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

Carboxysomes are protein microcompartments found in cyanobacteria, whose shell encapsulates rubisco at the heart of carbon fixation in the Calvin-cycle. Carboxysomes are thought to locally concentrate CO2 in the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, permeability coefficients have not previously been determined for these gases, or for Calvin-cycle intermediates such as bicarbonate (HCO3-), 3-phosphoglycerate (3-PGA), or ribulose-1,5-bisphosphate (RuBP). Starting from a high resolution cryo-EM structure of a synthetic beta-carboxysome shell, we perform unbiased all-atom molecular dynamics (MD) to track metabolite permeability across the shell. The synthetic carboxysome shell structure, lacking the BMC trimer proteins and encapsulation peptides, 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 CO2-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 approximately 10^-2 cm/s across the carboxysome shell, the shell proteins reflect enough CO2 back towards rubisco that 2650 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.