Enzyme catalyzed biotransformation of the energetic chemical octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is not known. The present study describes a xanthine oxidase (XO) catalyzed biotransformation of HMX to provide insight into the biodegradation pathway of this energetic chemical. The rates of biotransformation under aerobic and anaerobic conditions were 1.6 +/- 0.2 and 10.5 +/-0.9 nmol h(-1) mg protein(-1), respectively, indicating that anaerobic conditions favored the reaction. The biotransformation rate was about 6-fold higher using NADH as an electron-donor compared to xanthine. During the course of reaction, the products obtained were nitrite (NO2-), methylenedinitramine (MDNA), 4-nitro-2,4-diazabutanal (NDAB), formaldehyde (HCHO), nitrous oxide (N2O), formic acid (HCOOH), and ammonium (NH4+). The product distribution gave carbon and nitrogen mass-balances of 91% and 88%, respectively. A comparative study with native-, deflavo-, and desulfo-XO and the site-specific inhibition studies showed that HMX biotransformation occurred at the FAD-site of XO. Nitrite stoichiometry revealed that an initial single N-denitration step was sufficient for the spontaneous decomposition of HMX. Crown Copyright (C) 2003 Published by Elsevier Science (USA). All rights reserved.