Epoxidation of olf.fins by cytochromk P450 possible rolf, for hydroperoxo iron as the direct oxidant

The epoxidalion of olefinic compounds by P450 cytochromes is a common reaction with importance in xenobiotic metabolism and an as yet unclear role in unsuturated tatty acid metabolism. This reaction is generally believed to involve tbe insertion of an oxygen atom from a hypervalcnt oxenoicl iron complex. To probe the mechanism of the reaction, we have examined the cpoxidation and hydroxylation of small olefins and unsaturated fatty acids by P450 2E1 and an active site Thr mutant (T303A) of 2E1 in a reconstituted system. The <:J.v- and f/Yin.v-isomers of 2-hutene were found to undergo epoxidalion or hydroxylation to cf.v- and rran.vhutene 2,3-epoxide and rJ.s- and rr<m.s-2-butene-l-ol, respectively, without isomenJ.ation of the (VCt bond. 2E1 T303A is 4 limes as active as 2E1 in epoxidation. but onlv one-fourth as active in allylic hydroxylation. Similarly, epoxidation of 2-9-dodeeenoic acid by 2EI and 2E1 T303A occurs without isomcrination of the OrCio bond. With 2EI T303A. epoxidation and m-1 hydroxylation account for 83 and 11%, respectively, of the total réaction products, whereas with 2EI the corresponding values are 30 and 709; 2E1 T303A is only one-fourth as active as 2EI in overall metabolism of the fany acid. These results, together with the role proposed lor the active site Thr as a proton donor for cleavage of the peroxo O-O bond, suggest that epoxidaiions involve an electrophilic hydroperoxo rather than an oxene species.