H2O2-forming NADH oxidase with diaphorase (cytochrome) activity from Archaeoglobus fulgidus

An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from t he hyperthermophilic sulfate-reducing anaerobe Archaeoglobus fulgidus. N-te rminal sequence of the protein indicates that it is coded for by open readi ng frame AF0395 in the A.fulgidus genome. The gene AF0395 was cloned and it s product was purified from Escherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 4 7 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalen t oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaph orase activity in the presence of electron acceptors such as tetrazolium, a nd cytochrome c. During purification NoxA2 remains associated with the enzy me responsible for D-lactate oxidation, the D-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. To gether these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.