The Chloroperoxidase-Catalyzed Oxidation of Phenols. Mechanism, Selectivity, and Characterization of Enzyme-Substrate Complexes

Abstract

The reactivity of a series of para-substituted phenolic compounds in the peroxidation catalyzed by chloroperoxidase was investigated, and the results were interpreted on the basis of the binding characteristics of the substrates to the active site of the enzyme. Marked selectivity effects are observed. These operate through charge, preventing phenolic compounds carrying amino groups on the substituent chain to act as substrates for the enzyme, and through size, excluding potential substrates containing bulky substituents to the phenol nucleus. Also, chiral recognition is exhibited by chloroperoxidase in the oxidation of N-acetyltyrosine, where only the L isomer is oxidized. Kinetic measurements show that, in general, the efficiency of chloroperoxidase in the oxidation of phenols is lower than that of horseradish peroxidase. Paramagnetic NMR spectra and relaxation rate measurements of chloroperoxidase-phenol complexes are consistent with binding of the substrates close to the heme, in the distal pocket, with the phenol group pointing toward the iron atom. On the other hand, phenolic compounds which are not substrates for chloroperoxidase bind to the enzyme with a much different disposition, with the phenol group very distant from the iron and probably actually outside the active-site cavity.

Formation of reactive nitrogen species at biologic heme centers: a potential mechanism of nitric oxide-dependent toxicity.

“…Comparative experiments using chemical nitrating agents indicate that at low nitrite concentrations, the enzymatic nitration produces a regioisomeric mixture of nitrotryptophanyl derivatives resembling that obtained using nitrogen dioxide, whereas at high nitrite concentrations the product pattern resembles that obtained using peroxynitrite. values for the phenolic substrates, in the range from 0.1 to 1 mM, are one to two orders of magnitude smaller than the K M values we found for the oxidation of the same phenolic substrates to dimeric coupling products in the classical peroxidase-catalyzed reactions (18,19). This observation can be explained considering that a close proximity between the phenol and the heme, which is required for efficient electron transfer to compound I and II in the normal peroxidase reaction, is not needed in the nitration process.…”

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

“…In spite of the assumptions underlying the use of this equation, it proved to be useful in our previous studies on peroxidases (Casella, Poli, Gullotti, Selvaggini, Beringhelli, & Marchesini, 1994;Redaelli, Monzani, Santagostini, Casella, Sanangelantoni, Pierattelli, & Banci, 2002) and ascorbate oxidase (Gaspard et al, 1997). In the case of copper enzymes, it is generally assumed that the overall correlation time is determined by the electronic relaxation, and a τ C value of 3.5×10 −9 s has been estimated for a variety of copper enzymes (Bertini, Briganti, Luchinat, Mancini, & Spina, 1985;Williams & Falk, 1986).…”

Characterization of Recombinant Chloroperoxidase, and F103A and C29H/C79H/C87H Mutants

“…Therefore, there must be an interaction between glycols and steroids, and CPO (Libby, Thomas et al 1982;Geigert, Neidleman et al 1983). Stereospecific epoxidation and sulfoxidation of organic substrates also suggests binding at the active site of CPO (Casella, Gullotti et al 1992;Casella, Poli et al 1994). The binding of CPO to cis-β-methylstyrene has been modeled using information from the crystal structure as shown in Fig.…”

“…In addition, CPO has the potential to catalyze the synthesis of a wide range of chiral or prochiral products. Chloroperoxidase can catalyze many reactions that are important in synthesis such as sulfoxidation (Silverstein and Hager 1974;Doerge 1986), hydroxylation (Miller, Tschirret-Guth et al 1995) and epoxidation (Geigert, Lee et al 1986), enantioselectivly and in high yield (Casella, Poli et al 1994;Lakner and Hager 1996), such as the epoxidation of olefins (Ortiz de Montellano, Choe et al 1987), where traditional peroxidases have failed. Other reactions such as N-demethylation (Kedderis, Koop et al 1980) and dehalogenation (Osborne, Raner et al 2006) are also possible.…”