Methanogenesis
Methanogenic archaea release up to 1 Gt of methane (CH4) into the environment every year. Methane is a potent greenhouse gas with a greater warming potential than carbon dioxide. Thereby, given its ecologial and environmental relevance, we focus on understanding the molecular properties of core methanogenic proteins and supercomplexes that orchestrate methane production.
Depending on the species, methanogens thrive from different electron sources via an archaeal version of the Wood-Ljungdahl pathway. However, their core metabolism is governed by some of the unique systems that we study in our group. Among these, we are working with the methyl-coenzyme M reductase (Mcr), the greatest biological methane generator and one of the most abundant enzymes in Nature, specifically with its peculiar activation mechanism. Similarly, we have determined the overall architecture of the sodium-translocating methyltransferase complex (Mtr), which couples the transfer of a methyl group with the translocation of sodium ions to drive energy conservation which actually provides the methyl substrate for Mcr.
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Our research group is specialized in the manipulation of strictly anaerobic methanogens. We believe that the integration of anaerobic cultivation, genetic engineering, protein biochemistry, and high-resolution structural biology certainly prospect on expanding the catalogue of these extraordinary enzymatic machines.
I. Methyl-coenzyme M reductase and the activation of coenzyme F430.
The ability of Mcr to produce methane is only possible thanks to coenzyme F430: a nickel-coordinated prosthetic group with four pyrrole-derived (A-D), one gamma-lactam, and a cyclohexanone ring that rests deeply within both active sites of Mcr. Even though coenzyme F430 is a highly reduced molecule, the activity of Mcr relies on the oxidation state of the nickel ion. Although the redox states +2 and +3 are stable, Ni-F430 must be reduced to the +1 state for Mcr to be enzymatically active. Intriguingly, the activation of F430 requires a tremendous amount of energy (Eº' = -650 mV) that methanogens need to overcome in order to grow. Such activation process was previously demonstrated to be ATP-dependent and actioned by the small chaperone protein McrC (highly conserved in the Mcr operon of methanogenic archaea). However, the mechanistic details on the ATP-driven reduction of Ni-F430 were still pending.
We resolved the cryoEM structure of Mcr's activation complex in the absence of oxygen and after the incubation with ATP to an astonishing high resolution ranging from 1.8 to 2.1 Å! This model clearly showed the role of McrC not only to recruit a set of auxiliary proteins - or methanogenic marker proteins (Mmp) - that asymmetrically bind to one half of Mcr, but also comes with the previously proposed "A2 component": an ABC-transporter and very special ATPase that sits on the back of the Mcr core.
On top of the strict ATP-dependency of the activation complex, our high-resolution model enabled us to unveil a series of completely unexpected and very unique type of metal cofactors. At the core of McrC and the activating subunits, threee large iron-sulfur cluster shape a clear electron transfer pathway towards the most proximal Ni-F430. Altogether, the metal composition, molecular topology, and EPR spectroscopic features indicate that these cofactors are reminiscent of the L-cluster: precursor of the catalytic nitrogenase cluster or FeMoco/M-cluster.
In order to test whether our enzymatic preparation could perform the activation of Ni-F430, we followed the production of methane under ATP-depleted and ATP-rich conditions. The result: we observed a significant difference in the methane generated after the external addition of ATP.
The presence of L-clusters ([8Fe-9S-C]) provides new evolutionary insights between nitrogen fixation and the activation of methanogenesis. Our phylogenetic reconstructions imply that these cofactors likely had an origin in the reduction of coenzyme F430 before being later incorporated into nitrogenase and nitrogenase-like systems.
II. Architecture of the sodium pumping Mtr from methanogenic archaea
The N⁵-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr) couples the exergonic transfer of a methyl group from methyl-tetrahydromethanopterin to coenzyme M to form the last methanogenic intermediate methyl-coenzyme M. This methyl transfer between two methanogen-specific cofactors is coupled to the sodium ion transport across the membrane, forming the only energy-conserving step in hydrogenotrophic methanogenesis. This sodium-motive force drives chemiosmotic energy conservation and ATP synthesis via a Na⁺-dependent ATP synthase, forming the basis of what is often referred to as sodium-based bioenergetics in methanogenic archaea. Even though it is known that the full Mtr complex (a trimer of MtrABCDEFGH) presents a molecular weight >650 kDa, the available structural information had remain limited to only partial reconstructions. Not only the mechanisms governing the sodium-coupled methyl transfer are poorly understood, but also details on the regulation of Mtr (i.e., in response to environmental stress) have remained largely unknown.
Our efforts for the isolation of the Mtr complex from Methanosarcina mazei led to the cryoEM model of the full assembly with an outstanding resolution of 2.1 Å. This model offers previously unseen details of the characteristic cloverleaf-shaped architecture of Mtr. The Mtr core complex consists of a trimer of hetero-nonameric protomers, each composed of three multi-spanning transmembrane subunits (MtrCDE), four single-spanning transmembrane subunits (MtrABFG), and a dimeric cytosolic methyltransferase, MtrH. The trimerization interface is formed by the single-spanning transmembrane subunits MtrABFG, creating a dodecameric core.
Furthermore, we identified a hydrophilic charged pocket within the transmembrane domain of MtrE, which corresponds to the binding site for sodium ions in the Mtr complex. At this position, the cryoEM map consistently shows the sodium ion being octahedrally coordinated by six oxygen ligands within the expected distance range. This coordination site is highly conserved among Mtr homologues among methanogenic archaea.
A particularly exciting discovery was the identification of MtrI, a previously uncharacterised small protein that tightly associates to the MtrCDE cavity and is conserved within the order of Methanosarcinales. Surprisingly, the levels of MtrI increased in oxygen-exposed preparations compared to those strictly anaerobic - regardless of the growing conditions. MtrI was only found when the cobamide cofactor is in the oxidized Co(III) state, but rather absent in the reduced states Co(I) and Co(II). Thus, MtrI binding to the Mtr complex appears to be redox-dependent and it is likely associated with the evolutionary adaptation of Methanosarcinales species to oxygen exposure.
