Electronic and steric factors in regioselective hydroxylation catalyzed by purified cytochrome P-450

A reconstituted hydroxylation system consisting of electrophoretically homogeneous phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450 LM2), NADPH-cytochrome P-450 reductase, phospholipid, buffer, NADPH, and O2 was used to oxidize four cyclohexane derivatives: cyclohexene, methylcyclohexane, norcarane and norbornane. Cyclohexene gave only cyclohexene oxide and allylic cyclohexenol, while methylcyclohexane yielded all possible monohydric alcohols, but with 1 degrees:2 degrees:3 degrees ratios of 0.072:1:1.25. Norcarane yielded 2-norcaranol. While oxidation of norbornane produced exo-2- and endo-2-norborneols in a ratio of 3.4:1, replacement of all four exo-hydrogens by deuterium led to a reversal of the exo:endo ratio to 0.76:1. These and other observations are interpreted as evidence for a selective, hydrogen-abstracting enzyme-bound oxidant exhibiting a large intramolecular deuterium isotope effect. A transient substrate carbon radical is a probable intermediate in the hydroxylation process.