Resolution of multiple heme centers of hydroxylamine oxidoreductase from Nitrosomonas. 2. Mössbauer spectroscopy.

Abstract

Hydroxylamine oxidoreductase (HAO) isolated from Nitrosomas europaea is a complex protein of Mr 220000 with an (alpha beta)3 subunit structure. Each alpha beta 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 Mössbauer spectroscopy. In the fully reduced enzyme, approximately seven hemes per alpha beta 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 Mössbauer 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-450 moiety amounts to at least one Fe per alpha beta subunit. Together the Mössbauer results and the optical spectra suggest that the P-460 moiety is a heme. The Mössbauer 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.