RADICAL CLOCK SUBSTRATE PROBES AND KINETIC ISOTOPE EFFECT STUDIES OF THE HYDROXYLATION OF HYDROCARBONS BY METHANE MONOOXYGENASE

Four mechanistic probes, trans-2-phenylmethylcyclopropane (1), 2,2-diphenylmethylcyclopropane (2), trans-1,2-dimethylcyclopropane (3), and bicyclo[2.1.0] Pentane (4), were used as substrates to investigate the possible formation of radical intermediates in the catalytic cycle of the methane monooxygenase (MMO) system from Methylococcus capsulatus (Bath) in reactions conducted at 45-degrees-C. Hydroxylation of 1 gave two products in equal amounts, the alcohol trans-(2-phenyl-cyclopropyl)methanol (1a) and the phenol trans-2-(p-hydroxyphenyl)methylcyclopropane (1c). The two different products are attributed to discrimination by the enzyme of the two enantiomers of probe 1, one of which leads to hydroxylation of the aromatic ring and the other to formation of the primary alcohol. The absence of ring-opened product, 1-phenylbut-3-en-l-ol (1b), is noteworthy and suggests that the hydroxylation reaction may not proceed through formation of the cyclopropylcarbinyl radical or cation. The former species rearranges to the 3-butenyl radical with a rate constant of 4 X 10(11) S-1 at 45-degrees-C, which would require a rebound rate constant for MMO from M. capsulatus (Bath) in excess of 10(13) s-1. The possibility of a stereoelectronic barrier to ring opening in the active site of the enzyme is addressed through a semiquantitative analysis of the rate constant with Marcus theory. Studies with the other radical clock probes support the notion that a significant component of the hydroxylation reaction pathway does not proceed through a radical intermediate. Compound 2, the cyclopropylcarbinyl radical of which has a ring-opening rate constant of 5 x 10(11) s-1, yields exclusively (2,2-diphenylcyclopropyl)methanol (2a); 3 (k = 4.4 x 10(8) s-1) affords only trans-(2-methylcyclopropyl)methanol (3a); and 4 (k = 3 X 10(9) s-1) gives mainly a mixture of endo-and exo-2-hydroxybicyclo[2.1.0]pentane(4a and 4b). Kinetic isotope effect experiments were also carried out with derivatives of 1, mono-, di-, and trideuterated at the methyl position. No intermolecular isotope effect, assessed by comparing the rate of conversion of the undeuterated and trideuterated probes, was observed, a result consistent with C-H bond breaking not being involved in the rate-determining step of the enzymatic reaction. Intramolecular kinetic isotope effects of k(H)/k(D) = 5.15 and 5.03 were obtained from studies of the mono- and dideuterated probes, respectively, which indicates that the hydroxylation reaction involves a substantial C-H bond stretching component. The MMO hydroxylase from Methylosinus trichosporium OB3b gave both unrearranged and a small amount (3-5%) of rearranged products with 1, the ratio of which corresponds to a rebound rate constant of 6-9 X 10(12) s-1 at 30-degrees-C. These results are discussed in terms of several possible detailed mechanisms for the MMO hydroxylase reaction.