Structural basis of membrane potential coupled vectorial CO₂ hydration by the DAB2 complex in chemolithoautotrophs.

The fixation of dissolved inorganic carbon (DIC) such as CO2 and bicarbonate is fundamental to the global primary production. Many autotrophs depend on a diversity of CO2-concentrating mechanisms (CCMs) to overcome the inefficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the limited supply of DIC. While cyanobacterial CCMs are well characterized, analogous systems in chemolithoautotrophs, specifically active DIC uptake systems have long been overlooked. Here, we present the cryo-EM analysis of DAB2, an essential membrane protein complex for CO₂ uptake in Halothiobacillus neapolitanus. The cytoplasmic subunit DabA2 displays a β-carbonic anhydrase-like fold, while the transmembrane subunit DabB2 resembles the proton-conducting subunits of respiratory Complex I. Purified DAB2 binds CO₂ independent of protonmotive force (PMF); however, did not spontaneously hydrate CO2. Structural analysis reveals a deeply buried active site only accessible via gated substrate tunnels, suggesting substrate access and catalysis are tightly regulated. A distinct transmembrane helix of DabA2 forms the proton pathway and potentially couples proton translocation to catalysis. These features define a vectorial CO2 hydration mechanism that prohibits reverse bicarbonate dehydration. Our findings establish DAB2 as a prototype of a family of PMF-driven carbonic anhydrases, elucidating a distinct strategy for CO₂ capture in non-photosynthetic autotrophs.