Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation.Although the polynitroaromatic 2,4,6-trinitrotoluene (TNT) is rather recalcitrant, a number of microorganisms have been described to be able to use this compound as a nitrogen source (reviewed in reference 4). The presence of the three electrophilic nitro substituents on the aromatic ring tends to inactivate its system, thereby preventing oxidative attack by oxygenases that are frequently used by living organisms to catabolize aromatic compounds. Nevertheless, the nitro groups are easily reduced to hydroxylamino groups by nitroreductases (2,8,10,15). Ammonium has been proposed to be released from these derivatives upon a Bamberger-type rearrangement (3,6,8). On the other hand, the aromatic ring of the polynitroaromatic compound is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (13,14). This type of ring reduction is catalyzed by reductases, which are referred to here as type II hydride transferases.Our recent results show that the P. putida XenB protein, a type II hydride transferase of the Old Yellow Enzyme (OYE) family, reduces the nitro groups to produce hydroxylamines and also the aromatic ring to yield the transient production of the Meisenheimer monohydride complex (H Ϫ -TNT), which is further reduced to various isoforms of the Meisenheimer dihydride complex (2H Ϫ -TNT) (19). It is worth noting that nitrite is released in this reaction via the abiotic condensation of enzymatically produced hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes that yield diarylamines and stoichiometric amounts of nitrite, thereby making a mass balance possible for the first time (19).Several enzymes of the OYE family of flavoproteins (17) with type II hydride transferase activity have been described, i.e., the pentaerythritol tetranitrate (PETN) reductase of Enterobacter cloacae PB2 (5), the N-ethylmaleimide (NEM) reductase of several Escherichia coli strains (6, 18), and xenobiotic reductase B (XenB) of Pseudomonas fluorescens I-C (9). However, the end products formed from TNT by these type II hydride transferases remained unclear, although in the case of the XenB enzyme of P. fluorescens, Pak et al. (9) previously proposed a putative biphenyl structure as an end product based on the molecular mass. A product with a molecular mass similar to that found by Pak et al. (9) was found in cell extracts of E. ...