Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme that catalyzes the reversible hydration of CO₂ to form HCO₃^- and H⁺ using a Zn-hydroxide mechanism. The first part of catalysis involves CO₂ hydration, while the second part deals with removing the excess proton that is formed during the first step. Proton transfer (PT) is thought to occur through a well ordered hydrogen-bonded network of waters that stretches from the metal center of CA to an internal proton shuttle, His64. These waters are oriented and ordered through a series of hydrogen-bonding interactions to hydrophilic residues that line the active site of CA. Neutron studies were conducted on wild-type human CA isoform II (HCA II) in order to better understand the nature and the orientation of the Zn-bound solvent (ZS), the charged state and conformation of His64, the hydrogen-bonding patterns and orientations of the water molecules that mediate PT and the ionization of hydrophilic residues in the active site that interact with the water network. Several interesting and unexpected features in the active site were observed which have implications for how PT proceeds in CA.