EVIDENCE FOR A CROSS-LINK 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 hydroxglamine. Cytochrome P460 con tains an unidentified heme-like chromophore whose distinctive spectros copic properties are similar to those for the P460 heme found in HAO, The heme P460 of HAO has previously been shown by protein chemistry an d NMR structural analysis to be a c-heme with an additional covalent c rosslink between the C2 ring carbon of a tyrosine residue of the polyp eptide chain and a meso carbon of the porphyrin [Arciero, D,M, et al, (1993) Biochemistry 32, 9370-9378], The recent determination of the ge ne sequence for cytochrome P460 [Bergmann, D,J, and Hooper, A,B, (1994 ) FEES Lett, 353, 324-326] indicates that the heme in this protein als o possesses a c-heme binding site and provides the basis for determini ng whether an HAO-like crosslink exists to the porphyrin, Sequence ana lysis of a purified heme-containing tryptic chromopeptide from cytochr ome P460 revealed two predominant amino acid residues per cycle, Two p eptides present in the chromopeptide with the sequences NLPTAEXAAXHK a nd DGTVTVXELVSV, Comparison of the data to the gene sequence for the p rotein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding mot if, The gap in the sequence in the second peptide at cycle 7 is predic ted by the gene sequence to be a K, The results suggest that the lysin e residue is crosslinked in some manner to the porphyrin macrocycle, p ossibly mimicking the tyrosine crosslink found for the heme P460 of HA O, While a common role for the crosslinked residues in HAO and cytochr ome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pK(a) values for lys ine and tyrosine. (C) 1997 Federation of European Biochemical Societie s.