Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit)(-1), showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit)(-1) and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were > or =10 and > or =95 degrees C, respectively. At pH 7 and 83 degrees C, apparent Km values for NADH and O2 were 3 microM and 1.9 mM, respectively, and the specific activity at 1.4 mM O2 was 60 micromol min(-1) mg(-1); 62 % of NADH-derived reducing equivalents were recovered as H2O2 and the rest probably generated H2O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5'-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high Km, O2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.