Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modeling studies

Chloroperoxidase (CPO) is the most versatile of the known heme enzymes. It catalyzes chlorination of activated C-H bonds, as well as peroxidase, catalase and cytochrome P450 reactions, including enantioselective epoxidation. CPO contains a proximal heme-thiolate ligand, like P450, and polar distal pocket, like peroxidase. The substrate-binding site is formed by an opening above the heme that enables organic substrates to approach the activated oxoferryl oxygen atom. CPO, unlike other peroxidases, utilizes a glutamate acid-base catalyst, rather than a histidine residue.

The crystal structures of CPO complexed with exogenous ligands, carbon monoxide, nitric oxide, cyanide and thiocyanate, have been determined. The distal pocket discriminates ligands on the basis of size and pKa. The refined CPO-ligand structures indicate a rigid active-site architecture with an immobile glutamate acid-base catalyst. Molecular modeling and dynamics simulations of CPO with the substrate cis-beta methylstyrene and the corresponding epoxide products provide a structural and energetic basis for understanding the enantioselectivity of CPO-catalyzed epoxidation reactions.

The various CPO-ligand structures provide the basis for a detailed stereochemical mechanism of the formation of the intermediate compound I, in which Glu183 acts as an acid-base catalyst. The observed rigidity in the active site also explains the relative instability of CPO compound I and the formation of the HOCI chlorinating species. Energetics of CPO-substrate/ product molecular modeling provides a theoretical basis for the P450-type enantioselective epoxidation activities of CPO.