Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase

Abstract

The conversion of CO₂ by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, in situ methods following the microscopic details of CO₂ conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO₂, water, bicarbonate, and other reactants with β-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasatospora 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 combined with molecular dynamics simulations tracing CO₂ distributions in the active site of KsCCr identify an ‘activated’ water molecule forming the hydroxyl anion that attacks CO₂ and yields bicarbonate (HCO₃⁻). Computer simulations also 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.