Abstract
Experiments on chiral ethanes have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of configuration. It seem to rule out a radical mechanism for the hydroxylation chemistry, at least as mediated by this enzyme. The interpretation of subsequent experiments on n-propane, n-butane and n-pentane has been complicated by hydroxylation at both the pro-R and pro-S secondary “C-H” bonds. It has been suggested that these results merely reflect presentation of both the pro-R and pro-S “C-H” bonds to the hot “oxygen atom” species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of configuration. Now, we have augmented these earlier studies with experiments on [2-2H2]-, [1-2H3]-, [1-2H3,4-2H3]-butane and designed d,l form chiral deuterated butanes. Essentially equal amounts of (2R)-[3-2H2]butan-2-ol and (2R)-[2-2H]butan-2-ol are produced upon hydroxylation of [2-2H2]butane. The chemistry is regiospecific with full retention of configuration at the secondary carbon oxidized. Moreover, deuterium enriched in primary or secondary carbon of butane molecules presented higher hydroxylation reactivity due to the reduction of the van der Waal radius of the deuterium replacement resulting in the decrease of the parameter kM deduced from Michaelis-Menten Kinetics. In the case of the various chiral deuterated butanes, the distribution of products mirrors the stereochemical configurations of the chiral butane substrates, after allowance is made for the expected deuterium isotope effect on the kinetics of the “oxygen atom” insertion step. The extent of configurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of configuration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet “oxene” insertion across the “C-H” bond similar to that previously noted for singlet carbene insertion.