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Cyanobacteria are oxygenic photoautotrophs that efficiently fix atmospheric CO2, with the help of their CO₂-concentrating mechanism (CCM). The CCM combines inorganic carbon transporters with carboxysomes, self-assembling, proteinaceous organelles that co-encapsulate Rubisco and carbonic anhydrase (CA). Accumulated bicarbonate (HCO₃^-) diffuses into the carboxysome, where CA rapidly interconverts it to CO₂ for Rubisco to fix. CA activity must be confined within the carboxysome, as cytosolic CA activity would deplete HCO₃^-, thus short-circuiting the CCM. Due to the distinctly different redox environment between the cytosol and the carboxysome, CA activity has been proposed to be redox-regulated: active under oxidizing conditions, inactive under reducing. In this thesis I investigated carboxysomal CA regulation in both α- and β-carboxysomes and I identified interactions mediating the encapsulation of CA in α-carboxysomes. 

In Paper I, I describe in detail the stopped-flow spectroscopy-based method used to study carbonic anhydrase kinetics in Papers II and III.

In Paper II, I investigated the regulation of α-carboxysomal β-CA, CsoSCA. Through stopped-flow based kinetics measurements I demonstrated that its activity is redox-dependent, with reducing conditions inhibiting the enzyme. A vicinal cysteine pair acts as  the redox switch, as demonstrated by that mutations to alanines abolished activity. Phylogenetic and bioinformatics analysis showed this mechanism is conserved across species. Cryo-EM structures of the wild-type CsoSCA from Halothiobacillus neapolitanus c2 in both redox states plus the C283A-C284A mutant demonstrated that redox conditions regulate global conformational dynamics, which propagate close to the active site, controlling the catalytic readiness. 

In Paper III, I studied the kinetic behaviour of CcaA, the β-carboxysomal CA found in Synechocystis sp. 6803. Activity measurements in different redox states showed that, surprisingly, CcaA activity is redox-independent. To rationalize this, I performed a bioinformatics analysis that confirmed that, indeed, there are no cysteines that could act as a redox switch.

In Paper IV, we explored the encapsulation process of CsoSCA in Cyanobium sp. PCC 7001. We identified the Rubisco-binding short linear motif (SLiM) in the disordered N-terminus of CsoSCA, using a combination of turbidity and surface plasmon resonance (SPR)-based interaction assays. Through a cryo-EM structure, we found that there are two motifs binding to two separate pockets on Rubisco, with arginines at positions 4 and 20 being the critical interaction elements. 

Collectively, these findings advance our understanding of carboxysomal CA regulation and encapsulation and inform future biotechnological applications, including engineering carboxysome-based CCMs in plants and industrially relevant microorganisms.