Abstract text (incl. figure legends and references)

The prototypical hydrogen producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, catalyzes the conversion of formate to CO₂ and H₂ under anaerobic fermentative conditions. The enzyme is long known to share sequence homology with respiratory complex I, but an energy conserving character of FHL remains elusive.

We aim to get a better understanding of the FHL complex and the role it plays in bacterial bioenergetics by combining high-resolution electron cryo-microscopy (cryo-EM) with complementary biophysical methods.

Our cryo-EM structures reached resolutions of up to 2.6 Å and revealed important differences and similarities between FHL and complex I. We analyzed possible substrate proton pathways to the [NiFe] active site, which may play an important role in the mechanism of the enzyme. Furthermore, we identified an unpredicted metal-ion, which could play a regulatory role in binding the loosely attached formate dehydrogenase component.

Since FHL is an ancestor of respiratory complex I understanding its function on a molecular level sheds light on the evolution of energy conservation from early to modern life.