Reference

Tritiated Chiral Alkanes as Substrates for Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath): Probes for the Mechanism of Hydroxylation

Tritiated Chiral Alkanes as Substrates for Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath): Probes for the Mechanism of Hydroxylation. Valentine, Ann M.; Wilkinson, Barrie; Liu, Katherine E.; Komar-Panicucci, Sonja; Priestley, Nigel D.; Williams, Philip G.; Morimoto, Hiromi; Floss, Heinz G.; Lippard, Stephen J. (Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA). Journal of the American Chemical Society, 119(8), 1818-1827 (English) 1997 American Chemical Society. CODEN: JACSAT. 74364-75-3 is the cas registry number. This chemical is also mentioned in this article. ISSN: 0002-7863. DOCUMENT TYPE: Journal CA Section: 7 (Enzymes) The tritiated chiral alkanes (S)-[1-2H1,1-3H]ethane, (R)-[1-2H1,1-3H]ethane, (S)-[1-2H1,1-3H]butane, (R)-[1-2H1,1-3H]butane, (S)-[2-3H]butane, (R)-[2-3H]butane, and racemic [2-3H]butane were oxidized by sol. methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), and the abs. stereochem. of the resulting product alcs. was detd. to probe the mechanism of substrate hydroxylation. When purified hydroxylase, coupling protein, and reductase components were used, the product alc. displayed 72% retention of stereochem. at the labeled carbon for the ethane substrates and 77% retention for the butanes labeled at the primary carbon. A putative alkyl radical which would yield these product distributions would have a lifetime of 100 fs, a value too short to correspond to a discrete intermediate. Intramol. kH/kD ratios of 3.4 and 2.2 were detd. for ethane and butane, resp. When the hydroxylations were performed with purified hydroxylase but only a partially purified cellular ext. for the coupling and reductase proteins, different product distributions were obsd. These apparently anomalous results could be explained by invoking exchange of hydrogen atoms at the a carbon of the product alcs. The characteristics of this exchange reaction are discussed. Hydroxylation of [2-3H]butanes by the latter system yielded ~90% retention of stereochem. at the labeled carbon. The implication of these results for the catalytic mechanism of sMMO is discussed. Together with the mechanistic information available from a range of substrate probes, the results are best accounted for by a nonsynchronous concerted process involving attack of the C-H bond to form a pentavalent carbon transition state. .