Bacterial cytochrome P450eam catalyzes the hydroxylation of camphor to yield 5-exohydroxycamphor 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.
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