| Chloroperoxidase (CPO) from the fungus Caldariomyces fumago can catalyze many of the characteristic reactions of heme peroxidases, catalases, and cytochrome P450. In the present work, enantioselective epoxidation of alkenes by CPO has been investigated, with emphasis both on mechanistic aspects of epoxidation and its utility in chiral synthesis.;CPO inactivation during the epoxidation of cis-alkenes involves primarily alkene-independent reactions. A scheme is proposed in which the one-electron oxidized CPO intermediate, Compound II, accumulates during hydrogen peroxide turnover as a result of partial reduction of the first oxidized intermediate, Compound I. Compound II reacts with excess hydrogen peroxide to form the oxyferrous complex of CPO, Compound III, which decays by several pathways, one of which involves the release of superoxide. Superoxide anion then initiates CPO inactivation and degradation of the prosthetic heme.;A different mode of CPO inactivation was observed during epoxidation of monosubstituted alkenes, which were shown to react with CPO both as substrates for epoxidation and for a mechanism-based inactivation reaction. Inactivation yielded a green species in which the heme of CPO had become modified by addition of the alkene plus an oxygen atom. Specific ;The EPR spectrum of green CPO showed that the modified heme has a very strong octahedral crystal field splitting, resulting in a pure ;CPO inactivated with allylbenzene reverted spontaneously at room temperature to an active species spectrally indistinguishable from native CPO. At the same time, mass spectrometry revealed loss of the heme alkylation. The gain of activity occurred in two kinetic phases, and EPR showed that three ferric species were present at intermediate times during reactivation. Two species were assigned as green CPO and native CPO, while the third had g-values similar to native CPO, and was designated "quasi-native" CPO. The known reactions of N-alkylmetalloporphyrins were used to propose a scheme in which two distinct intramolecular heme dealkylation pathways regenerate native CPO from the green heme. Studies of the temperature dependence of reactivation gave an Eyring plot with activation parameters for green CPO reactivation consistent with an intramolecular mechanism of heme dealkylation.;To elucidate factors involved in the control of CPO heme N-alkylation by terminal alkenes, the epoxidation of a series of monosubstituted and 1,1-disubstituted terminal alkenes by CPO was investigated. Terminal alkenes containing a 2-methyl substituent did not inactivate CPO even when the corresponding monosubstituted alkene inactivated the enzyme. The inverse correlation between the epoxidation enantioselectivity and the tendency to heme N-alkylation suggested that CPO inactivation by terminal alkenes is controlled by steric properties of the alkene and the enzyme active site. (Abstract shortened by UMI.). |
