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

Abstract

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.

Keywords

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

Abbreviations

BES

2-bromoethanesulfonate

BPS

3-bromopropanesulfonate

CH3-S-CoM

methyl-coenzyme M

HS-CoB

coenzyme B

HS-CoM

coenzyme M

MCR

methyl-coenzyme M reductase

MCR-ox

MCR exhibiting the EPR signals ox1, ox2 or ox3

MCR-red1

MCR exhibiting the EPR signals red1a, red1c or red1m

MCR-red1a

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

MCR-red1c

MCR-red1 in the presence of coenzyme M

MCR-red1m

MCR-red1 in the presence of methyl-coenzyme M

MCR-red2

MCR exhibiting both the red1 and red2 EPR signals

Notes

Acknowledgements

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.

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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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