Covalent Adduct Formation as a Strategy for Efficient CO <sub>2</sub> Fixation in Crotonyl-CoA Carboxylases/Reductases
Rodrigo Recabarren, Matthias Tinzl, David Adrian Saez, Aharon Gómez, Tobias J. Erb, Esteban Vöhringer‐Martinez
Abstract
Increasing levels of CO 2 in the atmosphere have led to a growing interest into the various ways nature transforms this greenhouse gas into valuable organic compounds. Crotonyl-CoA carboxylases/reductases (Ccr’s) are the most efficient biocatalysts for CO 2 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 CO 2 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: In the direct mechanism, hydride transfer is immediately followed by CO 2 addition, whereas in the C2 mechanism a thermodynamically stable covalent adduct between the substrate and the NADPH cofactor is formed. This C2 adduct, which has been previously characterized experimentally, serves as a stable intermediate avoiding the reduction side reaction of the reactive enolate species, and it is able to react with CO 2 with similar kinetics as the direct reaction mechanism as confirmed by measured kinetic isotope effects. Thus, our results show that nature’s most efficient CO 2 -fixing enzyme uses the formation of a covalent adduct as a strategy to store the reactive enolate species. The emerging microscopic picture of the CO 2 -fixing mechanism confirms previous experimental observations and provides new insights into how nature handles highly reactive intermediates to fix this inert greenhouse gas.