Methanogenic archaea use complex enzyme systems to survive in anoxic environments with limited energy. A key mechanism for energy conservation is electron bifurcation, a reaction that "splits" the energy of a pair of electrons, thus making one electron more reductive at the cost of the other.

In a new study, researchers from the Max-Planck Institute for Terrestrial Microbiology and the Max-Planck Institute for Biophysics in Germany discovered a huge enzyme complex from a methanogenic archaea, which directly transfers electrons from electron bifurcation reactions to the reduction and fixation of carbon dioxide. Their detailed insights into this efficient energy conversion process may open new possibilities for sustainable biotechnology development. The findings were published in Science, entitled "Three-megadalton complex of methanogenic electron-bifurcating and CO2-fixing enzymes".

It is estimated that 1 billion tons of methane are produced annually by anaerobic microorganisms called methanogenic archaea. Since methane is a potent greenhouse gas, increased atmospheric methane concentrations threaten life and livelihoods. On the other hand, biomethane produced by anaerobic digestion of capture waste and wastewater may be a renewable fuel source. Therefore, understanding the mechanism of microbial methane formation has the potential to stimulate and support environmental protection efforts.

Methanogenic Archaea compete successfully by performing methanogenesis, which is one of the final steps in the anaerobic decomposition of organotrophs, usually under extreme conditions. Most methanogenic archaea produce methane from carbon dioxide (CO2) and hydrogen (H2) via a methanogenic cycle involving multiple enzymatic reactions. In typical methanogenic habitats, this reaction releases only a small amount of energy, so methanogens require efficient enzyme systems to survive in this energy-limited environment.

A particularly complex step in the methanogenic cycle is known as flavin-based electron bifurcation (FBEB). It has been hypothesized that methanogens transfer high-energy electron transfer in this reaction to immobilize carbon dioxide through a small electron carrier protein, ferredoxin, that freely diffuses in cells.

Surprisingly, in this study, these researchers found that transfer of electrons from FBEB to carbon dioxide reduction did not require such an electron carrier. They purified enzyme complex composed of formate dehydrogenase (Fdh), heterodisulfide reductase (Hdr) and formylmethanofuran dehydrogenase (Fmd) from the Methanospirillum hungatei. This species, as well as many other methanogens, often appear in anaerobic digesters that treat organic waste such as municipal wastewater or industrial waste.

These researchers described the function of this enzyme complex with an enzymatic assay and unraveled its structure by cryo-electron microscopy (Cryo-EM). Its structure shows that the enzymes that catalyze the last and first steps of the methanogenic cycle form a huge enzyme complex, which directly links these two steps—formic acid-driven FBEB and carbon dioxide reduction—and thus does not use the diffusible electron carrier protein ferredoxin.

Tomohiro Watanabe of the Max-Planck Institute for Terrestrial Microbiology, lead author of the paper, said, "Our structural analysis shows a huge enzyme complex. An electron transfer chain protein, namely polyferredoxin, forms a conductive pathway that directs high-energy electrons from FBEB directly to carbon dioxide reduction rather than through a soluble electron carrier. This means less opportunity to lose these precious electrons."

Structural comparison and previously published interaction assays suggest that this higher-order structure of Hdr and Fmd complexes may be common in different methanogenic archaea. These structures also provide new insights into the fine-tuning mechanism of FBEB. Bonnie Murphy, co-corresponding author of the paper, explained, "Cryo-electron microscopy methods allow us to use image classification to solve the structures of different conformational states present in the same sample. Under this new study, we find that the two different conformational states of this complex differ by the large rotation of the part we call the 'mobile arm'. By rotating between these two states, the complex controls the influx and egress of electrons into and out of the FBEB site."

Together, these findings contribute to our understanding of how energy metabolism in methanogenic archaea is fine-tuned for efficiency: by controlling electron influx and efflux into FBEB, and by allowing direct transfer of energetic electrons to fix carbon dioxide. Knowledge will help to design strategies to reduce greenhouse gas emissions and may enable electronic bifurcations to be more widely used in biotechnology.