Reaction of ferricCaldariomyces fumagochloroperoxidase withmeta-chloroperoxybenzoic acid: sequential formation of compound I, compound II and regeneration of the ferric state using one reactant

Abstract

The mechanism of the reaction between ferric Caldariomyces fumago chloroperoxidase (CCPO) and meta-chloroperoxybenzoic acid (mCPBA) has been examined. It has previously been established that an Fe(IV) -oxo porphyrin radical species known as Compound I (Cpd I) is formed by two-electron oxidation of the native ferric enzyme by a variety of oxidants including organic peracids and hydroperoxides. Cpd I can return to the ferric state either by direct oxygen insertion into an organic substrate (e.g. a P450 oxygenase-like reaction), or by two consecutive one-electron additions, the first resulting in an intermediate Fe(IV) -oxo species known as Compound II (Cpd II). There has been much debate over the role of Cpd II and the details of its structure. In the present study, both CCPO Fe(IV) -oxo intermediates are formed, but unlike most CCPO reactions, Cpd I and Cpd II are formed using the same reactant, mCPBA. Thus, the peracid is used as an oxo donor to produce Cpd I and then as a reductant to reduce Cpd I to Cpd II, and finally, Cpd II to the ferric state. The observation of saturation kinetics with respect to mCPBA concentration for each step is consistent with the formation of CCPO-mCPBA complexes in each phase of the reaction. The original reaction mechanism for ferric CCPO with mCPBA was hypothesized to involve a scrambling mechanism with a unique Fe -OOO-C(O)R intermediate formed with no observed Cpd II intermediate. The data reported herein clearly demonstrate the formation of Cpd II in returning the oxidized enzyme back to its native ferric state.