Previously, we reported that a salicylate 1-monooxygenase from Pseudomonas sp. ATCC 29352 biotransformed CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane) (C6H6N12O12) and produced a key metabolite with mol. wt. 346 Da corresponding to an empirical formula of C6H6N10O8 which spontaneously decomposed in aqueous medium to produce N2O, NH4+, and HCOOH [Appl. Environ. Microbiol. (2004)]. In the present study, we found that nitroreductase from Escherichia coli catalyzed a one-electron transfer to CL-20 to form a radical anion (CL-20(.-)) which upon initial N-denitration also produced metabolite C6H6N10O8. The latter was tentatively identified as 1,4,5,8-tetranitro-1,3a,4,4a,5,7a,8,8a-octahydro-diimidazo[4,5-b:4',5'-e ]pyrazine [IUPAC] which decomposed spontaneously in water to produce glyoxal (OHC-CHO) and formic acid (HCOOH). The rates of CL-20 biotransformation under anaerobic and aerobic conditions were 3.4 +/-0.2 and 0.25 +/- 0.01 nmol min(-1) mg of protein(-1), respectively. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.8 nitrite ions, 3.3 molecules of nitrous oxide, 1.6 molecules of formic acid, 1.0 molecule of glyoxal, and 1.3 ammonium ions. Carbon and nitrogen products gave mass-balances of 60% and 81%, respectively. A comparative study between native-, deflavo-, and reconstituted-nitroreductase showed that FMN-site was possibly involved in the biotransformation of CL-20. (C) 2004 Elsevier Inc. All rights reserved.