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 by autotrophic microorganisms 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 first cryo-EM structural analysis of DAB2, an essential membrane-associated protein complex for CO2 uptake in Halothiobacillus neapolitanus. The cytoplasmic subunit DabA2 displays a {beta}-carbonic anhydrase-like fold including a zinc ion, while the transmembrane subunit DabB2 resembles the proton-conducting subunits of respiratory Complex I. Purified DAB2 binds CO2 independent of proton motive force (PMF) however, did not spontaneously hydrate CO2. This suggests that CO2 hydration is PMF-dependent and may involve a gating mechanism. Structural analysis reveals an unconventional deeply buried active site only accessible via gated substrate tunnels, implying that substrate access, product release, and catalytic activation are tightly regulated. A unique transmembrane helix of DabA2 constitutes part of the proton conduction pathway and potentially couples proton translocation to enzymatic turnover. These features define a vectorial CO2 hydration mechanism that prohibits reverse bicarbonate dehydration and requires a proton gradient to initiate catalytic turnover. Our findings establish DAB2 as a prototype of a previously unrecognized family of PMF-driven carbonic anhydrases, elucidating a novel strategy for CO2 capture in non-photosynthetic autotrophs and expanding the mechanistic landscape of bacterial CCMs.