A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V max and K m for hydrazine are 6.2 ؎ 0.3 mol/min · mg and 5.5 ؎ 0.6 M, respectively. Hydrazine (25 M) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 M) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of "Candidatus Kuenenia stuttgartiensis" (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.The anaerobic ammonium oxidation (anammox) process is a fascinating subject of study, not only because it is an innovative technological advance for the removal of nitrogenous contaminants from wastewater (14,17,27), but also because it includes interesting and novel biochemical reactions. In the anammox reaction, ammonium and nitrite contribute in equimolar amounts to the formation of dinitrogen gas (N 2 ) according to the following equation (26): NH 4 ϩ ϩ NO 2 Ϫ 3 N 2 ϩ 2H 2 O; G°ϭ Ϫ358 kJ/mol ammonium. This biological reaction is performed under anoxic conditions by novel autotrophic bacteria, which have not yet been isolated but which are identified as deeply branching planctomycetes, based on nucleotide sequences of 16S rRNA genes (17,22,26). The first anammox bacterium to be identified, discovered in The Netherlands, has been provisionally named "Candidatus Brocadia anammoxidans" (15). An increasing number of 16S rRNA gene sequences from other putative anaerobic ammonium-oxidizing bacteria have been reported to date from laboratories (6,21,29,31) and from natural marine habitats such as the Black Sea (4, 5, 13, 16).We designed a continuous-flow reactor shielded from light in which to enrich cultures for bacteria having the ability to catalyze the anammox reaction from a seed of denitrifying sludge originating in Kumamoto City, Japan. In the reactor, the culture turned a distinctly red color and yielded large amounts of sludge that could remove ammonium and nitrite with a relatively low production of nitrate at ratios strongly resembling that of the anammox reaction (20). To investigate the bacterial composition of the enriched culture community, 16S rRNA gene sequences were amplified by PCR and cloned. The dominant clones had an identical sequence, which has 92.2% identity with the 16S rRNA gene of "Candidatus B...
