We propose to continue investigation of the chemical mechanisms of several types of oxidation by cytochrome P-450 enzymes. In each case our approach will be to use substrates specifically designed to probe key aspects of the overall mechanism including both the rate-limiting and product-determining steps. To aid in interpretation of results, reactions of these substrates using non- physiological oxidants (e.g. cumene hydroperoxide, iodosyl benzene) with P-450, non-P-450 peroxidase enzymes, and purely chemical model systems will also be studied. Three major types of probes will be employed, both singly and in concert: 1) manipulation of the one electron redox potential of the substrates, 2) kinetic deuterium isotope effects and stable isotope tracer studies and 3) cyclopropyl groups for detection of odd electron intermediates. The P-450 catalyzed processes we will focus on include 1) benzylic hydroxylation, 2) N-dealkylation of arylamines, 3) olefin epoxidation and 4) heteroatom desarylation. Collectively these studies will address two important contemporary issues regarding P-450 mechanisms. One is the dichotomy between electron-abstraction vs. hydrogen atom abstraction mechanisms as a function of the chemical properties of individual substrates. The other potential involvement of quantum- mechanical tunnelling to account for some of the non-classically large deuterium isotope effects seen with some P-450 reactions involving H- abstraction mechanisms. Since this latter area is a relatively new aspect of enzymology in general, our unique approach to it may have broader significance beyond cytochrome P-450.
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