Hydroxylamine oxidoreductase (HAO) from the autotrophic bacterium Nitrosomonas europaea catalyzes the 4-e(-) oxidation of NH2-OH to NO2-. The e(-) are transferred from NH2OH to an unusual 5-coordinate heme known as P460, which is the active site of HAO, and from there to an array of seven c-type hemes. NO., generated by laser flash photolysis of N,N'-bis (carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine, is found to act as a 1-e(-) donor to HAO. Most likely NO. binds P460 to yield a {Fe(NO)}(6) moiety, which then hydrolyzes to give the reduced enzyme and NO2-. The {Fe(NO)}(6) moiety is also a plausible final intermediate in the oxidation of NH2OH.