EVIDENCE FOR THE STRUCTURE OF THE ACTIVE-SITE HEME-P460 IN HYDROXYLAMINE OXIDOREDUCTASE OF NITROSOMONAS

Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine to nitrite in nitrification by Nitrosomonas europaea. I t has an alpha(n) subunit structure and eight covalently bound hemes p er subunit. Seven of these have visible spectra indistinguishable from heme c. The eighth, designated as P460, has unusual visible spectrosc opic features in the enzyme and in a heme-containing proteolytic fragm ent. Its structure has not been previously determined. Enzymatic diges tions of HAO were performed, and various proteolytic fragments were pu rified. Mass spectrometry confirmed the presence of authentic heme c i n some fragments, that is, iron protoporphyrin IX cross-linked by two thioether bonds to cysteine residues. It was possible to detect the pr esence of the P460 pigment in some fragments, based upon the sensitivi ty of this pigment to treatment of the holoenzyme with hydrogen peroxi de. A proteolytic fragment produced by sequential digestion with tryps in and pronase was shown to contain heme c and a hydrogen peroxide-sen sitive heme with an unusual visible, spectrum. This fragment contained two covalently cross-linked peptides. Mass spectrometry and NMR indic ated that the P460 heme was iron protoporphyrin IX covalently bonded b y two thioether bridges to peptide, but in addition there was a new, t hird covalent bond between a meso heme carbon and an aromatic ring car bon on a tyrosyl residue. The new covalent bond has been tentatively a ssigned to the C2 carbon of the tyrosyl ring and the 5-meso heme carbo n (IUPAC-IUB tetrapyrrole nomenclature), although this location requir es further proof.