Methyl-Coenzyme M Reductase
Devin Romines and David Marcey
© 2021 David Marcey

I. Introduction
II. Structure
III. Substrates and Reaction
IV. References


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I. Introduction

In methanogenic Archaea, the final step in the biomolecular pathway to generate methane is catalyzed by Methyl-Coenzyme M Reductase. This enzyme is a heterohexameric enzyme of ~300kD containing two identical active sites. The reaction it catalyzes combines two molecules, Methyl-Coenzyme M and Coenzyme B, into a heterodisulfide, releasing a methane molecule in the process. This anaerobic reaction (Figure 1) is the organism's only source of energy generation and produces approximately one billion tons of methane per year, some of which escapes into the atmosphere as a greenhouse gas. The reaction is facilitated by non-covalently bound Nickel porphinoid cofactors that promote the formation of a disulfide linkage between Methyl-Coenzyme M and Coenzyme B. Though the detrimental effects that uncontrolled greenhouse gases have on the environment are unquestionable, recent developments in technology have shown the potential for methane from anaerobic microbes to provide renewable energy.

This exhibit provides visualizations of some of the key structure-function relationships of this novel reductase, and is based on the work of Ermler, et al. (1995) and Grabarse, et al. (2001).

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II. Structure

Methyl-Coenzyme M Reductase is a hexamer containing two identical alpha subunits, two identical beta subunits, and two identical gamma subunits. These subunits are closely associated, with each subunit contacting every other subunit with the exception of the gamma subunits. The enzyme is present in multiple archaea and is highly conserved across many species, even distantly related ones. Composed mainly of alpha helices, the alpha and beta subunits are structurally very similar to one another (despite sharing only 14% sequence identity), suggesting that the enzyme may have originally existed as a tetramer that comprised four identical subunits encoded by two genes that subsequently diverged (see Figure 2). The gamma subunits may have been incorporated into the enzyme after the alpha and beta subunits diverged (Grabarse, et al., 2001).

These subunits form two active sites in the enzyme that are 50 apart. The first active site is comprised of the alpha, alpha', beta, and gamma subunits while the second active site is comprised of the alpha', alpha, beta', and gamma' subunits. Arrows show the entrance points and the cavities at the openings of the channel that leads to the active sites are indicated. Both active sites require four of the six subunits - this enzyme cannot exist as two separate enzymes and only functions in this two active site form.

Each active site is structured in the following manner. A 25 opening at the surface of the molecule allows for the substrates to enter into the active site. This 30 funnel-shaped channel leads to a cavity at the heart of the enzyme where the Nickel porphinoid resides. The funnel narrows to 8, restricting the molecules that can enter the active site region and facilitating the development of an anaerobic environment where the reaction can take place. The channel and cavity is visualized here by removing the alpha subunits. Arrows trace the path of one of the channels as viewed from several perspectives.

The Nickel porphinoids, also known as Cofactor F430s, are the heart of the active sites, and facilitate the formation of the disulfide linkage between Methyl-Coenzyme M and Coenzyme B, releasing a methane molecule in the process. These two cofactors are non-covalently anchored in the active site region by a network of hydrogen bonding. Here, H-bonds are indicated between one of the cofactors and sidechain or backbone atoms of Glnα332, Tyrα333, Glyα329, Valα32, Glnα'230, Valα'145, Valα'146, Glnα'147, Tyrβ367, Ileβ366, Serγ'154, Valγ155, Glyγ119, Serγ118, and Hisγ156.

The Nickel atoms in the two cofactors can exist in the Ni(I), Ni(II), or Ni(III) oxidation states. The Ni atoms are hexagonally coordinated, as shown here for one of the porphinoids. The four equatorial ligands are the nitrogen atoms of the tetrapyrrole ring. The fifth, axial ligand is the side chain oxygen of Glnα'147. The sixth ligand, also axial, is the thiol group of the Methyl-Coenzyme M when the substrates are bound and is the sulfonate oxygen of the heterodisulfide after the reaction has taken place (see below).

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III. Substrates and the Methane Generating Reaction

The first substrate to enter the active site region is Methyl-Coenzyme M. It is positioned nearly parallel to the Nickel porphinoid plane. The thiol group of this substrate interacts not only with the Nickel atom in the porphinoid, but also with the hydroxyl groups of Tyrα333 and Tyrβ367. The sulfonate group of the substrate anchors it to the active site by forming a salt bridge with the guanidium group of Argγ120 and by forming hydrogen bonds to the peptide nitrogen and peptide oxygen of Tyrα444 and Hisβ364 respectively. When bound, Methyl-Coenzyme M elicits a conformational change in the active site channel, allowing Coenzyme B to enter. This ensures that Coenzyme B does not enter the channel prior to Methyl-Coenzyme M.

Following the positioning of Methyl-Coenzyme M, the larger Coenzyme B enters the channel and is positioned by a network of molecular interactions. The molecule’s threoninephosphate moiety forms salt bridges with side chains of four positively charged amino acids, namely Argα270, methyl-Hisα'257, Lysα'256, Argα'225, and the amino of Glyβ369. Multiple van der Waals interactions are observed between the heptanoyl arm of Coenzyme B and a group of hydrophobic amino acids: Leuα320, Pheα330, Pheβ362, Pheβ361, and Pheα443. The thiol group of the molecule is stabilized by the side chain nitrogen of Asnα481 and the main chain peptide nitrogen of Valα482. These intermolecular interactions position Coenzyme B ~6 away from Methyl-Coenzyme M in preparation for their linkage and release of methane. The presence of Coenzyme B narrows the channel to a width of around 6, which denies other molecules access to the active site channel. The reaction is thus inaccessible to bulk solvent and occurs in an anaerobic environment.

The reaction that occurs between Methyl-Coenzyme M and Coenzyme B to produce their combined heterodisulfide and methane occurs in the hydrophobic, aromatic environment created by the sterochemistry of the active site channel and cavity of the enzyme, as well as the interactions that the substrates have with the enzyme to alter its conformation. When undergoing the reaction, the Methyl-Coenzyme M moves more than 4 from its starting position, making a 90o rotation up towards Coenzyme B. This results in the substrate interacting with new residues of the enzyme. As mentioned previously, one of the substrate's sulfonate oxygen atoms is axially coordinated with the Nickel atom and the hydroxyl group of Tyrα333. Another oxygen atom forms a hydrogen bond to the lactam ring of F430 and to the hydroxyl group of Tyrβ367. Figure 3 illustrates the mechanism by which the reaction progresses. The two sulfurs of the thiol groups of Methyl-Coenzyme M and Coenzyme B come close enough to allow van der Waals interactions when the methyl group of Methyl-Coenzyme M is near the Nickel atom. The formation of the heterodisulfide results in the release of a hydrogen ion from Coenzyme B that is accepted by the activated methyl group of Methyl-Coenzyme M, thus producing methane. Although not precisely understood, it is likely that the repulsion from the Nickel atom and the sulfonate oxygen of the heterodisulfide provides the force necessary for the heterodisulfide to exit the channel and for the methane to follow.

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IV. References

Ermler, U., Grabarse, W., Shima, S., Goubeaud, M., and Thauer, R. K. (1997). Crystal structure of methyl-coenzyme m reductase: the key enzyme of biological methane formation. Science, 278(5342), 1457-62.

Grabarse, W., Mahlert, F., Duin, E. C., Goubeaud, M., Shima, S., Thauer, R. K.( 2001). On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme m reductase upon substrate binding. Journal of Molecular Biology, 309(1), 315-330. https://doi.org/10.1006/jmbi.2001.4647

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