Aerobic Denitration of 2,4,6-Trinitrotoluene in the Presence of Phenazine Compounds and Reduced Pyridine Nucleotides

Phenazine-containing spent culture supernatants of Pseudomonas aeruginosa concentrated with a C-18 solid-phase extraction cartridge initiate NAD(P)H-dependent denitration of 2,4,6-trinitrotoluene (TNT). In this study, TNT denitration was investigated under aerobic conditions using two phenazine secondary metabolites excreted by P. aeruginosa, pyocyanin (Py) and its precursor phenazine-l-carboxylic acid (PCA), and two chemically synthesized pyocyanin analogs, phenazine methosulfate (PMS+) and phenazine ethosulfate (PES+) The biomimetic Py/NAD(P)H/O-2 system was characterized and found to extensively denitrate TNT in unbuffered aqueous solution with minor production of toxic aminoaromatic derivatives. To a much lesser extent, TNT denitration was also observed with PMS+ and PES+ in the presence of NAD(P)H. No TNT denitration was detected with the biomimetic PCA/NAD(P)H/O-2 system. Electron paramagnetic resonance (EPR) spectroscopy analysis of the biomimetic Py/NAD(P)H/O-2 system revealed the generation of superoxide radical anions (O-2(center dot-)). In vitro TNT degradation experiments in the presence of specific inhibitors of reactive oxygen species suggest a nucleophilic attack of superoxide radical anion followed by TNT denitration through an as yet unknown mechanism. The results of this research confirm the high functional versatility of the redox-active metabolite pyocyanin and the susceptibility of aromatic compounds bearing electron-withdrawing substituents, such as nitro groups, to superoxide-driven nucleophilic attack.