Peroxygenation mechanism for chloroperoxidase-catalyzed N-oxidation of arylamines

Abstract

The metabolism of three arylamine substrates by H2O2 in the presence of each of the peroxidative enzymes chloroperoxidase (CPX) and pea seed peroxygenase (PSM) was conducted with normal H2O2 and with 18O-labeled H2O2. The resulting C-nitroso aromatic metabolites were examined by GC-MS methods to determine the extent of 18O incorporation. The arylamine substrates were p-toluidine, 4-chloroaniline, and 3,4-dichloroaniline. For both enzymes, all three arylamines were found to give quantitative incorporation of 18O into their nitroso metabolites when [18O]H2O2 was the oxidant substrate. The introduction of the oxygen atom into 4-chloronitrosobenzene was found to occur during the first step of this process, since it was found that when (4-chlorophenyl)hydroxylamine was employed as the substrate, no significant incorporation of 18O occurred. These observations prove that CPX and PSM cause N-oxidation of primary arylamines via an oxygen transfer from the compound I activated forms of their heme functional groups. Therefore, these peroxidases are correctly called peroxygenases when acting in such a manner. A discussion of the reaction mechanisms for peroxidases and their relation to cytochrome P-450 oxidations is presented.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

“…CPO behaves as a catalase in terms of catalyzing the dismutation of hydrogen peroxide and the oxidation of alcohol (1). It mimics P450 cytochromes in catalyzing heteroatom dealkylation (4,5), benzylic hydroxylations (6), and oxygen transfer to alkenes (7)(8)(9), alkynes (10,11), sulfides (12,13), and arylamines (14,15). CPO is especially adept in the stereoselective epoxidation of alkenes (7-9, 16, 17) hydroxylation of alkynes (10,11) and in the production of chiral sulfoxides (12,13).…”

Peroxidative metabolism of carcinogenic N-arylhydroxamic acids: implications for tumorigenesis.

“…The MPO-derived oxidation products from drugs containing the free -NH2 group have been implicated in adverse effects such as agranulocytosis and generalized hypersensitivity reactions during drug therapy (25). The detection of the hydroxylamino derivatives from these drugs may be the result of peroxygenation, the mechanism recently elucidated for chloroperoxidase-catalyzed N-oxidation of p-substituted N-arylamines (26). It is thus becoming increasingly evident that the structure of N-arylamine and the type of the substituent on the aromatic ring are factors in determining the oxidation products.…”

The Influence of the Proximal Thiolate Ligand and Hydrogen Bond Network of the Proximal Helix on the Structural and Biochemical Properties of Chloroperoxidase

“…Stereoselectivity arises from the fact that to gain access to CPO's active site, the substrates need to properly orient in the channel that connects the surface of the enzyme to the heme pocket. Substrates for these two-electron oxidations include amines, alcohols, aldehydes, alkenes, alkynes, esters, anisoles, sulfides and indoles (111)(112)(115)(116)(117)(118)(119)(120)(121)(122)(123)(124)(125)(126). …”

“…In addition to the reactions described above, CPO is able to catalyze the oxidation of different monoterpenes in the presence of halide to yield halohydrins (167), regio-and stereoselective bromohydration of glycals to produce 2-deoxy-2-bromo saccharides (168), oxidation of unsubstituted and substituted indoles to yield oxindoles (125,169,170), Noxidation of arylamines to give nitroso metabolites (111,169,171), oxidation of alcohols to aldehydes (116), and oxidation of conjugated dienoic esters to yield a variety of products (123). Many of the above compounds can be converted into chemicals that serve as intermediates in the synthesis of flavors, fragrances, and drugs (125,167).…”