ACTIVE-SITE DYNAMICS OF XYLENE HYDROXYLATION BY CYTOCHROME-P-450 AS REVEALED BY KINETIC DEUTERIUM-ISOTOPE EFFECTS

The cytochrome P-450 catalyzed hydroxylation of o- and p-xylene and five deuterated derivatives of each has been investigated using phenobarbital-induced rat liver microsomes. All possible monohydroxylation products were observed but benzylic hydroxylation predominated strongly (88-96%). H/D discrimination was strongest when both isotopes were located on the same methyl group, less when they were located in different methyl groups on the same xylene molecule, and least when they were located in methyl groups on different molecules. Benzylic hydroxylation is subject to a large intrinsic (intramolecular) deuterium isotope effect (CH3/CD3 = 7.5-9.5), comprised of a large primary component (5.3-7.8) and a large normal alpha-secondary component (1.09-1.19). These isotope effects suggest a transition state for benzylic H-abstraction that is linear and symmetrical with substantial rehybridization toward planarity at the benzylic carbon and little residual C-H bond order remaining. In contrast aromatic hydroxylation of o- and p-xylene shows a small inverse alpha-secondary isotope effect(0.83-0.94). The D(V/K) isotope effect observed for benzylic hydroxylation in intermolecular competitions (ca. 1.9-2.3 for d0/d6 substrate mixtures) is substantially reduced by commitment to catalysis, with C(f) = (k(H) + k(r))k-1 = 3.6 for p-xylene and 5.9 for o-xylene. These results suggest a dynamic picture of catalysis with the following relative rates: methyl group rotation > substrate re-orientation within the Michaelis complex (i.e. isotopically sensitive branching to different products) > product formation (i.e. commitment to catalysis) > substrate dissociation prior to hydroxylation.