CYTOCHROME-P450 HYDROXYLATION OF HYDROCARBONS - VARIATION IN THE RATE OF OXYGEN REBOUND USING CYCLOPROPYL RADICAL CLOCKS INCLUDING 2 NEW ULTRAFAST PROBES

The oxidation of eight methyl-substituted and three alkyl-substituted cyclopropanes by rat liver microsomal cytochrome P450 and pure reconstituted rabbit P450 2B4 was studied. Alkane hydroxylation catalyzed by P450 is generally believed to proceed by hydrogen abstraction followed by reaction of the carbon-centered radical with an iron-bound hydroxyl radical, a process called oxygen rebound. Hydrogen abstraction from methylcyclopropanes generates cyclopropylcarbinyl radicals whose solution rate constants for ring opening are known [Bowry, V. W., et al. (1991) J. Am. Chem. Soc. 113, 5687-5698]. Rearranged products were only observed with the five substrates which, upon hydrogen abstraction, would generate a cyclopropylcarbinyl radical that undergoes ring opening with a rate constant greater-than-or-equal-to 2.0 x 10(9) s-1 in solution. Values of the rate constants for oxygen rebound (k(OH)) were calculated by determining the ratio of unrearranged products (cyclopropylmethanols) to rearranged products (alkenols). For each substrate this ratio was generally about the same for the oxidations catalyzed by microsomal P450 and by P450 2B4. It is concluded that all of the substrates are oxidized via an intermediate cyclopropylcarbinyl radical. Two ultrafast probes, trans-1-methyl-2-phenylcyclopropane and 1,1-diphenyl-2-methylcyclopropane, gave alcohol product ratios which yielded unreasonably high values for k(OH), viz., ca. 1.5 x 10(12) and ca. 7 x 10(12) s-1, respectively. It would appear to be likely that, with these two probes, the enzyme ''holds'' the phenyl group(s) in the intermediate cyclopropylcarbinyl radicals in a conformation which reduces the ring-opening rates relative to their rates in solution (in the least favorable conformation the rate of ring opening and hence the calculated k(OH) values would be reduced by ca. 3 orders of magnitude). Three of the substrates examined are unlikely to have the ring-opening rates of their cyclopropylcarbinyl radicals significantly influenced by the enzyme. These probes yield k(OH) approximately 2.4 x 10(11) s-1 for oxygen rebound to a primary alkyl radical and about an order of magnitude slower for rebound to a secondary alkyl radical.