Metabolic Swiching in Cytochrome P-450cam: Deuterium Isotope Effects on Regiospecificity and the Monooxygenase/Oxidase Ratio

Article abstract of DOI:10.1021/ja00246a038

Cytochrome p-450_cam, isolated and purified to homogenity from the soil bacteria Pseudomonas putida, has been shown to catalyze the hydroxylationof the substrate analogue norcamphor to form three distinct products and yields: 5-exo-hydroxynorcamphor (45percent), 6-exo-hydroxynorcamphor (47percent), and 3-exo-hydroxynorcamphor (8percent).Specific deuteriation of the norcamphor skeleton at the 5-,6-,and 3-positons drastically alters this product distribution, indicatinga substantial deuterium isotope effect.When the sum total of all oxygenated products formed in the presence of norcamphor is compared to the number of reducing equivalents consumed in the reaction (NADH), a striking unaccountability of electrons is observed.Thes are shown to reside in excess water produced by the four-electron reduction of atmospheric dioxygen by P-450_cam.Metabolismof specifically deuteriated norcamphor demonstrates a deuterium isotope effect on the branching ratio of substrate hydroxylation to excess water production and suggests that this oxidase activity of P-450_cam results from thetwo-electron reduction of a single oxygen-iron intermediate, ³⁺.