Novel reactivity of cytochrome P-450-CAM. Methyl hydroxylation of 5,5-difluorocamphor.

The interaction of the camphor hydroxylating P-450 isolated from Pseudomonas putida grown on camphor (P-450-CAM) with 5,5-difluorocamphor, a substrate analog in which the two methylene hydrogens at the normal site of hydroxylation have been replaced with fluorine, has been examined. This compound binds tightly to the enzyme with a dissociation constant and UV-visible absorption spectrum identical to that observed with d-camphor. In the presence of the reconstituted P-450-CAM system, 5,5-difluorocamphor is metabolized at a rate approximately one-third the rate of the physiological substrate, d-camphor, resulting in the formation of a hydroxylated product with a molecular weight of 204 as well as a minor (less than 3%) hydroxylated product of molecular weight 184. Isotopically labeled molecular oxygen (18O2) is incorporated into the major product while labeled oxygen from water (H218O) is not incorporated, clearly indicating that the hydroxyl oxygen originates from dioxygen. Proton NMR characterization (400 MHz) of the major product has led to its assignment as 5,5-difluoro-9-hydroxy-camphor, with supporting structural evidence provided by the mass spectral fragmentation pattern. The formation of 9-hydroxylated product represents the first example of methyl hydroxylation catalyzed by cytochrome P-450-CAM, indicates a change in regio-selectivity when the normal site of reaction is blocked, and supports the hypothesis that the delivery of the oxygen atom occurs from the exo side of the camphor molecule.