TRICHLOROETHYLENE OXIDATION BY PURIFIED TOLUENE 2-MONOOXYGENASE - PRODUCTS, KINETICS, AND TURNOVER-DEPENDENT INACTIVATION

Trichloroethylene is oxidized by several types of nonspecific bacteria l oxygenases. Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover dependent inactivation in vivo. In this work , the oxidation of trichloroethylene was studied with purified toluene 2-monooxygenase. All three purified toluene 2-monooxygenase protein c ompo nents and NADH were required to reconstitute full trichloroethyle ne oxidation activity in vitro, The apparent K-m and V-max were 12 mu M and 37 nmol per min per mg of hydroxylase component, respectively. T en percent of the full activity was obtained when the small-molecular- weight enzyme component was omitted, The stable oxidation products, ac counting for 84% of the trichloroethylene oxidized, were carbon monoxi de, formic acid, glyoxylic acid, and covalently modified oxygenase pro teins that constituted 12% of the reacted [C-14]trichloroethylene, The stable oxidation products may all derive from the unstable intermedia te trichloroethylene epoxide that was trapped by reaction with 4-(p-ni trobenzyl)pyridine. Chloral hydrate and dichloroacetic acid were not d etected, This finding differs from that with soluble methane monooxyge nase and cytochrome P-450 monooxygenase, which produce chloral hydrate , Trichloroethylene-dependent inactivation of toluene 2-monooxygenase activity was observed, All of the protein components were covalently m odified during the oxidation of trichloroethylene. The addition of cys teine to reaction mixtures partially protected the enzyme system again st inactivation, most notably protecting the NADH-oxidoreductase compo nent, This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase.