Structure-function studies to elucidate the mechanism of unique CO₂ uptake proteins in cyanobacteria

In cyanobacteria, unique CO₂-uptake (Cup) proteins function as a part of the CO₂-concentrating mechanism (CCM), which produces a high concentration of CO₂ around the main photosynthetic biomass-producing enzyme ribulose bisphosphate carboxylase/oxygenase (RubisCO). Cup proteins are bound to specialized NDH-1 complexes, suggesting the coupling of CO₂-hydration and redox activity of the photosynthetic electron transport (PET) system. The active sites of unique Cup proteins (CupA and CupB) incorporate arginine as a first-sphere Zn ligand, diverging from the typical histidine or cysteine residues found in standard carbonic anhydrases (CAs), a well-studied enzyme family which also hydrates CO₂. Here, point mutation studies of Cup in Synechococcus elongatus sp. PCC 7942 (hereafter Syn7942) revealed a critical Arg–Glu dyad for CupB stability: the loss of cellular CupB accumulation observed for mutations at CupB-E95 and CupB-Arg91 demonstrates their contribution to the enzyme’s structural integrity (Artier et al. 2022; Walker, Zhang, and Burnap 2024). These studies also found His89 of CupB (the constitutively expressed, low-affinity Cup protein) to play a catalytic role in the hydration of CO₂. We proposed that CupB-His89 acts as a vital proton-handling residue within the putative active site of CupB, reducing the likelihood of the back-reaction of Zn–OH hydration and pulling the reaction towards bicarbonate and proton production. To probe this hypothesis, we measured the redox state of the PQ pool and the flux of energy through PSI in both light- and dark-adapted cells. We observed the mutant CupB-H89Q to have greater flux of energy through PSI than the controls under all tested conditions, yet all mutants demonstrated an ~8-fold decrease in flux compared to WT Syn7942. These paradoxical results allowed us to investigate a phenomenon known as reduction-induced suppression of electron flow (RISE), first proposed by Shaku et al. in 2016. RISE suggests that a highly reduced PQ pool induces the suppression of electron flux at the Q-site of Cytochrome b₆f.