Kohn-Sham Density Functional Calculations Reveal Proton Wires in the Enolization and Carboxylase Reactions Catalyzed by Rubisco.

Ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) plays a fundamental role in the carbon cycle by fixing the atmospheric CO used in photosynthesis. Rubisco is all the more remarkable because it must catalyze some difficult multistep reaction chemistry involving proton transfers within the one active site. In the present study, we have used Kohn-Sham density functional theory at the B3LYP/6-31G* level with basis set superposition error and dispersion corrections (B3LYP-gCP-D3) to examine the possibility that the proton transfers can take place through molecular wires (including active-site water molecules) via the classical Grotthuss proton-shuttle mechanism. The results support an essential role for water molecules found in the crystal structures of Rubisco complexes as facilitators of proton transport in all the rate-limiting (catalytic) reaction steps through a network of short proton wires within the Rubisco active site. We suggest that completion of the initial product turnover (cycle) requires two excess protons produced in the initial carbamylation that is required for Rubisco activation. By use of proton wires, a large number of reaction steps may be accommodated within a single active site without necessitating the input of excessive conformational strain energy arising from the movement of residue side chains into positions where direct protonation of substrates can occur. The involvement of the identified types of proton wires in the kinetic mechanism is capable of providing a unique explanation for various experimental observations, including deuterium isotope effects and the results of site-directed mutagenesis experiments, and may thus provide a realistic solution to the problem of Rubisco's challenging chemistry.