JBIC Journal of Biological Inorganic Chemistry

, Volume 9, Issue 6, pp 691–705 | Cite as

Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues

  • Meike Goenrich
  • Felix Mahlert
  • Evert C. Duin
  • Carsten Bauer
  • Bernhard Jaun
  • Rudolf K. Thauer
Original Article


Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F430 as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent KM=20 mM; apparent Vmax=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent KM=5 mM; apparent Vmax=30 U/mg). Propyl-coenzyme M (apparent Ki=2 mM) and allyl-coenzyme M (apparent Ki=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I]0.5 V=2 µM), cyano-coenzyme M ([I]0.5 V=0.2 mM), 3-bromopropionate ([I]0.5 V=3 mM), seleno-coenzyme M ([I]0.5 V=6 mM) and trifluoromethyl-coenzyme M ([I]0.5 V=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D2O. The signal was abolished upon exposure of the enzyme to O2. 3-Bromopropanesulfonate ([I]0.5 V=0.1 µM), 3-iodopropanesulfonate ([I]0.5 V=1 µM), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O2. 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.


EPR spectroscopy Factor 430 Methanogenic archaea Methyl-coenzyme M reductase Nickel enzymes 







methyl-coenzyme M


coenzyme B


coenzyme M


methyl-coenzyme M reductase


MCR exhibiting the EPR signals ox1, ox2 or ox3


MCR exhibiting the EPR signals red1a, red1c or red1m


MCR-red1c or MCR-red1m after extensive washing by ultrafiltration in the absence of coenzyme M and methyl-coenzyme M


MCR-red1 in the presence of coenzyme M


MCR-red1 in the presence of methyl-coenzyme M


MCR exhibiting both the red1 and red2 EPR signals



This work was supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and by the Swiss National Science Foundation. We thank Reinhard Böcher for technical assistance. M.G. is grateful to the Claussen-Simon-Foundation for a fellowship.


  1. 1.
    Wolfe RS (2004) ASM News 70:15–18Google Scholar
  2. 2.
    Thauer RK (1998) Microbiology 144:2377–2406PubMedGoogle Scholar
  3. 3.
    Goubeaud M, Schreiner G, Thauer RK (1997) Eur J Biochem 243:110–114CrossRefPubMedGoogle Scholar
  4. 4.
    Becker DF, Ragsdale SW (1998) Biochemistry 37:2639–2647CrossRefPubMedGoogle Scholar
  5. 5.
    Mahlert F, Grabarse W, Kahnt J, Thauer RK, Duin EC (2002a) J Biol Inorg Chem 7:101–112Google Scholar
  6. 6.
    Krüger M, Meyerdierks A, Glockner FO, Amann R, Widdel F, Kube M, Reinhardt R, Kahnt J, Bocher R, Thauer RK, Shima S (2003) Nature 426:878–881CrossRefPubMedGoogle Scholar
  7. 7.
    Ermler U, Grabarse W, Shima S, Goubeaud M, Thauer RK (1997) Science 278:1457–1462CrossRefPubMedGoogle Scholar
  8. 8.
    Grabarse W, Mahlert F, Shima S, Thauer RK, Ermler U (2000) J Mol Biol 303:329–344CrossRefPubMedGoogle Scholar
  9. 9.
    Grabarse W, Mahlert F, Duin EC, Goubeaud M, Shima S, Thauer RK, Lamzin V, Ermler U (2001) J Mol Biol 309:315–330CrossRefPubMedGoogle Scholar
  10. 10.
    Grabarse W, Shima S, Mahlert F, Duin EC, Thauer RK, Ermler U (2001) Methyl-coenzyme M reductase. In: Messerschmidt A, Huber R, Poulos T, Wieghardt K (eds) Handbook of metalloproteins. Wiley, Chichester, pp 897–914Google Scholar
  11. 11.
    Jaun B, Pfaltz A (1986) J Chem Soc Chem Commun 1327–1329Google Scholar
  12. 12.
    Rospert S, Böcher R, Albracht SPJ, Thauer RK (1991) FEBS Lett 291:371–375CrossRefPubMedGoogle Scholar
  13. 13.
    Holliger C, Pierik AJ, Reijerse EJ, Hagen WR (1993) J Am Chem Soc 115:5651–5656Google Scholar
  14. 14.
    Finazzo C, Harmer J, Bauer C, Jaun B, Duin EC, Mahlert F, Goenrich M, Thauer RK, Van Doorslaer S, Schweiger A (2003) J Am Chem Soc 125:4988–4989PubMedGoogle Scholar
  15. 15.
    Finazzo C, Harmer J, Jaun B, Duin EC, Mahlert F, Thauer RK, Van Doorslaer S, Schweiger A (2003) J Biol Inorg Chem 8:586–593PubMedGoogle Scholar
  16. 16.
    Horng YC, Becker DF, Ragsdale SW (2001) Biochemistry 40:12875–12885CrossRefPubMedGoogle Scholar
  17. 17.
    Jaun B (1993) Methane formation by methanogenic bacteria: redox chemistry of coenzyme F430. In: Sigel H, Sigel A (eds) Metal ions in biological systems, vol XXXX. Dekker, New York, pp 287–337Google Scholar
  18. 18.
    Lin S-K, Jaun B (1992) Helv Chim Acta 75:1478–1490Google Scholar
  19. 19.
    Signor L, Knuppe C, Hug R, Schweizer B, Pfaltz A, Jaun B (2000) Chem Eur J 6:3508–3516CrossRefGoogle Scholar
  20. 20.
    Tada M, Masuzawa Y (1997) Chem Commun 2161–2162Google Scholar
  21. 21.
    Ghosh A, Wondimagegn T, Ryeng H (2001) Curr Opin Chem Biol 5:744–750CrossRefPubMedGoogle Scholar
  22. 22.
    Pelmenschikov V, Blomberg MR, Siegbahn PE, Crabtree RH (2002) J Am Chem Soc 124:4039–4049CrossRefPubMedGoogle Scholar
  23. 23.
    Pelmenschikov V, Siegbahn PE (2003) J Biol Inorg Chem 8:653–662CrossRefPubMedGoogle Scholar
  24. 24.
    Ahn Y, Krzycki JA, Floss HG (1991) J Am Chem Soc 113:4700–4701Google Scholar
  25. 25.
    Lin S-K, Jaun B (1991) Helv Chim Acta 74:1725–1738Google Scholar
  26. 26.
    Mahlert F, Bauer C, Jaun B, Thauer RK, Duin EC (2002b) J Biol Inorg Chem 7:500–513Google Scholar
  27. 27.
    Telser J, Horng YC, Becker DF, Hoffman BM, Ragsdale SW (2000) J Am Chem Soc 122:182–183CrossRefGoogle Scholar
  28. 28.
    Telser J, Davydov R, Horng YC, Ragsdale SW, Hoffman BM (2001) J Am Chem Soc 123:5853–5860CrossRefPubMedGoogle Scholar
  29. 29.
    Tang Q, Carrington PE, Horng YC, Maroney MJ, Ragsdale SW, Bocian DF (2002) J Am Chem Soc 124:13242–13256CrossRefPubMedGoogle Scholar
  30. 30.
    Singh K, Horng YC, Ragsdale SW (2003) J Am Chem Soc 125:2436–2443CrossRefPubMedGoogle Scholar
  31. 31.
    Piskorski R, Jaun B (2003) J Am Chem Soc 125:13120–13125CrossRefPubMedGoogle Scholar
  32. 32.
    Craft JL, Horng YC, Ragsdale SW, Brunold TC (2004) J Biol Inorg Chem 9:77–89CrossRefPubMedGoogle Scholar
  33. 33.
    Wasserfallen A, Nölling J, Pfister P, Reeve J, de Macario EC (2000) Int J Syst Evol Microbiol 50:43–53PubMedGoogle Scholar
  34. 34.
    Gunsalus RP, Romesser JA, Wolfe RS (1978) Biochemistry 17:2374–2377PubMedGoogle Scholar
  35. 35.
    Kobelt A, Pfaltz A, Ankel-Fuchs D, Thauer RK (1987) FEBS Lett 214:265–268CrossRefGoogle Scholar
  36. 36.
    Ellermann J, Hedderich R, Böcher R, Thauer RK (1988) Eur J Biochem 172:669–677PubMedGoogle Scholar
  37. 37.
    Rospert S, Voges M, Berkessel A, Albracht SPJ, Thauer RK (1992) Eur J Biochem 210:101–107PubMedGoogle Scholar
  38. 38.
    Rospert S, Linder D, Ellermann J, Thauer RK (1990) Eur J Biochem 194:871–877PubMedGoogle Scholar
  39. 39.
    Bonacker LG, Baudner S, Mörschel E, Böcher R, Thauer RK (1993) Eur J Biochem 217:587–595PubMedGoogle Scholar
  40. 40.
    Bradford MM (1976) Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  41. 41.
    Beinert H, Albracht SPJ (1982) Biochim Biophys Acta 683:245–277CrossRefPubMedGoogle Scholar
  42. 42.
    Schönheit P, Moll J, Thauer RK (1980) Arch Microbiol 127:59–65Google Scholar
  43. 43.
    Bonacker LG, Baudner S, Thauer RK (1992) Eur J Biochem 206:87–92PubMedGoogle Scholar
  44. 44.
    Reeve JN, Nölling J, Morgan RM, Smith DR (1997) J Bacteriol 179:5975–5986PubMedGoogle Scholar
  45. 45.
    Hedderich R, Thauer RK (1988) FEBS Lett 234:223–227CrossRefGoogle Scholar
  46. 46.
    Gunsalus RP, Wolfe RS (1978) FEMS Lett 3:191–193CrossRefGoogle Scholar
  47. 47.
    Wackett LP, Honek JF, Begley TP, Wallace V, Orme-Johnson WH, Walsh CT (1987) Biochemistry 26:6012–6018PubMedGoogle Scholar
  48. 48.
    Belay N, Daniels L (1988) Antonie van Leeuwenhoek J Microbiol Serol 54:113–125Google Scholar
  49. 49.
    Ellermann J, Rospert S, Thauer RK, Bokranz M, Klein A, Voges M, Berkessel A (1989) Eur J Biochem 184:63–68PubMedGoogle Scholar
  50. 50.
    Albracht SPJ, Ankel-Fuchs D, van der Zwaan JW, Fontijn RD, Thauer RK (1986) Biochim Biophys Acta 870:50–57CrossRefGoogle Scholar
  51. 51.
    Albracht SPJ, Ankel-Fuchs D, Böcher R, Ellermann J, Moll J, van der Zwaan JW, Thauer RK (1988) Biochim Biophys Acta 955:86–102CrossRefGoogle Scholar
  52. 52.
    Wackett LP, Honek JF, Begley TP, Shames SL, Niederhoffer EC, Hausinger RP, Orme-Johnson WH, Walsh C (1988) Methyl-S-coenzyme-M reductase: a nickel-dependent enzyme catalyzing the terminal redox step in methane biogenesis. In: Lancaster J Jr (ed) The bioinorganic chemistry of nickel. VCH, Weinheim, pp 249–274Google Scholar
  53. 53.
    Holliger C, Kengen SW, Schraa G, Stams AJ, Zehnder AJ (1992) J Bacteriol 174:4435–4443PubMedGoogle Scholar
  54. 54.
    Lin S-K (1992) Doctoral thesis, ETH, ZürichGoogle Scholar
  55. 55.
    Selmer T, Kahnt J, Goubeaud M, Shima S, Grabarse W, Ermler U, Thauer RK (2000) J Biol Chem 275:3755–3760CrossRefPubMedGoogle Scholar
  56. 56.
    Stubbe JA, van der Donk WA (1998) Chem Rev 98:705–762CrossRefPubMedGoogle Scholar
  57. 57.
    Wagner AF, Frey M, Neugebauer FA, Schafer W, Knappe J (1992) Proc Natl Acad Sci USA 89:996–1000PubMedGoogle Scholar
  58. 58.
    Knappe J, Wagner AF (2001) Adv Protein Chem 58:277–315CrossRefPubMedGoogle Scholar
  59. 59.
    Olson KD, Chmurkowska-Cichowlas L, McMahon CW, Wolfe RS (1992) J Bacteriol 174:1007–1012PubMedGoogle Scholar
  60. 60.
    Wondimagegn T, Ghosh A (2001) J Am Chem Soc 123:1543–1544CrossRefPubMedGoogle Scholar
  61. 61.
    Jaun B (1990) Helv Chim Acta 73:2209–2217Google Scholar
  62. 62.
    Craft JL, Horng YC, Ragsdale SW, Brunold TC (2004) J Am Chem Soc 126:4068–4069CrossRefPubMedGoogle Scholar
  63. 63.
    Duin EC, Signor L, Piskorski R, Mahlert F, Clay MD, Goenrich M, Thauer RK, Jaun B, Johnson MK (2004) J Biol Inorg Chem (in press)Google Scholar

Copyright information

© SBIC 2004

Authors and Affiliations

  • Meike Goenrich
    • 1
  • Felix Mahlert
    • 1
  • Evert C. Duin
    • 2
  • Carsten Bauer
    • 3
  • Bernhard Jaun
    • 3
  • Rudolf K. Thauer
    • 1
  1. 1.Max-Planck-Institut für Terrestrische Mikrobiologie and Laboratorium für Mikrobiologie, Fachbereich BiologiePhilipps-UniversitätMarburgGermany
  2. 2.Department of ChemistryAuburn UniversityUSA
  3. 3.Laboratorium für Organische ChemieEidgenössische Technische Hochschule ZürichZurichSwitzerland

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