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. It has an alpha n subunit structure and eight covalently bound hemes per subunit. Seven of these have visible spectra indistinguishable from heme c. The eighth, designated as P460, has unusual visible spectroscopic features in the enzyme and in a heme-containing proteolytic fragment. Its structure has not been previously determined. Enzymatic digestions of HAO were performed, and various proteolytic fragments were purified. Mass spectrometry confirmed the presence of authentic heme c in some fragments, that is, iron protoporphyrin IX cross-linked by two thioether bonds to cysteine residues. It was possible to detect the presence of the P460 pigment in some fragments, based upon the sensitivity of this pigment to treatment of the holoenzyme with hydrogen peroxide. A proteolytic fragment produced by sequential digestion with trypsin and pronase was shown to contain heme c and a hydrogen peroxide-sensitive heme with an unusual visible spectrum. This fragment contained two covalently cross-linked peptides. Mass spectrometry and NMR indicated that the P460 heme was iron protoporphyrin IX covalently bonded by two thioether bridges to peptide, but in addition there was a new, third covalent bond between a meso heme carbon and an aromatic ring carbon on a tyrosyl residue. The new covalent bond has been tentatively assigned to the C2 carbon of the tyrosyl ring and the 5-meso heme carbon (IUPAC-IUB tetrapyrrole nomenclature), although this location requires further proof.
Arciero
D M
DM
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Hooper
A B
AB
Cai
M
M
Timkovich
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006418:Heme / Q000737:chemistry*; D006736:Horses; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009606:Nitrosomonas / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry*; D011487:Protein Conformation
D006418:Heme; D010088:Oxidoreductases; C021401:hydroxylamine dehydrogenase