energy metabolism of methanogenic bacteria

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256 Biochimica et Biophysica Acta, 1018 (1990) 256-259 Elsevier BBAEBC00029 Energy metabolism of methanogenic bacteria Rudolf K. Thauer Laboratorium fiir Mikrobiologie, Fachbereich Biologie, Philipps-Universitiit, Marburg (F..R.G.) (Received 1 May 1990) Key words: Methanogenic bacterium; Archaebacterium; Energy conservation; Acetate fermentation; Hydrogen metabolism; Methanol disproportionation Introduction Methanogenic bacteria are strictly anaerobic archae- bacteria. Most of them can grow on H 2 and CO 2 as the energy source (reaction a). Some ferment acetic acid (reaction b) or methanol (reaction c) to CH4 and CO2. (a) CO 2 + 4 H 2 --~ CH 4 + 2 n20 AG o'= -131 kJ/mol CH 4 (b) CH3COO-+ H +~ CH 4 + CO2 AG O' = -36 kJ/mol CH 4 (c) 4 CH3OH -~ 3 CH 4 + CO 2 + 2 n20 AG O'= -107 kJ/mol CH 4 In methanogenic ecosystems the H 2 partial pressure is generally between 1 and 10 Pa. At these low H 2 concentrations the free energy change associated with reaction a is in the order of -30 kJ/tool. In vivo, approx. 50 kJ/mol are required to drive the synthesis of ATP from ADP and Pi- Thus, under physiological growth conditions in fermentations a and b, less than 1 mol ATP per mol CH 4 can be generated. The following mini-review first describes the reac- tions and enzymes involved in methane formation. The sites of energy conversion are then discussed. Only literature from the last 2 years (1989/90) is cited. For older work the reader is referred to two recent reviews on the subject [1,2]. Thermodynamic data were taken from Refs. 2-4. Methane formation from CO2 The reduction of CO 2 to CH 4 proceeds via carrier- bound Cl-intermediates. Methanofuran (MFR), tetra- hydromethanopterin (H4MPT) and coenzyme M (H-S- CoM) are the three carriers involved (Fig. 1). The sequence of reactions starts with the formation of a Abbreviations: MFR, methanofuran; CHO-MFR, formyl-MFR; H4MPT, tetrahydromethanopterin; H-S-CoM, coenzyme M. Correspondence: It. Thauer, Laboratorium ftir Mikrobiologie, Facli- bereich Biologie,Karl-von-Frisch Strasse, Philipps-Universit~it, D-3550 Marburg/Lahn, F.R.G. N-substituted carbamate from CO 2 and MFR followed by its reduction to formyl-MFR (CHO-MFR) [1,2]. (d) CO 2 + MFR --* CO2-MFR- + H + AG O'= + 8.1 kJ/mol (e) CO2-MFR-+ 2 [H] + H +-~ CHO-MFR + H20 E °' = - 456 mV (Reaction e; AG o'= +7.9 kJ/mol with H 2 as re- ductant, [HI = not yet identified electron donor). Reaction d proceeds in the absence of any enzyme. Whether the spontaneous rate is sufficient to account for the in vivo rates is not yet known. Reaction e is catalyzed by formyl-MFR dehydrogenase, which is a molybdenum-iron-sulfur protein. The enzyme contains non-covalently bound a pterin which, after oxidation with iodine or permanganate, shows fluorescence spec- tra identical to those obtained for the pterin cofactor from milk xanthine oxidase. The molybdenum to pterin ratio is 1 : 1 [5-8]. The physiological electron donor of CHO-MFR dehydrogenase is not yet known. The redox potential (E °') of the CO2/CHO-MFR couple is -497 mV, that of the H +/H 2 couple is -414 mV. CO 2 reduction to CHO-MFR under standard con- ditions is thus an endergonic reaction. This is even more so when the H E partial pressure is lower than 105 Pa. At a pressure of, e.g., 10 Pa, the redox potential (E') of the H+/H2 couple is only -300 mV. The electrons from H 2 must therefore be transported uphill approx. 200 mV before a reduction of CO 2 to CHO-MFR be- comes thermodynamically feasible. The electron carriers involved in this reversed electron transport have not been elucidated. Formyl-MFR is reduced to the oxidation state of formaldehyde via NS-formyl-H4MPT (CHO-H4MPT) and NS,Nl°-methenyl-H4MPT + (CH-H4MPT +) as in- termediates yielding NS,Nl°-methylene-H4MPT (CH2= H4MPT ) [1,2]. (For structures see Fig. 2.) (f) CHO-MFR + H4MPT --~ CHO-H4MPT + MFR AG O' = - 4.4 kJ/mol (g) CHO-HaMPT + H+~ CH=H4MPT++ H20 AG o' = - 4.6 kJ/mol (h) CH=H4MPT++ H 2 ~ CH2=H4MPT + H ÷ AG o' = - 5.5 kJ/mol 0005-2728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Page 1: Energy metabolism of methanogenic bacteria

256 Biochimica et Biophysica Acta, 1018 (1990) 256-259 Elsevier

BBAEBC00029

Energy metabolism of methanogenic bacteria Rudolf K. Thauer

Laboratorium fiir Mikrobiologie, Fachbereich Biologie, Philipps-Universitiit, Marburg (F..R.G.)

(Received 1 May 1990)

Key words: Methanogenic bacterium; Archaebacterium; Energy conservation; Acetate fermentation; Hydrogen metabolism; Methanol disproportionation

Introduction

Methanogenic bacteria are strictly anaerobic archae- bacteria. Most of them can grow on H 2 and C O 2 as the energy source (reaction a). Some ferment acetic acid (reaction b) or methanol (reaction c) to CH4 and CO2. (a) CO 2 + 4 H 2 --~ CH 4 + 2 n 2 0

AG o ' = - 1 3 1 k J /mo l CH 4 (b) CH3C OO-+ H + ~ CH 4 + CO2

AG O' = - 3 6 k J /m o l CH 4 (c) 4 CH3OH -~ 3 CH 4 + CO 2 + 2 n 2 0

AG O'= - 1 0 7 k J /mo l CH 4 In methanogenic ecosystems the H 2 partial pressure

is generally between 1 and 10 Pa. At these low H 2 concentrations the free energy change associated with reaction a is in the order of - 3 0 k J / tool . In vivo, approx. 50 kJ /mo l are required to drive the synthesis of ATP from ADP and Pi- Thus, under physiological growth conditions in fermentations a and b, less than 1 mol ATP per mol CH 4 can be generated.

The following mini-review first describes the reac- tions and enzymes involved in methane formation. The sites of energy conversion are then discussed. Only literature from the last 2 years (1989/90) is cited. For older work the reader is referred to two recent reviews on the subject [1,2]. Thermodynamic data were taken from Refs. 2-4.

Methane formation from CO 2 The reduction of CO 2 to CH 4 proceeds via carrier-

bound Cl-intermediates. Methanofuran (MFR), tetra- hydromethanopterin (H4MPT) and coenzyme M (H-S- CoM) are the three carriers involved (Fig. 1). The sequence of reactions starts with the formation of a

Abbreviations: MFR, methanofuran; CHO-MFR, formyl-MFR; H4MPT, tetrahydromethanopterin; H-S-CoM, coenzyme M.

Correspondence: It. Thauer, Laboratorium ftir Mikrobiologie, Facli- bereich Biologie, Karl-von-Frisch Strasse, Philipps-Universit~it, D-3550 Marburg/Lahn, F.R.G.

N-substituted carbamate from C O 2 and MF R followed by its reduction to formyl-MFR (CHO-MFR) [1,2]. (d) CO 2 + M F R --* CO2-MFR- + H +

AG O'= + 8.1 k J / m o l (e) CO 2 -MF R-+ 2 [H] + H +-~ CHO-MFR + H20

E °' = - 456 mV (Reaction e; AG o ' = +7.9 k J /m o l with H 2 as re- ductant, [HI = not yet identified electron donor).

Reaction d proceeds in the absence of any enzyme. Whether the spontaneous rate is sufficient to account for the in vivo rates is not yet known. Reaction e is catalyzed by formyl-MFR dehydrogenase, which is a molybdenum-iron-sulfur protein. The enzyme contains non-covalently bound a pterin which, after oxidation with iodine or permanganate, shows fluorescence spec- tra identical to those obtained for the pterin cofactor from milk xanthine oxidase. The molybdenum to pterin ratio is 1 : 1 [5-8]. The physiological electron donor of CHO-MFR dehydrogenase is not yet known.

The redox potential ( E ° ' ) of the CO2/CHO-MFR couple is - 4 9 7 mV, that of the H + / H 2 couple is - 4 1 4 mV. CO 2 reduction to CHO-MFR under standard con- ditions is thus an endergonic reaction. This is even more so when the H E partial pressure is lower than 105 Pa. At a pressure of, e.g., 10 Pa, the redox potential (E ' ) of the H + / H 2 couple is only - 3 0 0 mV. The electrons from H 2 must therefore be transported uphill approx. 200 mV before a reduction of CO 2 to CHO-MFR be- comes thermodynamically feasible. The electron carriers involved in this reversed electron transport have not been elucidated.

Formyl-MFR is reduced to the oxidation state of formaldehyde via NS-formyl-H4MPT (CHO-H4MPT) and NS,Nl°-methenyl-H4MPT + ( C H - H 4 M P T +) as in- termediates yielding NS,Nl°-methylene-H4MPT (CH2= H4MPT ) [1,2]. (For structures see Fig. 2.) (f) CHO-MFR + H4MPT --~ CHO-H4MPT + MFR

AG O' = - 4.4 k J /m o l (g) CHO-HaMPT + H + ~ C H = H 4 M P T + + H20

AG o' = - 4.6 k J / m o l (h) C H = H 4 M P T + + H 2 ~ CH2=H4MPT + H ÷

AG o' = - 5.5 k J /m o l

0005-2728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

Page 2: Energy metabolism of methanogenic bacteria

257

H 0 N ~ R ^

R 1 H H I ~u

NH; H 2 N ~ N L N H

H/S~SO;

Methonof uran Te t ra hydromethanopter in Coenzyme M

( MFR ) ( H4MPT ) ( H - S - C o M )

Fig. 1. C l - u n i t carriers involved in methanogenesis from CO 2. Structures of methanofuran (MFR), tetrahydromethanopterin (H4MPT) and coenzyme M (H-S-CoM). For the structures of R 1 and R 2 see Refs. 1 and 2.

The three enzymes mediating these reactions have been characterized. Interestingly, in Methanobacterium spe- cies the reduction of CH=H4MPT to CH2=HaMPT ( E ° ' = - 3 8 6 mV) with H 2 is mediated by only one protein exhibiting both CH2=H4MPT dehydrogenase and hydrogenase activity [9]. In Methanosarcina barkeri

two enzyme are involved, a coenzyme F420-dependent CH2=H4MPT dehydrogenase and a coenzyme F420-re- ducing hydrogenase. (For the structure of coenzyme F420 see Fig. 3).

CH2=H4MPT formed in reaction h is reduced to NS-methyl-H4MPT (CH3-H4MPT) (Fig. 2) (reaction i), which reacts with H-S-CoM to yield methylcoenzyme M (CHa-S-CoM) (reaction j) [1,2].

(i) CH2=H4MPT + F420H 2 ---* CH3-H4MPT + F420 AG O' = - 5.2 k J /mo l

(j) CH3-H4MPT + H-S-CoM ~ CH3-S-CoM + H4MPT AG o' = -- 29.7 k J / t oo l

(AG O' of reaction j is assumed to be identical to that of CH3-tetrahydrofolate + homocysteine conversion to te- trahydrofolate + methionine).

The reduction of CHE=HnMPT to CH3-H4MPT (E ° ' = - 3 2 3 mV) with coenzyme F42on 2 (E 0 ' = - 3 5 0

0 % _ . H H 0 ~H N - - -R 0 C N~- -R

HN N HN 5

N 5 - f o r m y l - H4MPT

(CHO - H4MPT)

N 5 , N 1° - rne theny l - H4MPT

(CH = H4MPT )

H H H 0 HC N - - R 0 HCH NlO'-- R

N 5 , N 10 - m e t h y l e n e - H4MPT N 5 - methy l - H4MPT

(CH 2 = H4MPT) (CH 3 - H4MPT)

Fig. 2. Structures of the tetrahydromethanopterin (H4MPT) bound Crintermediates. For structure of R see Refs. 1 and 2.

mV) is catalyzed by methylene-H4MPT reductase. The enzyme differs from methylene-tetrahydrofolate re- ductases in that a flavin prosthetic group is lacking [10-12]. Reaction j is catalyzed by methyl transferase. This is the only enzyme involved in CO 2 reduction to CH 4 which has, until now, not yet been purified. Evi- dence is available that the enzyme system contains a corrinoid protein which accepts the methyl group before it is further transferred to H-S-CoM [2,13]. In the test tube reaction j proceeds irreversibly. In vivo the reverse reaction is probably possible at the expenditure of en- ergy.

The final step in methanogenesis, the reduction of CH3-S-CoM to CH4, involves the reactions k and 1. The reduction of CH3-S-CoM with H-S-HTP (N-7- mercaptoheptanoylthreonine phosphate) (Fig. 3) is catalyzed by CH3-S-CoM reductase [1,14,15] and the reduction of CoM-S-S-HTP by heterodisulfide re- ductase [16]. (k) CH3-S-CoM + H-S-HTP ---, CH 4 + CoM-S-S-HTP

AG O' -- - 45 k J / m o l (1) CoM-S-S-HTP + 2 [H] - , H-S-CoM + H-S-HTP

E °' -- - 210 mV (Reaction 1; AG o' ---- - 4 0 k J / t o o l with H 2 as reductant, [HI = not yet identified electron donor).

In vivo both reactions proceed irreversibly. The CH 3- S-CoM reductase contains as prosthetic group a nickel porphinoid named coenzyme F430 (Fig. 4) [17]. In the active enzyme the nickel is probably in the monovalent oxidation state [18,19]. The CoM-S-S-HTP heterodi- sulfide reductase is an FAD-containing iron-sulfur pro- tein [20]. The physiological electron donor has not yet been elucidated; it is not F42oH 2.

Methanogenic bacteria generally contain a coenzyme F42o-reducing hydrogenase [21-23] and a viologen dye- reducing hydrogenase [24,25] catalyzing reactions m and n, respectively. The physiological electron acceptor of the latter enzyme is not known. Both hydrogenases are nickel-iron-sulfur proteins. The coenzyme F420-reducing hydrogenase additionally contains FAD [21-25]. (m) H E + F420 ---) F420H 2 AG O' = - 12 kJ /mo l (n) n 2 ~ 2 [H] E °' = - 4 1 4 mY ([H] = not yet identified reduced electron donor) In reactions m and n the reducing equivalents are provided for reactions e, i, and 1. CO 2 reduction to

Page 3: Energy metabolism of methanogenic bacteria

258

O H

I R

O H H H

H ~. N COO -

I I H ~ H R (~) __0 ~ "CH 3

Oxidized Reduced

Coenzyme F420 N - 7- Ivlercaptoheptanoyl - t hreoninephosphate

(H - S- HTP)

Fig. 3. Electron carriers involved in methanogenesis from CO 2. Structures of coenzyme F420 (oxidized and reduced form) and of N-7-mercaptohep- tanoylthreonine phosphate (H-S-HTP). For the structures of R see Ref. 1.

formylmethanofuran with H 2 (reactions e plus n) and CoM-S-S-HTP reduction with H 2 (reaction 1 plus n) probably involves an electron transport chain, the com- ponents of which remain to be elucidated.

Reactions d - m add up to reaction a and their free energy changes (AG ° ') add up to - 130.4 kJ /mol , which is by only 0.6 k J /mo l different from that calculated for reaction a. The free energy changes (AG ° ') given for the individual reactions can therefore be considered as being reasonable.

Methane formation from acetate The metabolism of acetate starts with its activation

to acetyl-CoA (reactions o and p) [26-28]. The methyl group of acetyl-CoA is then transferred to H4MPT in a reaction catalyzed by the carbon monoxide dehydro- genase complex (reaction q) [29,30]. The complex is composed of carbon monoxide dehydrogenase, a cor- rinoid protein and a methyl transferase. The carbon monoxide dehydrogenase, which is a nickel-iron-sulfur protein, probably catalyzes the cleavage of acetyl-CoA into an enzyme bound methyl group, CO 2 and two reducing equivalents. The corrinoid protein accepts the

HOOC o

HOOC COOH

H O O C L.,, , . . , . , / ,~ O L ~ i COOH

COOH

Fig. 4. Structure of coenzyme F430, the prosthetic group of methyl- coenzyrne M reductase catalyzing reaction k.

methyl group from carbon monoxide dehydrogenase, and the transferase mediates methyl transfer from the corrinoid protein to H4MPT. Evidence is available that the physiological electron acceptor of carbon monoxide dehydrogenase is ferredoxin. (o) C H 3 C O O - + A T p 4 + ~ CH3CO-P + ADP 3+

AG O' = + 13 k J /m o l (p) CH3CO-P + CoA-S-H ~ CH3CO-S-CoA + Pi

AG O' = - 9 k J /m o l (q) CH3CO-S-CoA + HaMPT

CH3-HaMPT + CO 2 + CoA-S-H + 2 [H] E 0, = _ 200 mV

Note that in reaction q four products are formed from two substrates. E ' is thus highly concentration-depen- dent. At 0.1 mM concentrations of all substrates and products, E ' = - 440 mV.

CH3-H4MPT formed in reaction q is converted to methane via reactions j, k, and 1 as in methanogenesis from CO 2. Interestingly, methanogenic bacteria contain carbonic anhydrase when growing on acetate [31]. A function of the enzyme in acetate transport has been proposed.

Methane formation from methanol The first step in this fermentation is the formation of

CH3-S-CoM from methanol and H-S-CoM [2]. (r) CH3OH + H-S-CoM ---, CH3-S-CoM + H20

AG O' = - 27.5 k J / m o l One out of four CH3-S-CoM generated in this reaction is oxidized to CO 2 involving reactions d- j in the reverse direction [26,32]. Via reactions e, h, and i 3 × 2 reducing equivalents are provided for the reduction of 3 mol CH3-S-CoM to CH 4 via reactions k and 1.

Sites of energy conversion In methanogenesis from CO 2 and H 2 only reactions

j, k, and 1, in which CH3-H4MPT is converted to CH 4, are exergonic enough to be coupled with energy con- servation. Evidence has recently been obtained with inside-out vesicles that the reduction of CoM-S-S-HTP with H 2 (reactions 1 plus n) is associated with the

Page 4: Energy metabolism of methanogenic bacteria

build-up of an electrochemical proton potential which drives the phosphorylation of ADP via a proton trans- locating ATPase [33-35]. More indirect evidence indi- cates that reaction j (or i) is a second coupling site, in which the free energy change associated with the methyl transfer reaction is conserved in an electrochemical sodium potential. Via a sodium-proton antiporter the sodium potential can be converted into a proton poten- tial and thus used to drive the synthesis of ATP [36,37].

As has been indicated above, the reduction of CO 2 to CHO-MFR with H 2 is an endergonic reaction. Evi- dence is available that this reaction is pushed by the electrochemical sodium potential generated in reaction j (or i) [38]. Under some conditions the driving force appears to be the electrochemical proton potential.

In methanogenesis from acetate 1 mol of ATP is hydrolyzed for the activation of acetate to acetyl-CoA (reactions o and p). Thus 'down stream' more than 1 mol ATP have to be generated. Three sites of energy conservation have been identified, reactions j, 1 plus n, and q. Evidence that reaction q is associated with energy conservation comes from the observation that the carbon monoxide dehydrogenase complex mediating reaction q also catalyzes the conversion of CO and H20 to CO 2 and 2 [H] (reaction s) and that the latter reaction is coupled with the build-up of an electrochem- ical proton potential which can drive the phosphoryla- tion of ADP [39,40]. (s) CO + H20 ~ CO 2 + 2 [H] E °' = - 524 mV

In methanogenesis from methanol reactions j and e are used in the opposite direction than in methane formation from CO 2. During methanol oxidation to CO 2 the formation of CH3-H4MPT from CH3-S-CoM via reaction e is driven by the electrochemical sodium potential [36] which is now generated during CHO-MFR oxidation to CO 2 via reaction e.

For a detailed discussion of the energetics, the reader is referred to the extended abstract by Gottschalk in this volume [41].

Acknowledgements

This work was supported by grants from the De- utsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.

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