Architecture of the 8 MDa Hdr-Vhu-Fwd super-assembly in class I methanogens

Methanogens are central to global carbon cycling and among the largest biological sources of methane, a potent greenhouse gas¹. At the heart of their energy metabolism lies the Hdr-Vhu-Fwd super-assembly, which couples H₂ oxidation with CO₂ reduction through flavin-based electron bifurcation. Here we present the cryogenic electron microscopy structure of the Hdr-Vhu-Fwd super-assembly from Methanococcus maripaludis, revealing an 8 MDa complex comprising 252 polypeptide chains and over 600 redox cofactors. Cryo-electron tomography further support that this super-assembly forms an intact structure within the cytoplasm of intact cells. This architecture comprises two hexameric HdrABC-Vhu rings linked by a tetrameric FwdF core, forming a continuous, circular electron chain. In this unique arrangement, 12 polyferredoxin subunits (VhuB) connect the Vhu-Hdr and Fwd complexes, thereby coupling electron bifurcation with CO₂ reduction and directly linking the last and the first step of methanogenesis. Moreover, we identify a modular variant of the complex in which the [NiFe]-hydrogenase Vhu is substituted by tungsten-containing formate dehydrogenase (FdhAB), indicating flexible integration of electron-input modules facilitating metabolic adaptation under diverse environmental conditions². Analysis of the taxonomic distribution reveals that this architecture is specific to class I methanogens and is distinct from the smaller Hdr-Fmd complex of class II³. Together, our study reveals that the the Hdr-Vhu-Fwd super-assembly has a modular and adaptable bioenergetic assembly, suggesting a lineage-specific architecture to adapt to diverse anaerobic niches.