Structural insights into the energy-converting methanogenic Mtr complex by Cryo-EM
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Methanogenic archaea are the primary biological source of methane emissions, with current global emissions estimated at approximately ~0.5 Gt/year. Through methanogenesis, they catalyze the terminal step of organic matter degradation under strictly anaerobic conditions, occurring in environments such as soils, wetlands, rice paddies, marine and freshwater sediments, landfills, sewage and wastewater treatment systems, as well as the digestive tracts of ruminants and termites. A substantial fraction of global methane emissions is biogenic in origin, arising from these anaerobic microbial processes in both natural and anthropogenic systems, and contributing significantly to total anthropogenic methane emissions. Due to its substantially higher radiative efficiency and Global Warming Potential relative to carbon dioxide (CO₂), methane is a major contributor to contemporary climate change.
More than a century of research on methanogenic archaea has established a detailed scientific framework for understanding their physiology and metabolism. In recent decades, the core pathway of methanogenesis has been extensively investigated. The enzymatic steps involved in the conversion of substrates to methane, the unique cofactors required, and the majority of the participating enzymes have been well characterized.
Despite these advances, several knowledge gaps remain, particularly with regard to energy conservation mechanisms in methanogenesis. For example, the molecular details of how the exergonic methyl transfer from methyl-tetrahydromethanopterin (CH₃–H₄MPT) to coenzyme M (CoM) is coupled to sodium ion translocation remain unresolved. This reaction, catalyzed by the membrane-bound Mtr complex, represents the only energy conserving step in hydrogenotrophic methanogenesis and is essential in the major methanogenic pathways that utilize only one substrate.
In this thesis, I present my work on the Mtr complex from the mesophilic, freshwater methanogen Methanosarcina mazei. By applying genetic tools in the native organism, one-step purification of the Mtr complex was enabled using a strep-tagged MtrE subunit, thereby isolating an intact complex. Cryo-EM of the complex purified under aerobic conditions yielded a full assembly, revealing its overall architecture. Unexpectedly, a small protein, termed MtrI, was bound at the membrane-embedded MtrCDE trimeric core. Mass spectrometry demonstrated that MtrI associates with the complex only upon exposure to oxygen and is absent under strictly anoxic conditions, suggesting a role as an oxygen-responsive binding factor.
Subsequent cryo-EM analysis of the anaerobically purified complex revealed an unexpected metallocluster located within the cavity formed by the MtrCDE trimer, at the position previously occupied by MtrI. The geometry of this cluster closely resembles nitrogenase FeMo/FeV/FeFe cofactors and was modelled as an [Fe₈S₉C] L-type cluster. Its location adjacent to the coenzyme M binding site and the corrinoid, together with its close proximity to the conserved sodium binding site, suggests a direct role in the coupling of methyl transfer to sodium translocation. Distinct conformational states of the complex further reveal helical motions implying a putative sodium translocation pathway.
Altogether, this thesis advances our understanding of the molecular mechanisms underlying energy conservation in methanogenic archaea.
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