Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea.
Cytochrome P460 and hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyze the oxidation of hydroxylamine. Cytochrome P460 contains an unidentified heme-like chromophore whose distinctive spectroscopic properties are similar to those for the P460 heme found in HAO. The heme P460 of HAO has previously been shown by protein chemistry and NMR structural analysis to be a c-heme with an additional covalent crosslink between the C2 ring carbon of a tyrosine residue of the polypeptide chain and a meso carbon of the porphyrin [Arciero, D.M. et al. (1993) Biochemistry 32, 9370-9378]. The recent determination of the gene sequence for cytochrome P460 [Bergmann, D.J. and Hooper, A.B. (1994) FEBS Lett. 353, 324-326] indicates that the heme in this protein also possesses a c-heme binding site and provides the basis for determining whether an HAO-like crosslink exists to the porphyrin. Sequence analysis of a purified heme-containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle. Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV. Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding motif. The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K. The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO. While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pKa values for lysine and tyrosine.
Arciero
D M
DM
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108, USA.
Hooper
A B
AB
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003580:Cytochromes / Q000737:chemistry*; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D009606:Nitrosomonas / Q000201:enzymology*
D003432:Cross-Linking Reagents; D003580:Cytochromes; C031690:heme C; D006418:Heme; C080650:cytochrome P-460; D008239:Lysine