Methyl (Alkyl)-Coenzyme M Reductases: Nickel F-430-Containing Enzymes Involved in Anaerobic Methane Formation and in Anaerobic Oxidation of Methane or of Short Chain Alkanes

Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. The active enzyme harbors the nickel(I) hydrocorphin coenzyme F-430 as a prosthetic group and catalyzes the reversible reduction of methyl-coenzyme M (CH₃-S-CoM) with coenzyme B (HS-CoM) to methane and CoM-S-S-CoB. MCR is also involved in anaerobic methane oxidation in reverse of methanogenesis and most probably in the anaerobic oxidation of ethane, propane, and butane. The challenging question is how the unreactive CH₃-S thioether bond in methyl-coenzyme M and the even more unreactive C-H bond in methane and the other hydrocarbons are anaerobically cleaved. A key to the answer is the negative redox potential (E_o') of the Ni(II)F-430/Ni(I)F-430 couple below -600 mV and the radical nature of Ni(I)F-430. However, the negative one-electron redox potential is also the Achilles heel of MCR; it makes the nickel enzyme one of the most O₂-sensitive enzymes known to date. Even under physiological conditions, the Ni(I) in MCR is oxidized to the Ni(II) or Ni(III) states, e.g., when in the cells the redox potential (E') of the CoM-S-S-CoB/HS-CoM and HS-CoB couple (E_o' = -140 mV) gets too high. Methanogens therefore harbor an enzyme system for the reactivation of inactivated MCR in an ATP-dependent reduction reaction. Purification of active MCR in the Ni(I) oxidation state is very challenging and has been achieved in only a few laboratories. This perspective reviews the function, structure, and properties of MCR, what is known and not known about the catalytic mechanism, how the inactive enzyme is reactivated, and what remains to be discovered.

Figure 1

Reaction catalyzed by methyl-coenzyme M reductase that in the active form has a greenish color derived from Ni(I)F-430. Ni(I)F430 has a λ_max = 382 nm and a weaker maximum at 754 nm. In the inactive state, MCR is yellow from Ni(II)F-430 that has an absorbance maximum at 430 nm. When MCR catalyzes the reduction of methyl-coenzyme M with coenzyme B in 100% D₂O, DCH₃ and some D₂CH₂ are formed. When MCR catalyzes CH₄ oxidation to methyl-coenzyme M in 100% D₂O, only CH₃–S-CoM is formed. It follows that the intermediates in the catalytic cycle do not exchange hydrogen with deuterium of the solvent. The hydrogen or deuterium atoms involved in the reaction are carried into the active site only via the thiol group of coenzyme B or, in the reverse reaction, via methane. The standard free energy change (ΔG^o) associated with ethane formation from ethyl-coenzyme M and coenzyme B is near −20 kJ/mol and thus by −10 kJ/mol less exergonic than ΔG^o = −30 kJ/mol associated with methane formation from methyl-coenzyme M and coenzyme B (see the paragraph on “Reversibility”).

Figure 2

Role of methyl-coenzyme M reductase (MCR) in the global carbon cycle. GPP, gross primary production. Terrestrial GPP ≈ 500 Gt CO₂/year. Marine GPP ≈ 300 Gt C/year. Methyl-X, methanol, methylthiol, methylamines, choline, and betaine. 3-PGA, 3-phosphoglycerate. Electron acceptors for anaerobic methane oxidation are highlighted in orange. For a review on the anaerobic oxidation of ethane, propane, and butane, see Singh et al., 2017.

Figure 3

Phylogenetic relation of McrA amino acid sequences present in Euryarchaeota (black, red, and orange branches) and in Bathyarchaeota (blue branches). The phylogenetic tree was constructed based on a maximum likelihood algorithm considering more than 450 amino acid positions. The scale bar indicates the number of amino acid substitutions per site. Bootstrap values higher than 90% are indicated by filled circles on the corresponding branch. Black branches, sequences from methanogens and methanotrophic archaea. Red branches, sequences from ca. Syntrophoarchaeum; SBU, ca. S. butanivorans; SCAL, ca. S. caldarius. Orange branch, sequence from ca. Argoarchaeum ethanivorans. Blue branches indicate Bathyarchaeota-related sequences. The identifiers refer to the locus tag of the gene sequences in the draft genomes. MrtA is synonymous to McrA isoenzymes II in the corresponding groups. The figure and legend are taken from Laso-Peres et al., 2016, (with permission of Nature-Springer), and updated by the orange branch with the branching order taken from Chen et al., 2019.

Figure 4

Energy metabolism of Methanothermobacter marburgensis. MFR, methanofuran. H₄MPT, tetrahydromethanopterin. F₄₂₀, coenzyme F₄₂₀. HS-CoM, coenzyme M. HS-CoB, coenzyme B. Fd, ferredoxin. MCR, methyl-coenzyme M reductase. Yellow dot, heterodisulfide reductase- hydrogenase complex (HdrABC-MvhADG) that couples the exergonic reduction of CoM-S–S-CoB with H₂ with the endergonic reduction of ferredoxin with H₂ via flavin-based electron bifurcation.^,− The redox potentials, in blue, are given under standard conditions.

Figure 5

Schematic drawing of the active site of inactive methyl-coenzyme M reductase (MCR) with F-430 in the Ni(II) oxidation state based on crystal structures up to 1.1 Å resolution of isoenzymes I from M. marburgensis. (A) MCR-ox1-silent, MCR in complex with coenzyme M (CoM-SH) and coenzyme B (CoB-SH). (B), MCR-silent, MCR with the heterodisulfide CoM-S–S-CoB bound. Sulfur in green, carbon yellow, nickel in blue, and oxygen in red. From Mahlert et al., 2002.

Figure 6

Transition state 1 according to the Siegbahn proposal. Two bonds are broken (C–S and S–H, drawn blue), and two bonds are formed (Ni–S and C–H, drawn red) in a single step. F-430 is depicted as “Ni”; R mimics CoM, and R′ mimics CoB. OAr depicts phenol groups used in the model for the tyrosine residues. From Scheller et al., 2013.

Figure 7

Schematic drawing of the catalytic cycle of methyl-coenzyme M reductase (MCR). The Ni(I) oxidation states are highlighted in greenish color, the Ni(II) oxidation states in yellow, and in the Ni(III) oxidation states in orange. TC, ternary complex. TS, transition state. IM, intermediate. *, increased reactivity of Ni(I)F-430 as a result of coenzyme B binding. k₊₁ = rate constant of the forward reaction (methane formation) (in black); k_–1 = rate constant of the back reaction (methane oxidation) (in dark blue); k_–2 = rate constant of the formation of CH₃–S-CoM from sterically contained methane (in red). The Ni(I) oxidation states exhibit characteristic EPR spectra: MCR-red1a, MCR in the absence of substrates; MCR-red1m, MCR in the presence of methyl-coenzyme M; MCR-red1c, MCR in the presence of coenzyme M; MCR-red1/2, MCR in the presence of coenzyme M and coenzyme B. In MCR-red1/2, part of the enzyme is in a Ni(III) hydride state (Figure 9). In the MCR-ox1 state, the thiol sulfur of coenzyme M is bound to the Ni(III) of F-430 (Figure 12). For details, see the text.

Figure 8

Electron nuclear double resonance (ENDOR) structure of methyl-coenzyme M bound to Ni(I) in the active site of methyl-coenzyme M reductase, exhibiting the EPR signal MCR-red1m. The Ni–S bond length is 3.94 Å, compared with a Ni–S bond length of 2.48 Å in the proposed transition state (Figure 6). From Hinderberger et al., 2008.

Figure 9

EPR spectra of methyl-coenzyme M reductase (MCR) with coenzyme M bound (MCR-red1c) and after additional binding of coenzyme B (MCR-red1/2). At saturating coenzyme B concentrations, the MCR-red1/2 spectrum is composed of 50% of the EPR spectrum MCR-red1cc, which is very similar to the EPR spectrum MCR-red1c and 50% of the MCR-red2, which in turn is composed of the axial MCR-red2a spectrum and the rhombic MCR-red2r spectrum. The active site nickel coordinations of MCR-red1cc, MCR-red2a, and MCR-red2r deduced from the spectra rather than the spectra are shown. Note that for MCR-red2a an alternative structure has been proposed, in which the acidic CoM-SH proton is making a hydrogen bond to the Ni ion. The color of MCR in the red1/2 state is greenish orange and that of MCR in the red1 cm³ state is greenish.^,, MCRs with nickel in the 3+ oxidation state have been shown to have a more orange than yellow color. MCR-red2r is expected to have a greenish color similar to that of other MCRs with nickel in the 1+ oxidation state.

Figure 10

Isotopic exchange of deuterium from D₂O into the methyl group of methyl-coenzyme M, consistent with formation of a σ-alkane-nickel complex as an intermediate. For clarity, the intermediates are drawn with the non-natural substrate ethyl-coenzyme M labeled with one ¹³C (drawn in red) in a deuterated medium. The ligand of coenzyme F-430 is shown schematically as bold lines. The bend in the Ni(III) intermediate symbolizes two cis coordination sites and a distorted equatorial macrocycle. The legend and figure are from Scheller et al., 2010 (reproduced with permission of Wiley-VCH Verlag GmbH & Co. KG). (See also refs (16) and (138)).

Figure 11

Proton inventory for methyl-coenzyme M reduction with coenzyme B to methane in H₂O/D₂O containing increasing amounts of deuterium as catalyzed by MCR I of M. marburgensis. The reaction was performed in the presence of Ti(III) and traces of cob(II)alamin to regenerate coenzyme B from CoM-S–S-CoB. The figure was taken with permission from the Ph.D. Thesis of S. Scheller (Figure 4) and slightly modified. When coenzyme B was limiting (not regenerated by reduction of CoM-S–S-CoB), then the apparent fractionation factor was found to be 0.297.

Figure 12

Reductive activation of inactive MCR-silent and MCR-ox1 to MCR-red1a. The active site nickel coordination deduced from the EPR spectra is shown. For coordination of CoM-S–S-CoB via the sulfonate group of CoM-SH in MCR-silent, see Figure 5B. A2 is a 60 kDa protein with two ATP-binding sites and A3a a 700 kDa nonhomogeneous iron–sulfur flavoprotein complex composed of several different proteins. The electrons can be provided by dithiothreitol.