Hydroxylamine oxidoreductase (HAO) isolated from Nitrosomas europaea is a complex protein of Mτ 220000 with an (αβ)3 subunit structure. Each aj3 subunit contains seven c-type hemes and approximately one unusual prosthetic group termed P-460. We have studied this enzyme in the oxidized and reduced states by using Mossbauer spectroscopy. In the fully reduced enzyme, approximately seven hemes per αβ subunit contributed to one spectrum characteristic of low-spin ferrous heme. The remainder of the iron (10-15% of the total) yielded an ill-defined absorption pattern. Carbon monoxide binds to the P-460 as shown by optical spectra. The Mossbauer spectra of reduced hydroxylamine oxidoreductase which had been exposed to CO showed a new spectral component, corresponding to one iron site, with parameters characteristic of a low-spin ferrous heme-carbonyl complex. It appears that this component is derived from the ill-defined spectrum observed in the reduced enzyme. This is the first direct evidence that the P-460 moiety amounts to at least one Fe per αβ subunit. Together the Mossbauer results and the optical spectra suggest that the P-460 moiety is a heme. The Mossbauer spectra of the oxidized (as isolated) enzyme suggest the presence of one or two low-spin ferric hemes which might be EPR undetectable because of either fast electronic spin relaxation or participation in a spin-coupled pair. The spectra gave no evidence for the presence of a ferrous site in oxidized HAO.