Covalent adduct formation as strategy for efficient CO2 fixation in crotonyl-CoA carboxylases/reductases

Increasing levels of CO2 in the atmosphere have led to a growing interest into the various ways nature transforms this greenhouse gas in valuable organic compounds. Crotonyl-CoA carboxylases/reductases (Ccrs) are the most efficient biocatalysts for CO2 fixation because of their oxygen tolerance, their high catalytic rate constants and their high fidelity. The reaction mechanism involving hydride transfer from the NADPH cofactor and addition of CO2 to the reactive enolate, however, is not completely understood. In this study, we use computer simulations in combination with high-level ab initio calculations to trace the free energy landscape along two possible reaction paths and identify a thermodynamically stable covalent adduct formed between the substrate and the NADPH cofactor in agreement with experimental observation. This C2 adduct serves as a stable intermediate avoiding the reduction side reaction of the reactive enolate species and it is able to react with CO2 with similar kinetics as the direct reaction mechanism as confirmed by measured kinetic isotope effects. Thus, our results show that nature's most efficient CO2-fixing enzyme uses the formation of a covalent adduct as a strategy to store the reactive enolate species. The emerging microscopic picture of the CO2-fixing mechanism confirms previous experimental observations and provides new insights into how nature handles highly reactive intermediates to fix the inert greenhouse gas.