Atomic View of Photosynthetic Metabolite Permeability Pathways and Confinement in Cyanobacterial Carboxysomes

Carboxysomes are protein microcompartments found in cyanobac- teria, whose shell encapsulates rubisco at the heart of carbon fixa- tion in the Calvin-Benson-Bassham (CBB) cycle. Carboxysomes are thought to locally concentrate CO2 and exclude O2 from the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, per- meability coefficients have not previously been determined for these gases, or for CBB cycle intermediates such as bicarbonate (HCO3 – ), 3-phosphoglyceric acid (3-PGA), or ribulose-1,5-bisphosphate (RuBP). Starting from a high resolution cryo-EM structure of a synthetic β-carboxysome shell, we perform unbiased all-atom molecular dynamics (MD) to track metabolite permeability across the shell. The synthetic carboxysome shell is found to have similar permeability coefficients for multiple metabolites, and is not selectively permeable to HCO3– relative to CO2. To resolve how these comparable permeabilities can be reconciled with the clear role of the carboxysome in the carbon-concentrating mechanism in cyanobacteria, complementary atomic-resolution Brownian Dynamics (ARBD) simulations estimate the mean first passage time for CO2 assimilation in a crowded model carboxysome. Despite a relatively high CO2 permeability of 10−2 cm/s across the carboxysome shell, the shell proteins reflect enough CO2 back towards rubisco that over 2600 CO2 molecules can be fixed by rubisco for every 1 CO2 molecule that escapes under typical conditions. The permeabilities determined from all-atom molecular simulation are key inputs into flux modeling, and the insight gained into carbon fixation can facilitate the engineering of carboxysomes and other bacterial microcompartments for multiple applications.