Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K m and k cat values of TNT reduction were 165 ؎ 43 M for TNT and 400 ؎ 94 s ؊1 , respectively. Cyanide, an inhibitor for the CO/CO 2 oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.2,4,6-Trinitrotoluene (TNT) is a chemical explosive that is a common contaminant of soils and groundwater at numerous Department of Defense facilities. For several decades, research has been conducted to develop ecologically sound means of remediating sites contaminated with this toxic compound. Studies investigating the potential for bioremediation (i.e., the use of microorganisms to metabolize hazardous wastes) have demonstrated that many aerobic and anaerobic microorganisms are capable of catalyzing the reduction of aryl nitro groups associated with TNT (3,6,[8][9][10]16), forming often uncharacterized products.Aerobic microorganisms contain nitroreductases that catalyze such reductions. An aryl nitro reductase purified from Neurospora crassa reduces several nitrobenzenes, 3,5-dinitrobenzoic acid, and TNT (22). In the presence of NADPH, enzymes in the crude extract of Pseudomonas sp. strain CBS3 catalyzed the reduction of p-nitrobenzoate and TNT (16). An NADH-or NADPH-dependent nitro reductase from Enterobacter cloacae also catalyzes the reduction of TNT (3).Some anaerobes reduce nitro groups to amines through a mechanism involving nitroso and hydroxyl-amino intermediates, while others, such as Clostridium acetobutylicum, reduce the nitro groups of TNT to hydroxyl-amino-nitrotoluenes without further reduction to the corresponding amines (6-8). The enzymes and/or proteins involved in these transformations have not been identified (9, 10), although ferredoxins, hydrogenases, CO dehydrogenases (CODHs), pyruvate-ferredoxin oxidoreductases, and sulfite reductases have all been implicated. For example, hydrogenase from Clostridium pasteurianum (in the presence of H 2 ) and partially purified CODH from Clostridium thermoaceticum (in the presence of CO), reduced 2,4-diamino-6-nitrotoluene to 2,4-diamino-6-hydroxylaminotoluene when ferredoxin was included in the reaction mixture (10). Reduction also occurred with reduced ferredoxin or methyl viologen in the absence of enzymes, although the rate was slower by orders of magni...
