Role of active site residues in peroxidase catalysis: studies on horseradish peroxidase

Abstract

Peroxidase catalyses oxidation of both aromatic and inorganic electrqn donors with H₂0₂ through the intermediate formation of compound I and compound II. Much information is now available on the mechanism of peroxidase-catalysed formation of enzyme intermediates as well as on the oxidation of electron donors using horseradish peroxidase (HRP) as a model enzyme. The recently available x-ray crystal structure of HRP has furnished valuable informations on the critical role of Arg-38, His-42, Phe- 179 and heme propionates of the heme distal pocket in peroxidase catalysis. Site-directed mutagenesis studies have clearly established that histidine-42 and arginine-38 are actively involved in the heterolytic cleavage of H₂0₂ during compound I formation. Ntv1R studies have indicated that inorganic donors such as iodide and thiocyanate bind at a site at equal distance from heme peripheral 1 and 8 CH₃ groups and the binding is controlled by protonation of an ionisable group of pK_a value around 4 presumably contributed by heme propionic acid. However, recent mutant and NMR studies have implicated the critical role of Phe-179 in aromatic donor binding which has been confirmed in the x-ray crystal structure also. Heme propionates also play an important role in donor oxidation by controlling the formation of compound I. Recent studies indicate that heme propionates maintain the proper orientation of the heme moiety with respect to its surrounding residues for catalytic formation of compound 1. They appear to control inorganic donor oxidation by regulating the entry of the donors at the active site through the formation of salt-bridge with the nearby positively charged residue. HRP also shows oxidase activity through the intermediate formation of compound I, II and III by endogenous H₂0₂ through a peroxidase-oxidase oscillatory reaction. Under certain condition, the enzyme also shows reductive reaction with pseudocatalytic decomposition of H₂O₂ to 0₂ . Except in the case of sulfoxidation of aryl thioethers by H₂0₂ in presence of molecular oxygen, HRP cannot show peroxygenase reaction by transferring the ferry I oxygen to the donor molecule due to steric hindrance caused by His-42 and Phe-41.