methane as fuel for anaerobic microorganisms

Methane as Fuel for Anaerobic MicroorganismsRUDOLF K. THAUER AND SEIGO SHIMA

Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

Methane has long been known to be used as a carbon and energy source by some aerobic alpha-and delta-proteobacteria. In these organisms the metabolism of methane starts with its oxida-tion with O2 to methanol, a reaction catalyzed by a monooxygenase and therefore restricted tothe aerobic world. Methane has recently been shown to also fuel the growth of anaerobic mi-croorganisms. The oxidation of methane with sulfate and with nitrate have been reported, butthe mechanisms of anaerobic methane oxidation still remains elusive. Sulfate-dependent methaneoxidation is catalyzed by methanotrophic archaea, which are related to the Methanosarcinalesand which grow in close association with sulfate-reducing delta-proteobacteria. There is evidencethat anaerobic methane oxidation with sulfate proceeds at least in part via reversed methano-genesis involving the nickel enzyme methyl-coenzyme M reductase for methane activation, whichunder standard conditions is an endergonic reaction, and thus inherently slow. Methane oxida-tion coupled to denitrification is mediated by bacteria belonging to a novel phylum and does notinvolve methyl-coenzyme M reductase. The first step in methane oxidation is most likely the exer-gonic formation of 2-methylsuccinate from fumarate and methane catalyzed by a glycine-radicalenzyme.

Key words: anaerobic oxidation of methane; methyl-coenzyme M reductase; nickel cofactorF430; glycyl-radical enzymes; methylsuccinate synthase; methanotrophic archaea; methanotrophicbacteria

Introduction

The anaerobic oxidation of methane (AOM) by mi-croorganisms has long been thought to be impossi-ble, not because the dehydrogenation of methane tomethanol (E◦′ =+0.166 V) with, for example, nitrate(E◦′ = +0.43 V) as electron acceptor is thermodynam-ically not possible (TABLE 1), but because the C-H bondin methane is not polarized, and therefore the abstrac-tion of a hydride in water at neutral pH is mechanis-tically not possible. This is only possible in superacidsor with strongly electrophilic metal-containing speciesin the absence of water.1,2 Therefore, the generalview is that in biological systems the first step inmethane oxidation must be its oxidation to a methylradical.

The dissociation energy of the C-H bond inmethane is +439 kJ/mol (TABLE 2), and thus largerthan that in other organic molecules. Only theC-H bond in benzene (−473 kJ/mol) is stronger.There is only one radical of biological relevance,the O-H radical (the dissociation energy of the O-H

Address for correspondence: Prof. Dr. R. Thauer, Max Planck Institutefor Terrestrial Microbiology, Karl-von-Frisch-Strasse, D-35043 Marburg,Germany. Voice: +49 6421 178101; fax: +49 6421 178109.

[email protected]

bond of water is 497 kJ/mol), that reacts withmethane under standard conditions in an exergonicreaction.

CH4 + OH (radical) = CH3 (radical) + H2O

�G◦ ′ = −58 kJ/mol (1)

In the dark the OH radical cannot be generated fromwater since the redox potential E◦′ =+2.33 V of theOH/H2O couple is more positive than that of all otherelectron acceptors that are stable in water. It can, how-ever, be formed by reduction of O2. Three electronsare required for the reduction, one of which can beprovided by the methyl radical.

O2 + 3e + 3H+ = OH(radical) + H2O

E◦ ′ = +0.29 V (2)

CH4 + O2 + 2e + 2H+ = CH3OH + H2O

E◦ ′ = +1.45 V (3)

Reaction 3 (reaction 1 + reaction 2) describes cor-rectly how methane is oxidized to methanol in aer-obic methanotrophic bacteria, while reactions 1 and2 are simplifications of the catalytic mechanism. Theydisregard that reaction 3 is catalyzed by metalloen-zymes and that the primary attack of the methane

Ann. N.Y. Acad. Sci. 1125: 158–170 (2008). C© 2008 New York Academy of Sciences.doi: 10.1196/annals.1419.000 158

Thauer & Shima: Methane as Fuel for Anaerobes 159

TABLE 1. Redox potentials E◦′ of electron accep-tors that could be used by microorganisms forthe anaerobic oxidation of methane

Redox couple n E◦′ (V)

CO2/CH4 8 −0.24S◦/H2S∗ 2 −0.27 (−0.12)a

SO2−4 /HS− 8 −0.22 (−200)b

SO3H−/HS− 6 −0.12APS/SO3H− 2 −0.06Glycine/acetate− + NH+

4 2 −0.01CH3SH/CH4 + H2S 2 +0.03Fumarate/succinate 2 +0.03AsO3−

4 /AsO−2 2 +0.13

Trimethylamine N-oxide/trimethylamine 2 +0.13Dimethylsulfoxide/dimethylsulphide 2 +0.16CH3OH/CH4 2 +0.17Fe(OH)3 + HCO3

−/FeCOc3 1 +0.2

NO2−/NH3 6 +0.33

NO2−/NO 1 +0.34

NO3−/NH3 8 +0.36

Mn4+/Mn2+ 2 +0.41NO3

−/NO2− 2 +0.43

2NO3−/N2 10 +0.76

2NO2−/N2 6 +0.95

2NO/N2O 2 +1.2N2O/N2 2 +1.36CH3(radical)/CH4 1 +2.1

E◦′at pH 7.0 are given for H2, CO2, CO, CH4, and O2 in thegaseous state at 105 Pa, for S◦ in the solid state, and for all othercompounds in aqueous solution at 1 M concentration. The valuesin brackets are E′ values calculated for physiological substrate andproduct concentrations. The E◦′ values were calculated from the�G◦′ values: �G◦′ =−nF�E, where n is the number of electronsand F = 96 487 J/mol/V. Except were indicated, �G◦′ valueswere taken from Thauer et al.91

aCalculated for a [HS−] = 0.1 mM.bCalculated for [sulphate] = 30 mM and [HS−] = 0.1 mM.c From Ehrenreich and Widdel.92

is by a high-valent metal-oxo species rather thanby a free OH radical.3–5 But they can best explainwhy both O2 and the reducing equivalents are re-quired for the oxidation of methane to methanol withO2.

In methanotrophic aerobic bacteria the methanolformed from methane is further oxidized to CO2. Thethree two-electron steps from methanol via formalde-hyde and formate to CO2 are not dependent on O2

and can proceed with electron acceptors readily avail-able in both aerobic and anaerobic organisms.6

CH3OH + H2O = CO2 + 6e + 6H+

E◦ ′ = −0.38 V (4)

TABLE 2. Gas-phase bond-dissociation energiesand radicals that are relevant to the enzymaticsystems discussed in this review

Radicals or relatedradicals of biological

Dissociation importance formedBond energy (kJ/mol) by bond dissociationa

H-OH 497 OH radical (+2.33 V)b

H-C6H5 473H-CH3 439H-H 436H-n-C4H9 425H-n-C3H7 423H-C2H5 423H-CH2COCH3 411H-s-C4H9 411H-s-C3H7 409H-CH2COOH 418c

H-CH2COCH3 411H-CH2CHO 395H-CH2CH2OH 390? 5′-Deoxyadenosine radical

(+1.4 V)H-SCH3 365 Thiyl radical (+1.33 V)H-CH2C6H5 376H-OOH 369 O2

− radical (+0.89 V)d

H-OC6H5 361 Tyrosyl radical (+0.94 V)H-C of glycine 350e Glycyl radical (+1.22 V)CH3-CH3 376CH2=CH2 352f

Bond dissociation energies, except where otherwise indicated,were taken from the Handbook of Chemistry and Physics, 84th Edition2003–2004, CRC Press.

aThe numbers in brackets are redox potentials E◦′ of theradical/undissociated compound couple in aqueous solution atpH 7. In the case of the glycyl radical, the pH was 10.5. Theredox potentials were taken from Stubbe and van der Donk.93

The redox potential of 5′-deoxyadenosyl radical formation is anestimate (see Wang and Frey94).

bThe OH radical can also be generated from H2O2 by a one-electron reduction: H2O2 + e− + H+ = OH + H2O (E◦′ =+0.38).

cFrom Sandala et al. 95 The C-H bond-dissociation energy wascalculated in this paper to be 408 kJ/mol. Since the H-C bond-dissociation energy for methane was calculated via the samemethod to be only 429 kJ/mol rather than 439 kJ/mol, the valuewas increased by 10 kJ/mol.

d O2− radical can also be generated from O2 by a one-electron

reduction: O2 + e− = O2− (E◦′ = −0.33 V).

eFrom Armstrong et al. 96; 334 kJ/mol in Sandala et al. 95

f Bond-dissociation energy for CH2=CH2 to CH2-CH2. Cal-culated from the energy of 728 kJ/mol for the dissociation ofCH2=CH2 to 2 CH2 minus the energy of 376 kJ/mol for thedissociation of CH3-CH3 to 2 CH3.

Because of these theoretical considerations, the factthat methane is oxidized to CO2 in marine sedimentsin the complete absence of O2 has long been ignored.The anaerobic oxidation in the sediments is coupled

160 Annals of the New York Academy of Sciences

to dissimilatory sulfate reduction.

CH4 + SO42− + H+ = CO2 + HS− + 2H2O

�G◦ ′ = −21 kJ/mol (5)

It has been shown that AOM with sulfate is a quan-titatively very important process in the global carboncycle (for an extensive review, see Ref. 7).

It was recently found that AOM is not restrictedto sulfate as electron acceptor. A year ago microbeswere enriched, which coupled methane oxidation withdenitrification.8

5CH4 + 8NO−3 + 8H+ = 5CO2 + 4N2 + 14H2O

�G◦ ′ = −765 kJ/mol CH4 (6)

3CH4 + 8NO2− + 8H+ = 3CO2 + 4N2 + 10H2O

�G◦ ′ = −928 kJ/mol CH4 (7)

In this article we describe what is currently knownabout how anaerobic microbes oxidize methane andwhich microbes are involved. The available evidenceindicates that AOM with sulfate proceeds via a differ-ent mechanism than AOM with nitrate, which is whythe two processes are discussed separately. Mostly, theliterature of the last two years will be considered. Forearlier references the reader is referred to two reviewson the subject by Shima and Thauer9 and by Thauerand Shima.10

Anaerobic Oxidation of Methanewith Sulfate

Rate, Yields, and Apparent Km

The microorganisms involved in AOM with sulfatecould, until now, only be grown in complex culturesconsisting of archaea and bacteria.11,12 Growth is ex-tremely slow and has a low yield. Doubling times ofseveral months and growth yields of 0.6 g (dry weight)per mol methane oxidized appear typical. Only 1% ofthe consumed methane is channeled into the synthe-sis of consortia biomass. The growth rate is almostlinearly dependent on the methane concentrationin the range tested (up to 1.4 × 106 Pa), indicatingthat the apparent K m for methane is above 10 mM.The specific rate of methane oxidation is 10 nmolmethane per min and mg cells (dry weight) at aCH4 partial pressure of 1.4 × 106 Pa.12 In situ mea-surements suggest that in marine sediments and inthe Black Sea chimneys fueled by methane hydrates13

the specific methane oxidation rates are of the sameorder.14–17

The Black Sea microbial mats catalyzing AOM inthe laboratory at a specific rate of 1 nmol per min(mU) per mg protein (at 1 × 105 Pa CH4) mediated theformation of methane from methanogenic substrates,such as H2/CO2, formate, methanol, methylamines,and/or acetate at specific rates less than 0.01 mU/mgprotein, indicating that the mats are dedicated to AOMrather than to methanogenesis.18 There is, however,evidence that the mats simultaneously oxidize andproduce methane.19 The contemporaneous activity ofAOM and methanogenesis also has been reported atthe Gulf of Mexico cold seeps.20

Involvement of Archaea andSulfate-Reducing Bacteria

Available evidence indicates that the archaea in-volved in AOM with sulfate are phylogenetically mostclosely related to methanogenic archaea of the or-der Methanosarcinales and that the archaea are theones that catalyze methane oxidation. Three lin-eages have been identified that are referred to asANME-1, ANME-2, and ANME-3.21–23 Whetherthese archaea also catalyze the reduction of sulfateis still a much-debated question. In their natural en-vironment they are always associated with sulfate-reducing bacteria (SRB) belonging taxonomically tothe delta group of proteobacteria.24–28 Whether thearchaea depend on the SRB for growth remains to beshown.

Syntrophy?H2, formate, and acetate have been considered to

be involved in interspecies electron transfer from themethanotrophic archaea to the SRB. None of thesecompounds have been found to inhibit AOM or tostimulate sulfate reduction in mats catalyzing AOMwith sulfate, which for thermodynamic reasons theyshould if they were intermediates, as exemplified forH2 in reactions 8 and 9.

CH4 + 2H2O = CO2 + 4H2

�G◦ ′ = +131 kJ/mol (8)

4H2 + SO42− + 2H+ = H2S + 4H2O

�G◦ ′ = −152 kJ/mol (9)

In this respect it is of interest that methanogenicarchaea have been shown to require a free energychange under physiological conditions (�G) of at least−10 kJ/mol and SRB one of at least −19 kJ/mol tosupport their metabolism in situ.29,30 The free energychange of −21 kJ/mol associated with AOM with sul-fate (reaction 5) is therefore probably not sufficiently


large to fuel the energy metabolism of two organismsin tandem.

Syntrophy of methanotrophic archaea and SRBcould also proceed by extracellular electron transferinvolving “nanowires,”31,32 which would, however, re-quire each archaeon to be in physical contact with anSRB. In the microbial mats investigated so far, espe-cially in those in which ANME-1 cells dominate, thisis not always the case. In the water column ANME-2also appear as single cells.33 The present view, there-fore, is that at least in some of the archaea, methaneoxidation and sulfate reduction occur in the same cells,although this is not backed up by metagenomic data.It has not yet been possible to demonstrate the pres-ence of gene homologues for the enzymes required fordissimilatory sulfate reduction in archaea of any of theANME clusters.

All attempts to grow the SRB in the absence of themethanotrophic archaea, with which they are associ-ated in the microbial mats, have failed so far. Com-binations of sulfate with H2, formate, acetate, lactate,glucose, and fructose were tried. Dissimilatory sulfatereduction with these electron donors is strongly ex-ergonic (e.g., reaction 9), and therefore should sustaingrowth. The hypothesis has therefore been put forwardthat the SRB in the mats might be fueled by the oxida-tion of the glycocalix surrounding the methanotrophicarchaea and gluing the cells in the mats together. Ifthis hypothesis is correct, growth of the SRB would bedependent on the presence of complex sugar mixtures,which are growth requirements difficult to mimic inthe laboratory. One might argue that sulfate reducersgenerally do not ferment sugars, one of the few ex-ceptions being Desulfovibrio fructosovorans. A look in thesequenced genomes indicates, however, that many ofthe sulfate reducers contain all the genes required tometabolize sugars.

Involvement of Methyl-Coenzyme MReductase in Anaerobic Oxidation

of MethaneMethanotrophic archaea have been shown to con-

tain gene homologues for methyl-coenzyme M re-ductase (MCR) and for other enzymes involved inmethanogenic archaea in methane formation fromCO2, suggesting that these enzymes are involved inAOM34,35 (for review, see Ref. 6). This is also indi-cated by the observation that AOM is inhibited bybromoethane sulfonate (BES),22 which is consideredto be a specific inhibitor of MCR.36

Recently, however, it has been shown that BES alsoeffectively inhibits growth of Xantobacter autotrophicus,metabolizing propylene by specific inactivation of one

of the enzymes involved in propylene degradation.37

Thus inhibition of the growth of an organism by BESis not necessarily an indication of the presence of MCR.Contrarily, noninhibition of the growth by BES cannotbe taken as evidence for the absence of MCR, sinceMCR is a cytoplasmic enzyme, and therefore BES hasto be transported into the cells in order to exert itsinhibitory effect. Uptake generally is by a coenzymeM transporter,38 which some methanogenic archaealack. These methanogens are resistant to inhibition byBES.

The most convincing evidence for the involvementof MCR in AOM comes from the biochemical analysisof microbial mats from the Black Sea, which catalyzeAOM and which comprise more than 50% archaeafrom the ANME-1 cluster.18 These mats were foundto contain high concentrations (10% of the proteinextracted from the mats) of two enzymes, Ni-proteinI and II. The nickel proteins were identified as MCRby their subunit composition, primary structure, andnickel cofactor F430. Metagenome analyses (see later inthe chapter) revealed that the genes coding for the twonickel-proteins belong to the genomes of the ANMEorganisms.18

The mcrA gene is a taxonomic marker formethanogenic archaea. Their phylogenetic relation-ship is reflected in the primary structures of the MCRfrom these organisms.39 Detection of the mcrA gene inmicrobial communities catalyzing AOM is also cur-rently used as an indication of the involvement ofMCR.34,35,40–42 But without knowing whether the mcrAgene is expressed, one has to be careful not to overin-terpret these findings.43

Methyl-Coenzyme M Reductase fromMethanogenic Archaea

MCR from methanogenic archaea is composed ofthree different subunits in an α2β2γ2 arrangement andcontains 2 mol of a nickel porphinoid, designated coen-zyme F430 (FIG. 1) as prosthetic group, which has to bein the Ni(I) oxidation state for the enzyme to be active(for review, see Ref. 44). The crystal structure of MCRwith F430 in the Ni(II) oxidation state was resolved to1.16 A. It revealed that the subunits are intertwinedsuch that they form two structurally interlinked activesites made up of the subunits α, α′β, and γ and α′, α,β′, and γ′, respectively. Each active site harbors oneF430 molecule buried deep within the protein and ac-cessible from the outside only via a 50-A-long channelmade up mainly of hydrophobic amino acid residues.Near to the active site are five modified amino acids: athioglycine, an N -methyl-histidine, an S-methyl cys-teine, a 5-(S)-methyl arginine, and a 2-(S)-methyl


FIGURE 1. Structure of F430, the prosthetic group of methyl-coenzyme M reductase from methanogenicarchaea. View from the β-face. In methyl-coenzyme M reductase from methanotrophic archaea of theANME-1 cluster, 172-methylthio-F430 with a molecular mass of 951 Da rather than F430 with a mass of905 Da is present.

glutamine.45 Labeling studies have shown that themethyl groups are biosynthetically derived from themethyl group of methionine and not from the methylgroup of methyl-coenzyme M.46 The five modifiedamino acids are highly conserved. However, in MCRfrom Methanosarcina the respective glutamine is notmethylated.47 Generally, the genes coding for thethree MCR subunits are coded in a transcription unitmcrBDCGA. The function of McrC and McrD, whichdo not copurify with MCR, is not known. In somemethanogens the mcrC and/or mcD genes are not lo-cated together with the mcrAGB genes.

Methyl-Coenzyme M Reductasefrom ANME-1 and ANME-2

The archaea present in the microbial mats catalyz-ing AOM in the Black Sea contain at least two differ-ent MCR, designated Ni-protein I and Ni-protein II,that can be separated by anion exchange chromatog-raphy.18 Ni-protein I contains a modified F430 with amolecular mass of 951 Da, whereas Ni-protein II con-tains the normal F430 with a molecular mass of 905 Da.

The structure of the modified F430 with a molecu-lar mass of 951 Da has recently been elucidated to be172-methylthio-F430 (S. Mayr, S. Shima, M. Kruger, R.Thauer, and B. Jaun, unpublished results). Ni-proteinI is present in a concentration of 7% of the extractedsoluble proteins, and Ni-protein II in a concentrationof up to 3%. Via the N-terminal amino acid sequencesof the three subunits, the encoding genes were identi-fied in a metagenome library of the mats. The codonusage and tetranucleotide signature of the three genes

in the cluster mcrBGA revealed that the three genescoding for Ni-protein I are located on the genome ofthe dominant ANME-1 archaeon present in the mats18

and those coding for Ni-protein II in the genome ofan ANME-2 archaeon (A. Meyerdierks, S. Shima, J.Kahnt, and M Kruger, unpublished results). Immuno-gold labeling of microbial mats with a specific antibodyrevealed that MCR was located in both ANME-1 andANME-2 archaea. The data also show that the MCR-like enzymes are not only encoded in the genomes ofANME-1 and ANME-2 archaea but are, in fact, ex-pressed at a high level.48

Ni-protein I from ANME-1 archaea differs fromNi-protein II from ANME-2 archaea not only in thatthe former harbors a modified F430 and the latter thenormal F430. In the α-subunit of Ni-protein I there is avaline, where there is a glutamine in Ni-protein II andin MCR from methanogenic archaea, which can bemethylated at C2. Two amino acids downstream of thevaline, there is a cysteine-rich sequence CCX4CX5Cnot present in Ni-protein II from ANME-2 and inMCR from methanogenic archaea. There is probablyanother interesting difference. In the sequence of Ni-protein I there appears to be a normal glycine, whilein that of MCR from methanogens there is principallya thioglycine. Whether the thioglycine is also absent inNi-protein II is not yet known.

Methyl-Coenzyme M ReductaseCatalyzed Reactions

MCR from methanogenic archaea catalyzes thereduction of methyl-coenzyme M (CH3-S-CoM)


FIGURE 2. Reaction catalyzed by methyl-coenzyme M reductase from methanogenic archaea.�G◦ =−30 kJ/mol for the MCR-catalyzed reaction is obtained from the difference in the free energychanges associated with several reactions.54,89 One of these reactions is the reduction of CoM-S-S-CoBwith H2. �G◦ for this reaction was revised recently due to the finding that the redox potential of theCoM-S-S-CoB/ HS-CoB + HS-CoM couple is −143 ± 10 mV rather than −200 mV.90 As a result, �G◦

for methyl-coenzyme M reduction with coenzyme B decreased from −45 kJ/mol to −33 kJ/mol. Thisvalue also has some uncertainty, since it is in part based on �G◦ =−28 kJ/mol associated with methyl-coenzyme M formation from methanol and coenzyme M, which was calculated from differences in bondenergies, which neglects differences in solvation energies.89 �G◦ for methyl-coenzyme M reduction isbest given as being −30 ± 10 kJ/mol.

with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB (�G◦′ =−30 kJ/mol). This is the methane-forming reaction in all methanogenic archaea. TheMCR catalyzed reaction is exergonic (FIG. 2). Un-der standard conditions (nongaseous substrates andproducts with a concentration of 1 M, CH4 at105 Pa pressure, and a pH of 7.0) the free en-ergy change (�Go′) associated with the reactionis estimated to be −30 kJ/mol methane. Thefree energy change under physiological conditions(�G′) is obtained from �G′ = �Go′ + RTln[Products]/[Substrates] = −30 + 5.7 log [Products]/[Substrates]. The equation predicts that the back reac-tion becomes exergonic when the product-to-substrateconcentration ratio is approximately105. Such a ratiois physiologically not unrealistic. At 105 Pa methane,the ratio is 105 when the intracellular concentrationof CoM-S-S-CoB is, for example, 1 mM (10−3 M) andthat of CH3-S-CoM and HS-CoB is 0.1 mM (10−4 M)each. Consistent with this, methanogenic archaea havebeen shown to be capable of very slow methane oxi-dation.49,50 In some of the earlier reports,51,52 how-ever, it has to be considered that the 14C methaneused to follow AOM was generated from 14CO2 by

methanogens and was therefore most likely contami-nated by 14CO.53

Rate of the Forward and Backward ReactionsMCR from Methanothermobacter marburgensis catalyzes

methane formation from methyl-coenzyme M with amaximal specific activity of approximately 100 U/mgprotein.54 Exponentially growing M. marburgensis canproduce methane at a specific rate of up to 5 U/mgprotein. Consistently, such grown methanogenic ar-chaea contain MCR at concentrations of 5–10% ofthe soluble cell proteins. Due to experimental reasons(equilibrium already reached after a few turnovers),the rate of the back reaction catalyzed by the enzymehas not yet been determined experimentally. The spe-cific rate can be estimated, however, employing theHaldane equation, which correlates the equilibriumconstant (K eq) of a reaction with the catalytic effi-ciency (kcat/K m) of an enzyme to catalyze the for-ward and the back reaction: K eq = catalytic efficiency(forward reaction)/catalytic efficiency (backwards re-action). The K eq for the MCR catalyzed reaction iscalculated from �Go′ = −RTlnK eq = −30 kJ/mol tobe near 105. Assuming the K m values of MCR for itssubstrates and products to be all, for example, 0.1 mM,


the Haldane equation predicts that MCR with a V max

for methyl-coenzyme M reduction of 100 U/mg cat-alyzes methane oxidation at a maximal specific rateof 1 mU/mg (10−5 × 100 U/mg). As indicated in thelegend to FIGURE 2, �Go′ =−30 kJ/mol of the MCRcatalyzed reaction is only known with an uncertaintyof ±10 kJ/mol. Therefore, and because the K m val-ues are most probably not all the same, the maximalspecific rate of methane oxidation could be as high as100 mU/mg and as low as 0.01 mU/mg.

Conformational Change Inducedby Coenzyme B

The active-site structure indicates that methyl-coenzyme M reduction to methane takes place in ahydrophobic pocket from which water is completelyexcluded. When entering the active site via the hy-drophobic channel, methyl-coenzyme M is strippedof water, and after the reaction the heterodisulfideis expelled into the water phase. The reaction mostprobably starts with a conformational change withinthe active site that is induced upon binding of coen-zyme B. This is indicated by the finding that uponaddition of coenzyme B to active MCR in the pres-ence of coenzyme M, the enzyme is converted fromthe MCRred1c state, exhibiting a Ni(I)-derived axialelectron paramagnetic resonance (EPR) signal, intothe MCRred2 state, exhibiting a Ni(I)-derived highlyrhombic EPR signal.55,56 In single-turnover experi-ments methane formation from methyl-coenzyme Mwas found to be dependent on coenzyme B.57

Coupling of the Two Methyl-Coenzyme MReductase Active Sites

There is evidence that the two active sites are struc-turally and functionally interlinked. The two α subunitsin the enzyme are intertwined such that a conforma-tional change in the one active site (made up of thesubunits α, α′, β, and γ) can be transmitted to the otheractive site (made up of the subunits α′, α, β′, and γ′)and vice versa.45 An indication for the coupling of thetwo active sites is the finding that at most 50% of theenzymes are converted from the MCRred1c state intothe MCRred2 state upon addition of coenzyme B.55

MCR thus shows “half-of-the-sites” reactivity. Basedon these findings, it has been proposed that the enzymeoperates according to a dual-stroke engine mechanism.This would allow the coupling of the endergonic stepsof the catalytic cycle in one active site to the exergonicsteps in the other site. The coupling is predicted tolower the activation energy for both the forward andthe backward reactions55 (FIG. 3).

Catalytic Mechanisms Discussed forMethyl-Coenzyme M Reductase

Three mechanisms for MCR are currently con-sidered.44,54 In mechanism 1 the methyl group ofmethyl-coenzyme M reacts with the Ni(I) in a nucle-ophilic substitution reaction, yielding methyl-Ni(III)and coenzyme M, which in turn react to methyl-Ni(II) and the thiyl radical of coenzyme M. Methyl-Ni(II) then reacts with a proton in an electrophilicsubstitution reaction to methane and Ni(II), and thecoenzyme M thiyl radical reacts with coenzyme B,yielding a disulfide anion radical, which is a strongreductant and which reduces Ni(II) back to Ni(I),thus closing the catalytic cycle. This mechanism ismainly supported by the findings that MCR-catalyzedmethyl-coenzyme M reduction proceeds with the in-version of stereoconfiguration,58 that enzyme boundNi(I)F430 reacts with 3-bromopropane sulfonate toalkyl Ni(III),59 and that free Ni(I)F430 in aprotic solventsreacts with methylbromide to form methyl-Ni(II)F430,which subsequently can be protonolyzed to methaneand Ni(II)F430.60 Also, the finding that MCR catalyzesthe reduction of ethyl-coenzyme M with less than 1%of the catalytic efficiency of methyl-coenzyme M re-duction is consistent with a nucleophilic substitutionas the first step in the catalytic cycle.55

Looking at the back reaction, the oxidation ofmethane, this mechanism has a problem. Methane oxi-dation would start by a nucleophilic attack of methaneon Ni(II) of F430. This is most unlikely since Ni(II)of F430 is not electrophilic enough to be able to attackmethane with a pKa of above 40. The low electrophilic-ity of F430(Ni II) is reflected by the low redox potentialE◦′ = <−0.6 V of the Ni(II)F430/Ni(I)F430 couple60,61

(FIG. 1).Density function calculations have revealed that

mechanism 1 is energetically not favorable,62,63 al-though the calculations have not taken into accountthat the two active sites could be energetically cou-pled. Based on their calculations, Ghosh64 and Sieg-bahn62,63 have proposed mechanism 2, in which, asthe first step in the catalytic cycle, the methyl thioetherbond in methyl-coenzyme M is reductively cleaved,yielding a Ni(II) thiolate and a methyl radical, whichin turn reacts with HS-CoB, yielding methane and aCoB-S. thiyl radical. The latter reacts with coenzymeM thiolate to form the disulfide anion radical, which,as in mechanism 1, is used to re-reduce the Ni(II)F430

in MCR to the Ni(I) oxidation state. Mechanism 2is backed up by the experimental finding that in ac-tive MCR coenzyme M reversibly coordinates withits thiol sulfur to Ni(I) of F430 when coenzyme B ispresent.65,66


FIGURE 3. Dual-stroke engine mechanism proposed for methyl-coenzyme M reductase. The mech-anism allows the coupling of endergonic steps of the catalytic cycle in the one active site to exergonicsteps in the other active site. The coupling is predicted to lower the activation energy both in the forwardand the back reaction.

But this mechanism also exhibits a flaw when look-ing at the back reaction. Methane oxidation wouldstart with the reaction of methane with the CoB-S·

thiyl radical. The bond-dissociation energy of the C-Hbond in methane is 439 kJ/mol, as compared to thatof the S-H bond of only 365 kJ/mol (TABLE 2), whichmakes the reaction of methane with a thiyl radicalyielding a methyl radical and a thiol thermodynami-cally very unfavorable. The activation energy for theback reaction would be unusually high (on the order of80 kJ/mol), but possibly could be lowered by couplingthe two active sites in MCR, as previously described.

Mechanism 3 is a modification of mechanism1. Methyl-Ni(III)F430 generated from Ni(I)F430 andmethyl-coenzyme M reacts with a proton, yieldingmethane and Ni(III)F430. Free Ni(III)F430 is a verystrong electrophile (E◦′ = > + 1 V),60 and this is prob-ably also true for enzyme bound Ni(III)F430, althoughthe EPR spectrum and the UV/visible spectrum of freeNi(III)F430 and those of Ni(III)F430 in MCRox are verydifferent, indicating major differences in the coordina-tion sphere.67,68 The back reaction, the oxidation ofmethane, would therefore start, with the electrophilicmetalation of methane by reaction of methane eitherend-on or side-on with the high-valent Ni(III) complexin MCR, as described for the activation of C-H bondsby other high-valent metal complexes.1

Why Methyl-Coenzyme M Reductase andNot Methylsuccinate Synthase?

As is outlined below, AOM with nitrate does notinvolve MCR. Available evidence indicates that the

first step is the formation of 2-methylsuccinate frommethane and fumarate catalyzed by a glycyl radical en-zyme. From bond-dissociation energy differences it canbe predicted that the formation of 2-methylsuccinatefrom methane and fumarate is an exergonic reactionwith �G◦′ between −10 and −20 kJ/mol69 (TABLE 2).The radical mechanism is, however, such that this stepcannot be coupled with energy conservation (see laterin this paper). Therefore, most if not all of the freeenergy generated during methane oxidation with sul-fate (�G◦′ =−21 kJ/mol) would be dissipated as heatin the first step, leaving not enough energy to drivethe phosphorylation of ADP in other steps. However,this argument holds true only for AOM with sulfateas an electron acceptor. With nitrate or nitrite (reac-tions 6 and 7) the free energy change associated withAOM would be more than sufficient to allow the for-mation of 2-methylsuccinate, and thus dissipation of15 ± 5 kJ/mol as heat in the first step.

Based on the same arguments, AOM with sulfateis predicted to start with a reaction operating underphysiological conditions near thermodynamic equilib-rium, and thus without the loss of energy. The MCR-catalyzed reaction perfectly fulfills this requirement.

Anaerobic Oxidation of Methanewith Nitrate

Only one convincing publication describing AOMwith electron acceptors other than sulfate has ap-peared. This is the paper by Raghoebarsing et al.,8 in


which they report that AOM can be coupled to NO3−

or NO2− reduction to N2 (reactions 6 and 7). The

mixed culture mediating these reactions consisted to10% of archaea related to the methanotrophic archaea(ANME) of the ANME clusters and to 90% of bacteriabelonging to a novel phylum. From their results the au-thors concluded that the archaea in the mixed culturecould be the organisms oxidizing CH4, and if so thatthen MCR would probably be the enzyme catalyzingthe first step in methane oxidation.

Noninvolvement of Methyl-Coenzyme MReductase

There was, however, one finding that already arguedstrongly against the conclusion that MCR could beinvolved in AOM with nitrate. The culture growingat a doubling rate for over 100 days oxidized methanewith a specific rate of 2 nmol/min and mg protein andan apparent K m for methane of <1 µM.8 The catalyticefficiency (kcat/K m) of AOM with nitrate was thusmore than 1000 times higher than that of AOM withsulfate (apparent K m for methane >1 mM), which, forreasons discussed earlier (Haldane equation), is notconsistent with MCR being involved in AOM withnitrate.70

But there was also a second finding, which is difficultto explain, assuming methane in the mixed culture isoxidized by the 10% archaea. In labeling experimentswith 13CH4 only the bacterial lipids and not the lipidsof the archaea became enriched in 13C indicating thatin the time span of the experiment (3 and 6 days) thearchaeal population did not proliferate.8

It was therefore not unexpected when it was foundthat during further cultivation of the mixed culture thepercentage of the archaea continuously decrease from10% to well below 1%, as determined microscopicallyby fluorescence in situ hybridization (FISH) of the ar-chaea. Parallelly, the F430 content of the cells (archaeaplus bacteria) decreased below the detection limit, ex-cluding that MCR is involved in AOM with nitrate inthe culture of concern.71

Mechanism of Anaerobic Oxidationof Methane with Nitrate

What could be the mechanism of AOM with ni-trate? Could methane directly react with one of theintermediates of denitrification, which are known tobe strong oxidants? Probably not! The NO/N2O cou-ple has a redox potential E◦′ of +1.17 V and theN2O/N2 couple has one of +1.36 V. But both re-dox potentials are still more negative than that of theCH3/CH4 couple, which is more positive than +2 V

(TABLES 1 and 2). But a direct reaction of CH4 witha nitrogen oxide cannot be completely excluded. Innonaqueous solutions, methane can react with NO+

and NO+2 by electrophilic substitution and insertion,

respectively.72,73

An indication comes from the observation that thedenitrifying cultures also catalyzed the oxidation ofethane, propane, and butane.71 This finding suggeststhat methane oxidation could proceed via the mecha-nism of anaerobic oxidation of propane and of longerchain alkanes in sulfate-reducing and -denitrifyingbacteria.

In recent years sulfate-reducing and -denitrifyingbacteria have been characterized that can grow onalkanes with more than one carbon (for reviews, seeRefs. 74–76). None of these organisms can metab-olize methane and only a few can oxidize ethane.The first step in the anaerobic oxidation of alkaneshas been shown to be the exergonic formation of 2-alkylsuccinate from alkane and fumarate. The func-tionalized alkane is then degraded via β-oxidation re-generating fumarate.77–81

2-Alkylsuccinate formation is catalyzed by a glycylradical enzyme. In the proposed catalytic mechanismthe glycyl radical (for the EPR signal, see Ref. 82)extracts an H atom from the alkane via a thiyl rad-ical, preferentially from the C2 position, yielding analkyl radical, which reacts with fumarate, yielding a 2-alkylsuccinyl radical. This radical reacts with the cys-teine residue to 2-alkylsuccinate, regenerating the thiylradical, and thus the glycyl radical of the enzyme.83–85

The dissociation energy of the C-H bonds of C2 ofpropane and of other alkanes is near +410 kJ/mol andthus 60 kJ/mol higher than that of the C-H bond in theglycine residue of the alkylsuccinate synthase (TABLE 2).The difference of 60 kJ/mol evidently can be overcomein the transition state of the enzyme; otherwise, thereaction would not be catalyzed. The question is, couldsuch a mechanism also work for AOM?

In the reaction of methane with fumarate to 2-methylsuccinate, a methyl radical (439 kJ/mol) wouldhave to be formed at the expense of a glycyl radical(350 kJ/mol). The difference in dissociation energiesis almost 90 kJ/mol, which appears to be too large tobe overcome in the transition state of the enzyme. Ithas to be considered, however, that glycyl radical en-zymes are, just like MCR, functional dimers and showhalf-of-the-site reactivity. By coupling the endergonicsteps of the catalytic cycle in one active site with exer-gonic steps in the catalytic cycle of the second activesite, the relatively large difference in dissociation en-ergies might be overcome.


Metabolic Cycles Regenerating FumarateThe formation of 2-methyl succinate from fu-

marate and methane is metabolically attractive sincefumarate can theoretically be regenerated from 2-methylsuccinate via at least two metabolic cycles inwhich CH4 is oxidized to CO2 and in which onlyknown biochemical reactions and enzymes are in-volved (for more information, see the References in,e.g., Erb et al.86).

(i) Fumarate + CH4 → 2-methylsuccinate →mesaconate + 2H → citramalate → citramalyl-CoA → pyruvate + acetyl-CoA → → → oxaloac-etate → citrate → isocitrate → 2-oxoglutarate +CO2 + 2H → succinyl-CoA + CO2 + 2H →succinate → fumarate + 2H.

(ii) Fumarate + CH4 → 2-methylsuccinate → 2-methylsuccinyl-CoA → 2-ethylmalonyl-CoA →butyryl-CoA + CO2 → crotonyl-CoA + 2H→ 3-hydroxybutyryl-CoA → 4-hydroxybutyryl-CoA → 4-hydroxybutyrate → succinate semi-aldehyde + 2H → succinyl-CoA + 2H→succinate→fumarate + 2H.

The following reactions could provide the cells withprecursors for biosynthesis: fumarate + CH4 → 2-methylsuccinate → mesaconate + 2H → citramalate→ citramalyl-CoA → pyruvate + acetyl-CoA → →→ oxaloacetate. Oxaloacetate would be formed frompyruvate via carboxylation and pyruvate formed fromacetyl-CoA via reductive carboxylation.

Why Not Methyl-Coenzyme M ReductaseInvolved in Anaerobic Oxidation of Methane

with Nitrate?MCR from methanogenic archaea is only ac-

tive when its prosthetic group F430 is in the Ni(I)oxidation state. The redox potential (E◦′) of theNi(II)F430/Ni(I)F430 couple has been determined tobe below −0.6 V.60,61 Due to the negative redox po-tential, which is more than 0.2 V below that of thehydrogen electrode at pH 7.0, and due to the fact thatin the enzyme F430 is not completely electrically in-sulated from electron acceptors present in the solvent,Ni(I)F430 in MCR slowly oxidizes to Ni(II)F430, evenunder strictly anoxic conditions. The rate of MCRinactivation increases with increasing redox potetialin its environment.44 Considering that re-reduction ofMCR in the cells requires ATP,54 the negative redoxpotential of F430 could preclude the operation of MCRin cells, in which the redox potential of the termi-nal electron acceptors is more positive than 0 V as inthe case of the nitrate/nitrite couple (E◦′ =+0.43 V),the NO2

−/NO couple (E◦′ =+0.34 V), the NO/N2O

couple (E◦′ = +1.17 V), and the N2O/N2 couple(E◦′ =+1.36 V). This is probably one reason whyAOM coupled to denitrification does not involveMCR. On the contrary, the redox potential of theadenylylsulfate (APS)/sulfite couple (E◦′ = −0.06 V)and that of the sulfite/H2S couple (E◦′ = −0.12 V) in-volved in dissimilatory sulfate reduction are evidentlysufficiently negative to allow the function of MCR inthe presence of these electron acceptors.

Following these arguments one can predict that, iffound, AOM with sulfur could involve MCR sincethe redox potential of the S◦/H2S couple is −0.12 Vat physiological HS− concentrations (TABLE 1). And,AOM with Fe(III) (+0.2 V) or Mn(IV) (+0.41 V), ifonce found, is predicted not to involve MCR.

Alkane Carboxylation and theBiological Formation of Ethane

and Propane

Recently evidence has been published that in someSRB the anaerobic oxidation of alkanes could be initi-ated by the subterminal carboxylation of the alkaneat the C3 position.87 The carboxylation of alkanesis, under standard conditions, an endergonic reac-tion (�G◦′ =+28 kJ/mol). For it to proceed underphysiological conditions, the fatty acid concentrationwould have to be in the µM range. Carboxylationof methane to acetate is thermodynamically even lessfavorable (�G◦′ =+36 kJ/mol). The findings by Soet al.87 should therefore be taken only as a reminderthat there could be more than two ways to anaerobi-cally oxidize methane.

In this respect the finding that ethane and propaneare biologically formed in the deep marine subsurfaceis of interest. Reduction of acetate to ethane is one fea-sible mechanism. Propane is enriched in 13C relative toethane. The amount is consistent with derivation of thethird C from CO2. At typical sedimentary conditions,the reactions yield free energy sufficient for growth.88

Conclusion

Until now AOM by microorganisms has only beenreported with sulfate or nitrate/nitrite as electron ac-ceptors. AOM with sulfate involves archaea and mostprobably MCR, whereas AOM with nitrate involvesbacteria and possibly a glycyl radical enzyme. How-ever, it has to be considered that the organisms medi-ating AOM have not yet been grown in pure cultureand that therefore most conclusions are only based on


circumstantial evidence. From thermodynamics ofAOM with different electron acceptors one can predictthat AOM should also be possible with S◦, arsenate,Fe(III), and Mn(IV). Organisms mediating these re-actions will have to looked for in appropriate niches

Acknowledgments

This work was supported by the Max Planck Societyand by a grant from the Fonds der Chemischen Indus-trie. We thank Martin Kruger and Fritz Widdel fromthe Max Planck Institute for Marine Microbiology inBremen for a fruitful collaboration.

Conflict of Interest

The authors declare no conflicts of interest.

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