Evidence for Two Different Active Oxygen Species in Cytochrome P450 BM3 Mediated Sulfoxidation and N-Dealkylation Reactions

Abstract

Herein, we report the results from two experiments that are consistent with sulfoxidation and N-dealkylation involving two different enzyme substrate complexes and thus two different active oxygen species that do not interchange. The first experiment involves the use of a mutant that may increase the amount of the hydroperoxy-iron species (FeIIIO2H).1 This mutant increases the amount of sulfoxidation relative to the amount of N-dealkylation by 4-fold. In a second experiment, deuterium substitution on the N-methyl groups of substrate does not result in an increase in sulfoxidation. This later result is consistent with N-dealkylation and sulfoxidation being mediated by two different active oxygen species. While the data indicate two active oxygen species, they do not distinguish between the different possibilities for the active oxygen species.

KINETIC ISOTOPE EFFECTS IMPLICATE A SINGLE OXIDANT FOR CYTOCHROME P450-MEDIATEDO-DEALKYLATION,N-OXYGENATION, AND AROMATIC HYDROXYLATION OF 6-METHOXYQUINOLINE

“…Thus, for both the reactions to occur from both oxidants, it is required that the relative energy of formation of 3 and 4 be identical for both oxidizing species. In fact, mutants that purportedly enhance Cpd 0 have been shown to alter product ratios and rates of reaction in every case, indicating a difference in the energetics of the two oxidants Vatsis and Coon, 2002;Volz et al, 2002;Jin et al, 2003;Chandrasena et al, 2004) and consistent with computational results (Ogliaro et al, 2002).…”

“…In separate studies, Hutzler et al (2003) have suggested that two different oxidants may be involved in O-dealkylation and N-dealkylation. Jin et al (2003) came to the conclusion that Cpd 0 could epoxidize alkenes but not hydroxylate the high-energy aliphatic C-H bond on camphor, and Volz et al (2002) suggested that Cpds 0 and 1 may be the sole oxidants involved in N-dealkylation and sulfoxidation, respectively.…”

“…Three things led us to consider the possibility that N-oxygenation may occur through Cpd 0. 1) Sulfoxi- dation, a reaction closely related to N-oxygenation, has been shown by isotopically sensitive branching experiments to occur from a second oxidant (Volz et al, 2002). 2) N-Oxygenation possesses a relatively low energy barrier in comparison to aromatic hydroxylation (Dowers et al, 2004), and low barrier reactions have a higher probability of occurring through a reaction with Cpd 0, given its low reactivity.…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…The enzymes catalyze a broad range of reactions such as hydroxylation [5], decarboxylation [6], epoxidation [7], reductive dehalogenation [8], amine/oxygen dealkylation [9], and sulfoxidation [10], employing molecular oxygen and electrons [e.g. from NAD(P)H].…”

Is the Ferric Hydroperoxy Species Responsible for Sulfur Oxidation in Cytochrome P450s?

“…[8] Studies of the kinetics of wildtype and mutant P450 BM3 catalyzed oxidation of a subtrate with both amine or thioether moieties led to the postulate that two separate oxidants affect amine and thioether oxidation; the oxidation of sulfur was greater for the mutant in which Cpd 0 activity is potentiated. [8] An alternative explanation of these results that does not implicate Cpd 0 was proposed in which different spin states of Cpd I masquerade as two different oxidants. [3] Whether Cpd 0 does play a role in wildtype P450 catalyzed sulfoxidation was thus unclear.…”