A wealth of H(2)O-producing NADH oxidase (NOX) homologues have been discovered in the genomes of the hyperthermophilic Archaea, including two homologues in the genome of Pyrococcus furiosus which have been designated as NOX1 and NOX2. In order to investigate the function of NOX1, the structural gene encoding NOX1 was cloned from the genome of P. furiosus and expressed in Escherichia coli, and the resulting recombinant enzyme (rNOX1) was purified to homogeneity. The enzyme is a thermostable flavoprotein that can be reconstituted only with FAD. rNOX1 catalyzes the oxidation of NADH, producing both H(2)O(2) and H(2)O as reduction products of O(2) (O(2) + 1-2NADH + 1-2H(+) --> 1-2NAD(+) + H(2)O(2) or 2H(2)O). To our knowledge, this is the first NADH oxidase found to produce both H(2)O(2) and H(2)O. The enzyme exhibits a low K(m) for NADH (< 4 microm), and shows little or no reaction with NADPH. Transcriptional analyses demonstrated that NOX1 is constitutively expressed regardless of the carbon source and a single promoter was identified 25 bp upstream of the nox1 gene by primer extension. Although P. furiosus is a strict anaerobe, it may tolerate oxygen to some extent and we anticipate NOX1 to be involved in the response to oxygen at high temperatures.