Rubisco proton production drives the elevation of CO2 within condensates and carboxysomes

Membraneless organelles containing the enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), formed via liquid-liquid phase separation, are a common feature of organisms utilizing CO2 concentrating mechanisms (CCMs). In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while microalgae have evolved liquid-like Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the function of carboxysomes to the generation of elevated CO2 within the organelle via a co-localized carbonic anhydrase (CA), and inwardly diffusing HCO3-. Here we present a novel concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction, and have evaluated this in a reaction-diffusion, compartment model to investigate functional advantages of Rubisco condensation and how these may lead to carboxysome evolution. Applying diffusional resistance to reaction species within a modelled condensate allows Rubisco-derived protons to drive the conversion of HCO3- to CO2 via co-condensed CA, enhancing both localized [CO2] and Rubisco rate. Protonation of RuBP and PGA plays an important role in modulating internal pH and CO2 generation. Application of the model to potential evolutionary intermediate states prior to contemporary carboxysomes revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully-formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.