Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase Bfrom Pseudomonas fluorescens I-C

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified Xen B, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluoresc ens I-C, was evaluated by using natural abundance and [U-C-14]TNT preparati ons. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydro xylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subseq uent reactions of these metabolites were nonenzymatic and resulted in predo minant formation of at least three dimers with an anionic mit of 376 as det ermined by negative-mode electrospray ionization mass spectrometry and the release of similar to0.5 mol of nitrite per mol of TNT consumed. The extent s of the initial enzymatic reactions were similar in the presence and in th e absence of O-2, but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP(+), Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed tha t both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonat ed dihydride-Meisenheimer complex of TNT were required precursors for the d imerization and nitrite release reactions. The mit 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing e vidence for an aryl amine functional group. In combination, the experimenta l results are consistent with assigning the chemical structures of the mit 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl, A mech anism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.