Action at a distance: The remarkable coupling of CO ₂ uptake to electron transfer in specialized cyanobacterial NDH-1 complexes

Cyanobacteria achieve highly efficient photosynthesis using a CO₂-concentrating mechanism relying on specialized Type I (NDH-1) complexes. Among these, NDH-1₃ and NDH-1₄ catalyze redox-coupled hydration of CO₂ to bicarbonate, supporting carbon fixation in carboxysomes. The mechanism of coupling electron transfer to CO₂-hydration by these variant NDH-1 complexes remains unknown. We engineered a <i>Synechococcus</i> PCC7942 strain that expresses exclusively the high flux/low affinity NDH-1₄ complex, enabling the observation of the coupling of CO₂ hydration to cyclic electron flow in isolation from the other NDH-1 isoforms normally present in cells. We found that inhibition of the CupB protein by the carbonic anhydrase inhibitor ethoxzolamide (EZ) suppressed CO₂ uptake, slowed photosystem I rereduction, and abolished proton pumping as probed by acridine orange fluorescence. These effects were absent in strains lacking Cup proteins, confirming specificity. The results demonstrate that CO₂ hydration and electron transfer through NDH-1₄ are tightly coupled via proton translocation across the thylakoid membrane. These findings provide direct evidence for the bidirectional interaction in bioenergetic coupling between the plastoquinone reduction and the CO₂ uptake at the distal Zn-site over a span of ~150 Å and support a proton-removal hypothesis involving the proton transfer pathways from the Zn-site of CO₂ hydration to an energetically coupled proton loading site evolutionarily repurposed from the ancestral proton pumping mechanism to enable energetic CO₂ uptake.