Atomic view of photosynthetic metabolite permeability pathways and confinement in synthetic carboxysome shells

Rubisco activity is essential for all life on earth, capturing atmospheric CO2 and incorporating it into photosynthesis-driven metabolism. Cyanobacteria encapsulate rubisco into bacterial microcompartments called carboxysomes to elevate the local CO2 concentration and enhance rubisco kinetics. The permeability across carboxysome shells is a key parameter to understand the CO2 concentration mechanism in cyanobacteria, as it limits the concentration gradient between the inside and outside of the carboxysome. Through molecular simulation, we track the motion of photosynthetic metabolites across the synthetic carboxysome shell to determine a permeability coefficient, and further compare them to the rate of CO2 leakage and turnover within model carboxysomes. This mechanistic insight is paramount to design and engineer such organelles for carbon fixation, bioenergy, and sustainability applications. 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, or ribulose-1,5-bisphosphate. Starting from a high-resolution cryogenic electron microscopy structure of a synthetic β-carboxysome shell, we perform unbiased all-atom molecular dynamics to track metabolite permeability across the shell. The synthetic carboxysome shell structure, lacking the bacterial microcompartment 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 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 toward rubisco that 2,650 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.