Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea Jo ¨ rg Kahnt 1 ,Ba ¨ rbel Buchenau 1 , Felix Mahlert 1 , Martin Kru ¨ ger 2 , Seigo Shima 1 and Rudolf K. Thauer 1 1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany 2 Bundesanstalt fu ¨ r Geowissenschaften und Rohstoffe, Hannover, Germany Methane is formed in methanogenic archaea from methyl-coenzyme M by reduction with coenzyme B. This reaction is catalyzed by methyl-coenzyme M reductase (MCR). The 300 kDa enzyme is composed of three different subunits in an a 2 b 2 c 2 arrangement and contains 2 mol of the nickel tetrapyrrole coen- zyme F 430 , tightly bound. The prosthetic group has to be in the Ni(I) oxidation state for the enzyme to be active. Some methanogenic archaea contain two MCR isoenzymes, designated MCR I and MCR II, the syn- thesis of which is differentially regulated [1]. There is circumstantial evidence that MCR is also involved in the anaerobic oxidation of methane with sulfate by methanotrophic archaea of the ANME-1, ANME-2 or ANME-3 clusters [2–4]. The crystal structure of MCR I from Methanother- mobacter marburgensis has been resolved to 1.16 A ˚ [5–8]. The structure revealed two identical F 430 -binding sites, roughly 50 A ˚ apart. Each F 430 is buried deeply within the protein complex and is accessible from the protein surface only via a 50 A ˚ long channel, which at its narrowest part is only 6 A ˚ in diameter. The channel and the coenzyme-binding sites are formed mainly by hydrophobic residues of subunits a, a¢, b and c , and a¢, a, b¢ and c¢, respectively (the prime superscript indi- cates the second identical subunit). Surprisingly, in the active site region, five modified amino acids were found: thioglycine a445, forming a thioxo peptide (thioamide) bond with tyrosine a446, S-methylcyste- ine a452, 2-(S)-methylglutamine a400, 1-N-methylhisti- dine a257 (3-methylhistidine according to IUPAC nomenclature) and 5-(S)-methylarginine a271 (Fig. 1). The modifications are introduced after translation, as the DNA sequence of the encoding mcrA gene shows Keywords methanogenic archaea; methanotrophic archaea; methylated amino acids; methyl- coenzyme M reductase; thioxo peptides Correspondence R. Thauer, Max-Planck-Institute fu ¨ r terrestrische Mikrobiologie, Karl-von-Frisch- Strasse, D-35043 Marburg, Germany Fax: +49 6421 178109 Tel: +49 6421 178101 E-mail: thauer@mpi-marburg.mpg.de Website: http://www.mpi-marburg.mpg.de/ (Received 11 June 2007, revised 23 July 2007, accepted 26 July 2007) doi:10.1111/j.1742-4658.2007.06016.x Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marbur- gensis 2 was shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophi- lus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats contain- ing mainly methanotrophic archaea of the ANME-1 cluster were also ana- lyzed. Abbreviation MCR, methyl-coenzyme M reductase. FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS 4913 no unusual codons or unusual codon usages at the positions in which the five modified amino acids were found. Via in vivo labeling experiments with l-(methyl- D 3 )-methionine, it was shown that the methyl groups in the four methylated amino acids are introduced cotranslationally or post-translationally by specific S-adenosylmethionine-dependent protein methylases [9]. How the sulfur is transferred into the carboxamide group of glycine in the peptide chain remains to be shown. Neither the functions of the five modifications nor whether the modifications are present in MCRs from all methanogens 3 are known. Comparison of primary structures deduced from the DNA sequences reveals that the five amino acid positions are conserved in MCRs from all methanogenic archaea [9]. However, in the gene for the a-subunit of MCR from methano- trophic archaea of the ANME-1 cluster, there is a codon for a valine, whereas in mcrA from methano- genic archaea, there is a codon for a glutamine [2]. Methanogenic archaea and methanotrophic archaea all belong to the kingdom of Euryarchaeota. They are classified on the basis of their 16S RNA sequence in five orders, Methanobacteriales, Methanopyrales, Met- hanococcales, Methanomicrobiales and Methanosarci- nales [10]. The phylogenetic distance between the archaeal orders is as large as between, for example, proteobacteria and Gram-positive bacteria. The phy- logeny is reflected in the primary structure of the MCR a-subunit, which can therefore be used to clas- sify methanogens [11]. In the work reported here, we have analyzed by MS the MCR from at least one representative of each of the five orders of methanogenic archaea and from two methanotrophic archaea of the ANME-1 cluster. We have included in the analysis both MCR I and MCR II from Methanothermobacter marburgensis (growth temperature optimum 65 °C) and MCR I from a mesophilic (37 °C) and a hyperthermophilic (85 °C) Methanococcus species. The analysis revealed that the thioxo peptide bond is conserved in all MCRs investigated, but that there are differences in the post- translational methylations. Specifically, Cys a452 is not methylated in the enzyme from the hyperthermophilic Methanocaldococcus jannaschii and Methanopyrus kandleri, and Gln a400 is not methylated in Methano- sarcina barkeri. Results Up to now, the crystal structures of three methyl-coen- zyme M reductases have been resolved, MCR isoen- zyme I from Methanothermobacter marburgensis , MCR from Methanosarcina barkeri,andMCRfromMethano- pyrus kandleri [7]. In case of the enzyme from Methano- thermobacter marburgensis and Methanosarcina barkeri, the resolution was high enough to identify the post-translational modifications; in the case of the Methanopyrus kandleri enzyme, it was not. Attempts to obtain diffracting crystals of MCR II from Methano- thermobacter marburgensis and of the MCR from the other methanogens mentioned below were not success- ful. We therefore searched for post-translational modifications in the active site region by subjecting the a-subunit of MCRs to tryptic digestion, followed by separation and sequencing of the peptides of interest. Either before or after the tryptic digestion, any oxidized cysteine residues were reduced with dithiothreitol and subsequently alkylated with 4-vinylpyridine. Either of two methods for sequencing the alkylated tryptic peptides were employed: (a) the tryptic peptide was subjected to partial hydrolysis with aminopepti- dase M and the partial hydrolysate was then analyzed O - O NH 3 + NH + N H 3 C H 3 C S O - O NH 3 + H 2 NN O - NH O NH 3 + CH 3 H H 2 N O - OO NH 3 + H 3 C N S H N 1 -Methylhistidine S-Methylcysteine 5-(S)-Methylarginine 2-(S)-Methylglutamine Thiopeptide bound thioglycine Fig. 1. Post-translationally modified amino acids in the a-subunit of methyl-coenzyme M reductase isoenzyme I from Methanothermo- bacter marburgensis. Two of these modified amino acids, 2-(S)- methylgutamine and 5-(S)-methylarginine, have until now not been found in any other protein. 1-N-Methylhistidine ¼ 3-methylhistidine according to IUPAC nomenclature. 1 Methanogenic archaea methyl-coenzyme M reductase J. Kahnt et al. 4914 FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS by MALDI-TOF MS (Fig. 2); or (b) the tryptic pep- tide was subjected to MALDI-TOF MS ⁄ MS (Fig. 3). As a control, the five modifications within the active site region of MCR I from Methanothermobacter mar- burgensis were found using both methods. Where indi- cated, proteases other than trypsin were used for peptide generation. Tryptic peptide containing thioglycine and S-methylcysteine or cysteine In the tryptic digest of the MCR a-subunit from all methanogens investigated, a peptide containing a thi- oxo peptide was found (Table 1). The sequence LGFY-thioxoglycine-YDLQDQC is highly conserved in the nine tryptic peptides analyzed. Very few varia- tions were found, namely a phenylalanine instead of a tyrosine directly before the thioglycine (Methanoculleus thermophilus and Methanosarcina barkeri) or directly after the thioglycine (Methanosarcina barkeri), and an alanine instead of an aspartate two positions from the thioglycine (Methanopyrus kandleri). The cysteine fol- lowing the conserved glutamine (Q) is methylated in the two isoenzymes from Methanothermobacter mar- burgensis and in the enzymes from Methanococcus vol- tae, Methanoculleus thermophilus and Methanosarcina barkeri, but is not methylated in the enzyme from Met- hanopyrus kandleri and Methanocaldococcus jannaschii. The thiocarbonyl group in thiopeptides can undergo a slow reaction with water, in which the 32-sulfur is replaced by a 16-oxygen. This explains why, in some spectra, a parallel sequence shifted by 16 Da to smaller masses was seen (Fig. 2). In two MCRs from methanotrophic archaea of the ANME-1 cluster [11,12] a tryptic peptide with the N-terminal sequence LGFYGYDL QDQCTAC 5 was found (amino acids modified in other MCR are in bold type), but the glycine was not thioxylated and the cysteine was not methylated (Table 1). As the MCR was isolated from microbial mats from the Black Sea and could not be tested for activity, the possibility can- not be excluded that the enzyme once had a thioxo group but lost it by spontaneous hydrolysis. Peptides containing N-methylhistidine, 5-methyl- arginine or 2-methylglutamine The methylated histidine (H257) was found in the a-sub- unit of MCRs from all methanogenic archaea and of the two MCRs from the methanotrophic archaea of the Intensity (%) 1325.5 1453.61 1566.74 1637.8 1800.92 2037.05 D Q L A Y Y F Thio Gly G L 1873.94 * * * * * Mass (m/z) 0 100 2184.15 2241.19 2353.28 1210.42 Fig. 2. MALDI-TOF MS of the peptide mixture generated via aminopeptidase M from the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanopyrus kandleri. The purified enzyme was denatured in SDS, digested with trypsin, and then alkylated with 4-vinylpyri- dine. Subsequently, the tryptic peptides were separated by HPLC and analyzed by MALDI-TOF MS. The peptide with a mass of 2353.28 Da (predicted to contain the thioglycine) was partially hydrolyzed by aminopeptidase M, and the partial hydrolysate was analyzed by MALDI-TOF MS. From the peptide ladder, the N-terminal amino acid sequence was found to be LGFY-thioglycine-YALQD. The mass peaks labeled with an asterisk have a mass that is 16 Da smaller than those of the respective thioglycine-containing peptides. The thiocarbonyl group in thiopep- tides can undergo a slow reaction with water in which the 32-sulfur is replaced by an 16-oxygen. ThioGly, thioglycine. J. Kahnt et al. 1 Methanogenic archaea methyl-coenzyme M reductase FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS 4915 ANME-1 cluster (Table 2). The sequence around the methylated histidine is shown in Fig. 4. The methylated arginine (R271) is also highly con- served in the a-subunit of MCRs from methanogenic archaea. However, in the MCR from the methano- trophic archaea of the ANME-1 cluster, the arginine in the respective tryptic peptide was not methylated (Table 2). The glutamine (Q400) is not methylated in the a-subunit of MCR from Methanosarcina barkeri.In Table 1. 10;1110;11 Amino acid sequence of the MCR a-subunit tryptic peptide containing the thioglycine. G*, thioglycine; C, S-methylcysteine. Amino acids modified in most MCR are indicated by bold type. MCR from Sequence of the thioglycine-containing tryptic peptide Methanothermobacter marburgensis (MCR I) LGFYG*YDLQDQC*GASNVFSIR a,b Methanothermobacter marburgensis (MCR II) LGFYG*YDLQDQC*GASNSLSIR a Methanopyrus kandleri LGFYG*YALQDQCGAANSLSVR a Methanocaldococcus jannaschii (MCR I) LGFYG*YDLQDQCGAANSLSFR b Methanococcus voltae LGFYG*YDLQDQC*GASNSLAIR a,c Methanoculleus thermophilus (MCR I) LGFFG*YDLQDQC*GSANSLSIR c Methanosarcina barkeri LGFFG*FDLQDQC*GATNVLSYQGDEGLPDELR c Methanotrophic archaea of the ANME-1b cluster LGFYGYDLQDQCTACGSYSYQSDEGMPFEMR c,d a Sequence determined via aminopeptidase M and MALDI-TOF MS. b Sequence taken from Selmer et al. [9]. c Sequence determined via MALDI-TOF MS ⁄ MS. d Sequence of peptide obtained from two different MCRs isolated from the Black Sea mats. Intensity (%) Mass ( m/z ) 0 1257.15 R GF S A Pyridyl- ethyl Cys L N A QS D Q L D Y 321.43 608.27 521.31 408.34 722.27 793.26 864.25 921.23 1964.0 1891.63 1728.36 1613.4 1500.38 1372.26 1129.15 174.59 Thio Gly 2127.99 Y F G L 2446.36 100 Fig. 3. MALDI-TOF MS ⁄ MS of the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanocaldococcus jannaschii. The purified enzyme was denatured in SDS, alkylated with 4-vinylpyridine, and then subjected to SDS ⁄ PAGE. After in-gel digestion of the a-sub- unit with trypsin, the generated peptides were analyzed by MALDI-TOF MS. The peptide with a mass of 2446.36 Da (predicted to contain the thioglycine) was then analyzed by MALDI-TOF MS ⁄ MS. From the fragment pattern, the following amino acid sequence is deduced: LGFY-thioglycine-YDLQDQ-pyridylethylCys-GAANSLSFR. ThioGly, thioglycine. 1 Methanogenic archaea methyl-coenzyme M reductase J. Kahnt et al. 4916 FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS the two MCRs from the ANME-1 cluster, there is a valine instead of a glutamine (Table 2). Discussion Methanogenic archaea are dependent on methane formation for growth, and thus on a functional methyl- coenzyme M reductase [1,13]. Therefore, a genetic analysis of methanogenic archaea with respect to the function of the post-transcriptional modifications of MCR is presently not possible [14,15]. A reversed genetic approach is also not in sight, because the genes required for the biosynthesis of the nickel cofactor F 430 [16] and for the post-translational modifications [9] are not yet known [17,18]. Also, the in vitro reconstitution of active enzyme from its subunits and cofactor has proven very difficult; very little activity is recovered [19]. Therefore, the function of the post-translational modifications can at present only be approached by comparison of MCRs from archaea differing in phylo- genetic relationship and ⁄ or growth conditions. An early hypothesis was that the post-translational or cotranslational modifications modulate the kinetic properties (K m and V max ) of MCR, which is why we started the analysis with isoenzyme II from Methano- thermobacter marburgensis, which differs considerably in K m and V max from isoenzyme I [20]. However, this hypothesis had to be abandoned when we found that the two isoenzymes were identical with respect to their post-translational modifications (Table 2). His a257 is methylated in all analyzed MCRs, indi- cating an essential function (Table 2). The residue 1-N-methylhistidine is part of the coenzyme B-binding site. Its unmethylated nitrogen atom Ne2 donates the shortest of the three hydrogen bonds of the protein to the phosphate group of coenzyme B. The distance of 2.65 A ˚ indicates the presence of a strong hydrogen bond with fully overlapping orbitals. Owing to the positive inductive effect, the pKa of the methylated amino acid is expected to increase slightly (imidazole and N-methylimidazole differ in their pKa by 0.1), and this will affect the strength of coenzyme B binding. A more important function of the methyl group may involved improved orientation of the histidine inside the coenzyme B-binding site. With the methylation, the histidine residue is prevented from forming two alter- nate nitrogen positions by rotating around the angle v 2 , due to occlusion by a peptide oxygen from the pre- ceding residue [21]. The functions of the three other methylations are less obvious, in part because they are not conserved in all MCR enzymes (Table 2). The reduction of methyl- coenzyme M with coenzyme B takes place in a hydro- phobic pocket in the complete absence of water. It involves radical intermediates that are very reactive [5,18]. This is probably why the active site of this enzyme is lined up primarily with unreactive aromatic and aliphatic amino acid residues. The methyl groups of S-methylcysteine, 5-methylarginine and 2-methylglu- tamine are oriented towards the active site chamber Table 2. Amino acid modifications found in the a-subunit of MCRs from methanogenic archaea by MS analysis of tryptic peptides. Where indicated, the a-subunit was subjected to proteolysis by chymotrypsin, endoproteinase AspN or BrCN rather than by trypsin. The data for MCR I from Methanothermobacter marburgensis were taken from Selmer et al. [9]. NF, respective peptide not found. MCR from Thio-Gly S-Methyl-Cys N-Methyl-His 5-Methyl-Arg 2-Methyl-Gln Methanothermobacter marburgensis (MCR I) ++ + + + Methanothermobacter marburgensis) (MCR II) ++ + + a + Methanopyrus kandleri + Cys + + b + Methanocaldococcus jannaschii (MCR I) + Cys + + c + a Methanococcus voltae ++ + NF + Methanoculleus thermophilus (MCR I) + + NF + a + Methanosarcina barkeri ++ + + a Gln Methanotrophic archaea of the ANME-1 cluster (two MCRs) Gly Cys + Arg Val a a-Subunit cleaved with chymotrypsin. b a-Subunit cleaved with BrCN. c a-Subunit digested with AspN. J. Kahnt et al. 1 Methanogenic archaea methyl-coenzyme M reductase FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS 4917 and have no contact with solvent. The function of the methylations could thus be to protect the respective amino acid residues from reaction with the radical intermediates. As the methylations provide the amino acids with additional hydrophobic interactions, another function could be to restrict the conforma- tional flexibility of the residues, as has been discussed for post-transcriptional tRNA modifications [22–24]. The observed differences in methylation are difficult to explain, because they may be compensated for by differences in positioning or properties of nearby resi- dues, which are not directly evident. A function for the thioxo peptide bond (for proper- ties see Pfeifer et al. [25] and Artis & Lipton [26]) was proposed in the catalytic cycle of MCR. In the final step of the cycle, a disulfide anion radical of coen- zyme M and coenzyme B is formed, which is oxidized to the heterodisulfide by reducing the active site’s nickel back to the Ni(I) oxidation state [5,18]. The thi- oxo group is positioned such that it could be involved in electron transport from the disulfide anion radical to the nickel. The redox potential of the thioxo pep- tide ⁄ radical couple is estimated to lie between that of the disulfide ⁄ disulfide anion radical couple () 1.7 V) [27] and the F 430 [Ni(II)] ⁄ F 430 [Ni(I)] couple () 0.64 V) [18]. It has also been suggested that upon a one-elec- tron reduction of the thioxo peptide bond, a change from the trans to the cis conformation (similar to the light-induced switch of the thioxo peptide bond from trans to cis [28,29]) could occur, and that this confor- mational change could be involved in coupling the two active sites [4,30,31]. The finding of the thioxo peptide Fig. 4. Partial amino acid sequence of the a-subunit of MCRs from six methanogenic archaea of five different orders and from two methano- trophic archaea of the ANME-1 cluster. The numbering is that for the a-subunit of MCR I from Methanothermobacter marburgensis. The amino acids, which in MCR from Methanothermobacter marburgensis are post-translationally modified, are highlighted in bold. The ANME- 1a sequence (2216) was provided by A. Meyerdierks, Max Planck Institute for Marine Microbiology in Bremen, and the ANME-1b sequence was taken from Lyoyd et al. [40]. 1 Methanogenic archaea methyl-coenzyme M reductase J. Kahnt et al. 4918 FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS bond in MCRs from all methanogenic archaea sup- ports this hypothesis, whereas the finding that the two MCRs isolated from the Black Sea microbial mats lacks this bond does not. As the thioxo peptide bond can lose its sulfur by hydrolysis, and as the MCR extracted from the microbial mats from the Black Sea was inactive, the function of the thioxo peptide bond as proposed above cannot yet be dismissed. This ques- tion will remain open until methanotrophic archaea can be grown in the laboratory and until active MCRs can be isolated from them for analysis of the presence of the thioxo peptide bond. Methylated amino acids are found in many proteins, although the presence of 5-methylarginine and 2-meth- ylglutamine has, until now, only been reported for MCR [9]. A thioxo peptide bond in a protein has not been encountered before. However, such a bond has been found, for example, in methanobactin from meth- ane-oxidizing bacteria [32] and thioviridamide from Streptomyces olivoviridis [33]. How these nonriboso- mally synthesized polypeptides are thioxylated is not known. A mechanism similar to that described for the sulfuration of the C-terminal glycine in ThiS and MoaD, involved in thiamine biosynthesis and molyb- dopterin biosynthesis, respectively, and for the sulfura- tion of uridine in tRNA to thiouridine can be envisaged [34–36]. This mechanism probably also applies for the synthesis of pyridine-2,6-bis(thiocarb- oxylate) from dipicolinic acid in Pseudomonas stutzeri [37–39]. Experimental procedures Methanothermobacter marburgensis (DSMZ 2133) (growth temperature optimum 65 °C), Methanopyrus kandleri (DSMZ 6324) (98 °C), Methanococcus voltae (DSMZ 1537) (37 °C), Methanocaldococcus jannaschii (DSMZ 2661) (85 °C), Methanoculleus thermophilus (DSMZ 3915) (57 °C) and Methanosarcina barkeri (strain Fusaro) (DSMZ 804) (38 °C) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Ger- many). The first three methanogenic archaea were grown on H 2 and CO 2 , Methanoculleus thermophilus on isopropa- nol, H 2 and CO 2 , and Methanosarcina barkeri on methanol. The microbial mats containing methanotrophic archaea of the ANME-1 cluster were from the Black Sea [2]. MCR was purified from cells by published procedures [1]. Of the methanogenic archaea mentioned above, Methano- thermobacter marburgensis, Methanocaldococcus jannaschii and Methanoculleus thermophilus contain two MCR iso- enzymes, whereas Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri do not have MCR isoenzymes. For determination of the amino acid modifications in the a-subunit of MCR, the a-subunit was subjected to proteo- lysis by trypsin, and then the tryptic peptides, after separa- tion, were analyzed by MALDI-TOF MS. Where indicted, the a-subunit was subjected to proteolysis by chymotrypsin (sequencing grade; Roche, Mannheim, Germany), endopro- teinase AspN (sequencing grade; Roche) or BrCN rather than by trypsin. MALDI-TOF MS analysis of tryptic peptides after partial digestion with aminopeptidase M Purified MCR (2 mg of protein in 25 lL) was supplemented with SDS and dithiothreitol to final concentrations of 1.5% and 200 mm, respectively, and then incubated for 10 min at 95 °C to fully denature the protein. Subsequently, the solu- tion was diluted 10-fold with 100 mm NH 4 HCO 3 (pH 8.3), supplemented with 0.2 mg of trypsin (diphenyl carbamyl chloride 6 -treated, 11392 U mg )1 ; Fluka, Buchs, Switzerland), and incubated for 12 h at 37 °C. Then, excess 4-vinylpyri- dine (60 mm) was added to alkylate the cysteine-derived thiol groups. After incubation for 30 min at 70 °C, the non- reacted vinylpyridine was removed by evacuation to dryness in a Savant-SpeedVac SC110; (Life Sciences International, Frankfurt, Germany) 7 and the dried material containing the tryptic peptides was redissolved in 400 lLofH 2 O. After removal of SDS using a Detergent-Out column (Genotech, St Louis, MO, USA), the peptides were separated by RP- HPLC and analyzed by MALDI-TOF MS. The peptide whose molecular mass matched with that predicted for the modified amino acid containing tryptic peptide was partially hydrolyzed with 10 mU of aminopeptidase M (Roche) at pH 7.1 for 30 min at room temperature, and the partial hydrolysate was then analyzed via MALDI-TOF MS (Voyager DE-RP; ABI, Darmstadt, Germany; 337 nm N 2 laser). From the peptide ladder, the N-terminal amino acid sequence of the peptides was obtained as shown in Fig. 2 for the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanopyrus kandleri. MALDI-TOF MS ⁄ MS analysis of tryptic peptides Purified MCR (0.15 mg) was denatured in SDS (0.15%) containing dithiothreitol (6 mm), alkylated with excess 4-vi- nylpyridine (30 mm) as described above, and then subjected to SDS ⁄ PAGE followed by staining with Coomassie Blue. The band corresponding to the a-subunit was cut out, di- stained and dried under vacuum, and the dried material was suspended in 30 lLof10mm NH 4 HCO 3 (pH 8.3) con- taining 0.6 lg of trypsin (sequence grade, modified; Pro- mega, Madison, WI, USA) and 10% acetonitrile. After incubation at room temperature for 12 h, the trypsin digest was extracted, and the extract was applied to a C18 Pep- Map 100 nano LC column 8 (Dionex, Idstein, Germany) for J. Kahnt et al. 1 Methanogenic archaea methyl-coenzyme M reductase FEBS Journal 274 (2007) 4913–4921 ª 2007 The Authors Journal compilation ª 2007 FEBS 4919 the separation of the peptides, which were subsequently analyzed by MALDI-TOF MS. The peptide whose mass matched that predicted for the modified amino acid con- taining tryptic peptide was analyzed by MALDI-TOF MS ⁄ MS (Ultraflex; Bruker, Bremen, Germany). From the fragment pattern, the amino acid sequence was obtained as exemplified in Fig. 3 for the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanocaldococcus jannaschii. Acknowledgements This work was supported by the Max Planck Society and the Fonds der Chemischen Industrie. References 1 Thauer RK (1998) Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiology 144, 2377– 2406. 2 Kru ¨ ger M, Meyerdierks A, Glockner FO, Amann R, Widdel F, Kube M, Reinhardt R, Kahnt J, Bocher R, Thauer RK et al. (2003) A conspicuous nickel protein in microbial mats that oxidize methane anaerobically. Nature 426, 878–881. 3 Shima S & Thauer RK (2005) Methyl-coenzyme M reductase (MCR) and the anaerobic oxidation of meth- ane (AOM) in methanotrophic archaea. Curr Opin Microbiol 8, 643–648. 4 Thauer RK & Shima S (2007) Methyl-coenzyme M reductase in methanogens and methanotrophs. 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In case of the enzyme. the a-subunit of MCRs from methanogenic archaea. However, in the MCR from the methano- trophic archaea of the ANME-1 cluster, the arginine in the respective