Introduction: During mixed-acid fermentation, a number of Enterobacteriaceae produce hydrogen (H₂) using the formate hydrogenlyase (FHL) complex, a progenitor of complex I of the respiratory chain. The FHL complex serves to maintain pH homeostasis of the cytoplasm and possibly to translocate nH⁺. The complex"s soluble arm, which is oriented towards the cytoplasm, facilitates two reactions: a formate dehydrogenase (FdhH) catalyses the oxidation of formate to CO₂ and a H⁺; a hydrogenase domain receives two electrons from this oxidation via iron-sulfur clusters and reduces 2H⁺ to H₂. This soluble arm is also connected to the membrane domain, which includes a variable number of transmembrane subunits. While the well-studied FHL-1 complex in E. coli includes two membrane subunits (HycCD), the less well-explored FHL-2 complex has five membrane subunits (HyfB-F). The enterobacterium Trabulsiella guamensis has a single FHL-2_Tg complex.

Goals: Despite their shared phylogeny with complex I, the potential role of the membrane subunits of FHL-2_Tg in proton pumping and their contribution to the catalytic reactions of the soluble arm remain unresolved. To explore their possible contribution to proton translocation and catalysis, an intact, active FHL-2_Tg complex must be isolated.

Materials & Methods: We created plasmids carrying the hyf genes from T. guamensis and introduced them into a hydrogenase-deficient strain of E. coli. The FHL-2_Tg was synthesized and purified anaerobically from this strain using a StrepTag-based system. The analyses included activity assays, gas chromatography, Blue-Native & SDS-PAGE, as well as western blots.

Results: We successfully purified an FHL-2_Tg complex anaerobically that disproportionates formic acid to H₂ and CO₂. Although the aerobically purified complex lacks this activity, the individual enzymatic components retain activity when measured using viologen dyes. Structural and biochemical analyses of this complex will be reported.

Summary: Conditions have been optimized for the purification of an active FHL-2_Tg complex, marking the first step towards examining the potential proton-pumping activity of this complex.