines indicate the cyclic pathway (steps A–C). The tioned in the primitive (a) and chemoautotrophic cell independent of broken arrow indicates the priming reaction that is not part of the surface-catalyzed reactions (b) (Ferry and House 2006). The stippled cyclic pathway. b THMPT Tetrahydromethanopterin. By permission areas represent the lipid membranes. Pi represents inorganic phos- (Ferry and House 2006) Ann Microbiol (2010) 60:1–12 9 complex of acetate-utilizing Methanosarcina species (The thioesters from CO and a methyl group derived from co- Comprehensive Microbial Resource. J. Craig Venter Institute, evolution of enzymes leading to the reduction of CO to the http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi). Fur- methyl level that is the extant Wood-Ljungdahl pathway thermore, a CO dehydrogenase was purified from the (Fig. 9b). Thus, it is proposed that the Wood-Ljungdahl autotrophic methanogen Methanococcus vannielii that con- pathway first evolved as an energy-yielding pathway that tains nickel and is composed of subunits with molecular was adapted for biosynthesis of cell carbon, and evolution weights similar to the CdhAE component (DeMoll et al. of the first autotroph, once the pre-biotic organic soup was 1987). These results are consistent with synthesis of acetyl- exhausted for incorporation into cell material (Ferry and CoA in autotrophic methanogens catalyzed by a CO House 2006). It is further proposed that the pathway shown dehydrogenase with properties similar to the CdhA-E in Fig. 9b was the foundation for evolution of methanogenic complex of acetate-utilizing species. pathways. Evolution Conclusions It is proposed that Earth’s atmosphere at the time of the origin of life contained significant amounts of CO (Holland Methanogens play an essential role in the global carbon 1984; Kasting 1990; Kharecha et al. 2005) and that the cycle by serving as terminal organisms in anaerobic micro- CdhA-E complex of methanogens and homologs in the bial food chains that convert complex organic matter to Bacteria domain evolved early during the origin and evo- CH and CO . They utilize simple molecules for growth 4 2 lution of primitive life forms (Lindahl and Chang 2001). In that include acetate and several one-carbon compounds. the chemoautotrophic origin of life (Russell et al. 1988; Although first reported in 1931, comparatively little is Wachtershauser 1988; Russell and Hall 1997), the primary known today of the ecology, physiology, and biochemistry pre-biotic initiation reaction for carbon fixation was the of CO utilization by methanogens. Only a few species surface-catalyzed synthesis of an acetate thioester from are reported to metabolize CO, and it is not yet known CO and H S driven by a geochemical energy source. A unequivocally if CO is a viable energy source for prominent feature of the chemoautotrophic theory is the methanogens in diverse environments. However, recent evolution of biological CO fixation pathways of which investigations of M. acetivorans suggest this methanogen one is a primitive form of the extant Wood–Ljungdahl utilizes CO as a growth substrate in nature converting CO, pathway (Wachtershauser 1997; Pereto et al. 1999; Martin abundant in the native habitat, to CH and acetate via an and Russell 2003; Russell and Martin 2004) in which unusual pathway. CO is also an important intermediate 2CO molecules are reduced to acetyl-CoA implying early during growth of methanogens on acetate and assimilation evolution of primitive forms of enzymes with catalytic of CO for cell carbon. Clearly, more research is warranted capabilities of the CdhA-E complex of methanogens and to determine the extent of CO utilization by methanogens homologs in the Bacteria domain. A role for a primitive in native environments. form of CdhC has been proposed in a modification of the Acknowledgements Research in the laboratory of J.G.F has been chemoautotrophic theory (Ferry and House 2006). In the supported by the NIH, DOE, NSF, and NASA. modified theory, the primitive CdhC catalyzes C-S bond formation (reaction A in Fig. 9a) regenerating the CH COSR thioester from acetate and HS-R that is the energy source References in the first energy-yielding metabolic cycle. Reaction A in Fig. 9a is driven by the conversion of FeS and H Sto pyrite Abbanat DR, Ferry JG (1990) Synthesis of acetyl-CoA by the and H a previously proposed energy source in the carbon monoxide dehydrogenase complex from acetate-grown chemoautotrophic theory (Wachtershauser 1988). The theory Methanosarcina thermophila. 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Published: Feb 2, 2010

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