The Unusual Reactivities of Amphitrite ornata Dehaloperoxidase and Notomastus lobatus Chloroperoxidase Do Not Arise from a Histidine Imidazolate Proximal Heme Iron Ligand

Notomastus lobatus chloroperoxidase (NCPO) and Amphitrite ornata dehaloperoxidase (DHP) catalyze the halogenation of phenols and dehalogenation of halophenols, respectively. Both enzymes require peroxide for activity and have recently been shown to contain histidine (His) as their proximal heme iron ligand. DHP is the only heme enzyme known to catalyze peroxide-dependent defluorination reactions, and NCPO is the first His-ligated heme-containing peroxidase capable of chlorinating halogen acceptors using chloride as the halogen donor. To probe the roles of the proximal His ligands in the mechanism of action of these two novel peroxidases, they have been examined by resonance Raman and infrared spectroscopy to determine the vibrational properties of their deoxyferrous and ferrous-CO derivatives. Previous studies of His-ligated heme proteins have revealed characteristic distinctions between dioxygen-binding globins and peroxidases. In the latter, the spectral properties are indicative of strong hydrogen bonding of the His imidazole N-1 hydrogen, giving histidinate character to the His, whereas the globins are thought to contain a predominately neutral His. The partially anionic peroxidase proximal His is thought to facilitate O-O bond cleavage of bound peroxide en route to the key oxo-ferryl compound I peroxidase intermediate. Surprisingly, the vibrational data reported herein for DHP and NCPO are similar to those previously reported for neutral His/imidazole-ligated globins and heme complexes. The vibrational data indicate that these enzymes do not activate bound peroxide through a mechanism dependent on a push effect imparted by a partially ionized proximal histidine as proposed for typical heme peroxidases.