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

The conversion of CO 2 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 CO 2 conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO 2 , water, bicarbonate, and other reactants with β-carbonic anhydrase from Escherichia coli ( Ec CA) and crotonyl-CoA carboxylase/reductase from Kitasatospora setae ( Ks Ccr), two of the fastest CO 2 -converting enzymes in nature. Our data reveal that Ks Ccr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics simulations tracing CO 2 distributions in the active site of Ks CCr identify an 'activated' water molecule forming the hydroxyl anion that attacks CO 2 and yields bicarbonate (HCO 3 - ). 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 CO 2 .