The tritiated chiral alkanes(
S)-[1-
2H
1,1-
3H]ethane,(
R)-[1-
2H
1,1-
3H]ethane,(
S)-[1-
2H
1,1-
3H]butane,(
R)-[1-
2H
1,1-
3H]butane,(
S)-[2-
3H]butane,(
R)-[2-
3H]butane, and racemic[2-
3H]butane were oxidized by solublemethanemonooxygenase (sMMO) from
Methylococcuscapsulatus (Bath), and the absolute stereochemistry ofthe resultingproduct alcohols was determined in order to probe the mechanism ofsubstrate hydroxylation. When purifiedhydroxylase, coupling protein, and reductase components were used, theproduct alcohol displayed 72% retention ofstereochemistry at the labeled carbon for the ethane substrates and77% retention for the butanes labeled at theprimary carbon. A putative alkyl radical which would yield theseproduct distributions would have a lifetime of 100fs, a value too short to correspond to a discrete intermediate.Intramolecular
kH/
kDratios of 3.4 and 2.2 were determinedfor ethane and butane, respectively. When the hydroxylations wereperformed with purified hydroxylase but onlya partially purified cellular extract for the coupling and reductaseproteins, different product distributions were observed.These apparently anomalous results could be explained by invokingexchange of hydrogen atoms at the
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carbon ofthe product alcohols. The characteristics of this exchangereaction are discussed. Hydroxylation of[2-
3H]butanesby the latter system yielded ~90% retention of stereochemistry atthe labeled carbon. The implication of theseresults for the catalytic mechanism of sMMO is discussed. Togetherwith the mechanistic information availablefrom a range of substrate probes, the results are best accounted for bya nonsynchronous concerted process involvingattack on the C-H bond by one or more of several pathways discussedin the text.