Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenylcyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.The soluble methane monooxygenase (sMMO) 1 from Methylococcus capsulatus (Bath), the hydroxylase component of which contains a dinuclear nonheme iron center, effects the mixed function oxidation of substrate,Equation 1 (1-3). In methanotrophic bacteria from which sMMO is isolated, methane is the natural substrate, providing both carbon and energy for the growth of the cell. The kinetically difficult selective transformation of methane to methanol is carried out at the active site of a 251-kDa hydroxylase enzyme (MMOH) (4 -7), the structure of which has been determined by x-ray crystallography. Electrons are supplied from NADH via a 38.5-kDa reductase protein (MMOR) (4,8), and the reactivity of the system is modulated by a 15.8-kDa coupling protein (MMOB) (9, 10). 2In addition to methane, sMMO can oxidize a wide variety of substrates (12)(13)(14)(15). This property has allowed the use of radical clock substrate probes to derive mechanistic information about the hydroxylation step in the enzyme mechanism. Radical clocks were first described in 1980 (16) and calibrated (17) for use in mechanistic studies of proteins (18,19) and related model compounds (20,21). Specifically, these substrates can reveal whether radical intermediates are formed during a reaction and, if so, can establish their lifetime and the rate constant for radical rebound. Fig. 1 depicts the oxidation of a radical clock substrate probe, which uses the ring opening of a cyclopropylcarbinyl radical. If hydrogen atom abstraction from the probe occurs, the resulting radical can either rebound to form the unrearranged alcohol or undergo ring opening before rebound to afford the rearranged alcohol. If the ring opening rate is known (17), the rebound rate, k oxidation , can be calculated according to Equation 2. k oxidation ϭ k ring opening ͑unrearranged alcohol / rearranged alcohol) (Eq. 2)The hypothesis that substrate radical intermediates form in the sMMO s...