The NADH oxidase from Pyrococcus furiosus - Implications for the protection of anaerobic hyperthermophiles against oxidative stress

A wealth of H2O-producing NADH oxidase (NOX) homologues have been discovere d in the genomes of the hyperthermophilic Archaea, including two homologues in the genome of Pyrococcus furiosus which have been designated as NOX1 an d NOX2. In order to investigate the function of NOX1, the structural gene e ncoding NOX1 was cloned from the genome of P. furiosus and expressed in Esc herichia coli, and the resulting recombinant enzyme (rNOX1) was purified to homogeneity. The enzyme is a thermostable flavoprotein that can be reconst ituted only with FAD. rNOX1 catalyzes the oxidation of NADH, producing both H2O2, and H2O as reduction products of O-2, (O-2 + 1-2NADH + 1-2H(+) --> 1 -2NAD(+) + H2O2 or 2H(2)O). To our knowledge, this is the first NADH oxidas e found to produce both H2O2 and H2O. The enzyme exhibits a low K-m for NAD H (< 4 <mu>M), and shows little or no reaction with NADPH. Transcriptional analyses demonstrated that NOX1 is constitutively expressed regardless of t he 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.