Infrared Spectroscopy Reveals Metal independent Carbonic Anhydrase Activity in Crotonyl CoA Carboxylase/Reductase

The conversion of CO2 by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, methods following the conversion of CO2 at the active site in situ are limited. Here, we used infrared spectroscopy to study the interaction of CO2, water, and other reactants with β-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasaospora setae (KsCcr), two of the fastest CO2-converting enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues allows identifying an ‘activated’ water molecule, forming the hydroxyl anion that attacks CO2 and yields bicarbonate (HCO3–). Molecular dynamics simulations are analyzed to trace CO2 in the active site of KsCcr and explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ infrared spectroscopy combined with molecular dynamics simulations provides a simple, yet powerful new approach to investigate the atomistic reaction mechanisms of different enzymes with CO2.