Most CO2 from the atmosphere is assimilated in photosynthetic organisms by the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) enzyme as part of the Calvin cycle. Despite its relevance and many efforts in the last few decades, the mechanistic picture of the catalytic CO2 fixation reaction is still under debate. Here, we combine QM/MM molecular dynamics simulations with high-level electronic structure methods and the projector-embedding approach to provide reference values for the activation and reaction free energies of the catalytic CO2 fixation reaction. Our results show that carboxylation is protonation state dependent and irreversible, making the back reaction (decarboxylation reaction) highly unfavourable. The carbamylated lysine residue, Kcx201, coordinated to the magnesium (II) cation in the active site plays a central role shuffling protons from and to the substrate creating the proper reactive enolate species that adds CO2. The emerging microscopic picture that involves several protonation equilibria and the free energy profile of the CO2 fixation reaction provides insights that may be used in the future to improve enzymatic efficiency in RuBisCO.