Stereochemistry and deuterium isotope effects in camphor hydroxylation by the cytochrome P450cam monoxygenase system.
Bacterial cytochrome P450cam catalyzes the hydroxylation of camphor to yield 5-exo-hydroxycamphor in vivo and in a reconstituted system with oxygen, pyridine nucleotide, flavoprotein dehydrogenase, and putidaredoxin. Product is also formed when the ferric form of the hemoprotein is mixed with the exogenous oxidants iodosobenzene, m-chloroperbenzoic acid, and hydrogen peroxide. In this paper we show that when the P450cam-dependent hydroxylation reactions are studied with camphor analogues containing deuterium at either the 5-exo or 5-endo position, a very small intermolecular isotope on the overall reaction velocity is observed and a significant intramolecular isotope effect is documented. We suggest the existence of an intermediate substrate-carbon radical and demonstrate that abstraction can occur from either the exo or endo position at carbon 5 on the camphor skeleton, with the oxygen stereospecifically added to only the Re face to give 5-exo-hydroxycamphor as the unique product. Using these substrates, we observed nearly identical hydrogen/deuterium isotope ratios in the product alcohol for the pyridine nucleotide/atmospheric dioxygen as well as exogenous oxidant supported hydroxylations, suggesting that these reactions share a common hydrogen-abstracting species. The relatively small magnitude of the measured intramolecular isotope effect can be rationalized with a model involving a reversible hydrogen-abstraction step and/or the involvement of heavy-atom motion in the reaction coordinate.
D002164:Camphor / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009243:NAD; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase