Structural Basis of Membrane Potential Coupled Vectorial CO ₂ Hydration by the DAB2 Complex in Chemolithoautotrophs

Abstract The fixation of dissolved inorganic carbon (DIC) such as CO 2 and bicarbonate by autotrophic microorganisms is fundamental to the global primary production. Many autotrophs depend on a diversity of CO 2 -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 CO 2 uptake in Halothiobacillus neapolitanus . The cytoplasmic subunit DabA2 displays a β-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 CO 2 independent of proton motive force (PMF) however, did not spontaneously hydrate CO 2 . This suggests that CO 2 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 CO 2 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 CO 2 capture in non-photosynthetic autotrophs and expanding the mechanistic landscape of bacterial CCMs.