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

Our structural model reveals the electron-transfer pathway extending from H₂ oxidation at the [NiFe] active site in VhuADGU to CO₂ reduction by the tungstopterin active site in FwdBC (Fig. 4). A central component to this chain is the bifurcating FAD in HdrA of the HdrABC homodimeric subcomplex (Extended Data Figs. 6a and 8e). Here the electron flow diverges into high- and low-potential branches of the bifurcation reaction³¹ in the HdrABC subcomplex (Fig. 4b,c). In the high-potential pathway, electrons flow from the bifurcating FAD through a chain of [4Fe-4S] clusters in HdrA and HdrC to HdrB, where two non-cubane [4Fe-4S] clusters form the active site for CoM-S-S-CoB reduction. A set of conserved residues promotes proton transfer from the surface to the active site niche (Extended Data Fig. 6d–f). Concurrently, the low-potential pathway directs the electron flow from the bifurcating FAD to the inserted ferredoxin domain of HdrA (Extended Data Figs. 6c and 8a,b). Located in the C-terminal Fd-like region, the two [4Fe-4S] clusters are directly linked to the polyferredoxin VhuB that connects HdrA with FwdF. The FwdF subunit mediates electron transfer over a series of [4Fe-4S] clusters to tungstopterin cofactor in FwdBC.

To probe the electronic coupling within this super-assembly, we used in situ Fourier-transform infrared spectroscopy³². For this, the carbonyl absorbance bands of the [NiFe]-hydrogenase active-site cofactor³³ of VhuADGU are followed in time as a function of the gas composition across the sample film, that is, 10% CO₂ in N₂ with or without 10% H₂. In the absence of heterodisulfide, Fig. 4d and Extended Data Fig. 9 show how VhuADGU becomes reduced after contact with 10% H₂ as the one-electron-reduced Ni-L state (1,902 cm⁻¹) is populated over the oxidized Ni-SI state (1,918 cm⁻¹). The Ni-C state, a characteristically stable intermediate of [NiFe]-hydrogenase³⁴, is not observed (Supplementary Fig. 1). The concentration of CO₂ bound to formate dehydrogenase FdhAB (2,338 cm^–1) is unaffected by the reaction with H₂ and, when the atmosphere was switched back to 0% H₂, the hydrogenase remained in the reduced Ni-L state. However, in the presence of chemically synthesized heterodisulfide (Supplementary Fig. 2), a marked decrease of CO₂ was observed after reduction by 10% H₂ (Fig. 4d and Extended Data Fig. 9), indicative of CO₂-to-formate conversion through FdhAB under electron-bifurcating conditions. Without H₂, the CO₂ concentration remained stable. The electronic coupling becomes additionally evident when VhuADGU reverts back into a redox equilibrium between Ni-SI, Ni-L, and the two-electron-reduced Ni-R state (1,938 cm⁻¹) in the absence of H₂ (Supplementary Table 5). Importantly, this behaviour was not observed when heterodisulfide was absent from the reaction mixture (Fig. 4d and Supplementary Fig. 1), indicative of impaired electron efflux toward Hdr and Fwd.