JBIC Journal of Biological Inorganic Chemistry

, Volume 9, Issue 6, pp 691–705

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


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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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