Spectroscopic investigation of the nickel-containing porphinoid cofactor F430. Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent, red and ox forms

  • Evert C. Duin
  • Luca Signor
  • Rafal Piskorski
  • Felix Mahlert
  • Michael D. Clay
  • Meike Goenrich
  • Rudolf K. Thauer
  • Bernhard Jaun
  • Michael K. Johnson
Original Article


Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F430, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F430 in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F430, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F430M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle π orbitals that are unique to Ni(I) forms of F430. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F430 macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.


Methyl-coenzyme M reductase Nickel enzymes Factor 430 Methanogenic archaea Magnetic circular dichroism spectroscopy 



cofactor 430


penta-methylated form of cofactor 430


F430M with the nickel atom in the +1 oxidation state


F430M with the nickel atom in the +2 oxidation state


F430M with the nickel atom in the +3 oxidation state


methyl-coenzyme M reductase


MCR exhibiting the MCR-ox1 EPR signal


EPR silent form of MCR obtained from the MCRox1 form


MCR exhibiting the EPR signals red1c and/or red1m


MCRred1 in the presence of coenzyme M


MCRred1 in the presence of methyl-coenzyme M


MCR exhibiting both the red1 and red2 EPR signals


EPR silent form of MCR obtained from the MCRred1 form


EPR silent form of MCR



This work was supported by the Max-Planck-Gesellschaft (R.K.T), by the Fonds der Chemischen Industrie (R.K.T.), and by grants from the National Institutes of Health (GM60329 and GM62542 to M.K.J.), the National Science Foundation (MCB98008857 to M.K.J) and the Swiss National Science Foundation (20-66773 to L.S, R.P, B.J). M.G is a recipient of a scholarship of the Claussen-Simon-Stiftung. We thank Dr. Richard C. Conover for help in fitting the VHVT MCD saturation magnetization data and the reviewers for many insightful comments and suggestions.

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Copyright information

© SBIC 2004

Authors and Affiliations

  • Evert C. Duin
    • 1
  • Luca Signor
    • 2
  • Rafal Piskorski
    • 2
  • Felix Mahlert
    • 3
  • Michael D. Clay
    • 4
  • Meike Goenrich
    • 3
  • Rudolf K. Thauer
    • 3
  • Bernhard Jaun
    • 2
  • Michael K. Johnson
    • 4
  1. 1.Department of Chemistry and BiochemistryAuburn UniversityAuburnUSA
  2. 2.Laboratorium für Organische ChemieETH ZürichZürichSwitzerland
  3. 3.Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie, Fachbereich BiologiePhilipps-UniversitätMarburgGermany
  4. 4.Department of Chemistry and Center for Metalloenzyme StudiesUniversity of GeorgiaAthensUSA

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