MECHANISTIC STUDIES ON THE CYTOCHROME P450-CATALYZED DEHYDROGENATION OF 3-METHYLINDOLE

The mechanism of 3-methyleneindolenine (3MEI) formation from 3-methyli ndole (3MI) in goat lung microsomes was examined using stable isotope techniques. 3MEI is highly electrophilic, and its production is a prin cipal factor in the systemic pneumotoxicity of 3MI. Noncompetitive int ermolecular isotope effects of V-D = 3.3 and (D)(V/K) = 1.1 obtained a fter deuterium substitution at the S-methyl position indicated either that hydrogen abstraction from the methyl group was not the initial ra te-limiting step or that this step was rate-limiting and was masked by a high forward commitment and low reverse commitment to catalysis. An intramolecular isotope effect of 5.5 demonstrated that hydrogen atom abstraction was probably the initial oxidative and rate-limiting step of 3MI bioactivation or that deprotonation of an aminium cation radica l, produced by one-electron oxidation of the indole nitrogen, was rate -limiting. However, a mechanism which requires deprotonation of the am inium cation radical is probably precluded by an unusual requirement f or specific base catalysis at a site in the cytochrome P450 enzyme oth er than the heme iron. The pattern of O-18 incorporation into indole-3 -carbinol from O-18(2) and (H2O)-O-18 indicated that approximately 80% of the indole-3-carbinol was formed in goat lung microsomes by hydrat ion of 3MEI. However, the inverse reaction, dehydration of indole-3-ca rbinol, did not significantly contribute to the formation of 3MEI. The se results show that 3MEI was formed in a cytochrome P450-catalyzed de hydrogenation reaction in which the rate-limiting step was presumably hydrogen atom abstraction from the 3-methyl position. The ratio of the amounts of 3MEI to indole-3-carbinol formed (50:1) indicated that deh ydrogenation of 3MI is an unusually facile process when compared to th e dehydrogenation of other substrates catalyzed by cytochrome P450 enz ymes.