Elucidating the Role of Primary and Secondary Sphere Zn2+Ligands in the Cyanobacterial CO2Uptake Complex NDH-14: The Essentiality of Arginine in Zinc Coordination and Catalysis

Abstract The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO 2 -concentrating mechanism (CCM). Specialized Type-1 NDH complexes function as a part of this mechanism to couple photosynthetic energy generated by redox reactions of the electron transport chain (ETC) to CO 2 hydration. This active site of CO 2 hydration incorporates an arginine side chain as a Zn ligand, diverging from the typical histidine and/or cysteine residues found in standard CAs. In this study, we focused on mutating three amino acids in the active site of the constitutively expressed NDH-1 4 CO 2 hydration complex in Synechococcus sp. PCC7942: CupB-R91, which acts as a zinc ligand, and CupB-E95 and CupB-H89, both of which are in close interaction with the arginine ligand. These mutations aimed to explore how they affect the unusual metal ligation by CupB-R91 and potentially influence the unusual catalytic process. The most severe defects in activity among the targeted residues are due to a substitution of CupB-R91 and the ionically interacting E95 since both proved essential for the structural stability of the CupB protein. On the other hand, CupB-H89 mutations show a range of catalytic phenotypes indicating a role of this residue in the catalytic mechanism of CO 2 -hydration, but no evidence was obtained for aberrant carbonic anhydrase activity that would have indicated uncoupling of the CO 2 -hydration activity from proton pumping. The results are discussed in terms of possible alternative CO 2 hydration mechanisms.