The use of intramolecular isotope effects to distinguish between deprotonation and hydrogen atom abstraction mechanisms in cytochrome P-450- and peroxidase-catalyzed N-demethylation reactions

Intramolecular isotope effects were determined for the N-demethylation of N-methyl-N-trideuteriomethylaniline catalyzed by two isozymes of cytochrome P-450 and several peroxidases in order to differentiate between deprotonation and hydrogen atom abstraction steps. Lactoperoxidase, hemoglobin, myoglobin, and two isozymes of horseradish peroxidase catalyzed the hydroperoxide-dependent N-demethylation at initial rates ranging from 20 to 1700 min-1. These hemeproteins exhibited large and comparable intramolecular isotope effects (kH/kD = 8.6 to 10.1). In contrast, two isozymes of cytochrome P-450 as well as chloroperoxidase (v = 1.5 to 1700 min-1) gave low isotope effects (kH/kD = 1.7 to 3.1) under identical conditions. Catalase exhibited an intermediate intramolecular isotope effect (kH/kD = 5.4). These results have been interpreted to indicate that most of the hemeproteins investigated catalyze N-demethylation reactions via alpha-carbon hydrogen atom abstraction, while the reactions catalyzed by cytochrome P-450 and chloroperoxidase proceed via alpha-carbon deprotonation.