Our previous report showed the existence of microaerophilic Bifidobacterium species that can grow well under aerobic conditions rather than anoxic conditions in a liquid shaking culture. The difference in the aerobic growth properties between the O 2 -sensitive and microaerophilic species is due to the existence of a system to produce H 2 O 2 in the growth medium. In this study, we purified and characterized the NADH oxidase that is considered to be a key enzyme in the production of H 2 O 2 . Bifidobacterium bifidum, an O 2 -sensitive bacterium and the type species of the genus Bifidobacterium, possessed one dominant active fraction of NADH oxidase and a minor active fraction of NAD(P)H oxidase activity detected in the first step of column chromatography for purification of the enzyme. The dominant active fraction was further purified and determined from its N-terminal sequence to be a homologue of b-type dihydroorotate dehydrogenase (DHOD), composed of PyrK (31 kDa) and PyrDb (34 kDa) subunits. The genes that encode PyrK and PryDb are tandemly located within an operon structure. The purified enzyme was found to be a heterotetramer showing the typical spectrum of a flavoprotein, and flavin mononucleotide and flavin adenine dinucleotide were identified as cofactors. The purified enzyme was characterized as the enzyme that catalyzes the DHOD reaction and also catalyzes a H 2 O 2 -forming NADH oxidase reaction in the presence of O 2 . The kinetic parameters suggested that the enzyme could be involved in H 2 O 2 production in highly aerated environments.Obligatory anaerobes such as Clostridium, sulfate-reducing bacteria, and Bacteroides species are known as anaerobes that show inhibited growth in the presence of oxygen; however, recent research has revealed that these anaerobes show various types of oxic growth, depending on the concentration of oxygen, over a range of 0.1 to 21% in association with the expression of oxygen metabolic enzymes (5, 8, 11, 12, 17-20, 26, 30, 38, 44, 48).The genus Bifidobacterium contains probiotic species beneficial for the human intestine, but its sensitivity to O 2 decreases its viability during the stages of food processing, storage, and incorporation into the human intestine (42, 45). Several approaches have been taken to determine the mechanisms of bifidobacterial aerobic growth inhibition (10, 21, 43). de Vries and Stouthamer proposed that O 2 -sensitive Bifidobacterium species produce H 2 O 2 through the reaction of NADH oxidase detected in cell extracts (10); however, the molecular-level mechanisms of growth inhibition and H 2 O 2 production have since remained essentially unknown.In our previous study, we classified Bifidobacterium species into oxygen-sensitive species and microaerophilic species (21,22). Many species belonging to the genus Bifidobacterium show oxygen-sensitive growth profiles under highly aerated conditions (21). These species grow in liquid medium when shaken under 5% O 2 conditions, but growth is inhibited under 10 to 20% O 2 conditions with an accumulation...

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