Cytochrome P460 of Nitrosomonas europaea -: Formation of the heme-lysine cross-link in a heterologous host and mutagenic conversion to a non-cross-linked cytochrome c′

The heme of cytochrome P460 of Nitrosomonas europaea , which is covalently crosslinked to two cysteines of the polypeptide as with all c -type cytochromes, has an additional novel covalent crosslink to lysine 70 of the polypeptide [Arciero, D.M. & Hooper, A.B. (1997) FEBS Lett. 410 , 457-460]. The protein can catalyze the oxidation of hydroxylamine. The gene for this protein, cyp , was expressed in Pseudomonas aeruginosa strain PAO lac I, resulting in formation of a holo-cytochrome P460 which closely resembled native cytochrome P460 purified from N. europaea in its UV-visible spectroscopic, ligand binding and catalytic properties. Mutant versions of cytochrome P460 of N. europaea in which Lys70 70 was replaced by Arg, Ala, or Tyr, retained ligand-binding ability but lost catalytic ability and differed in optical spectra which, instead, closely resembled those of cytochromes c '. Tryptic fragments containing the c -heme joined only by two thioether linkages were observed by MALDI-TOF for the mutant cytochromes P460 K70R and K70A but not in wild-type cytochrome P460, consistent with the structural modification of the c -heme only in the wild-type cytochrome. The present observations support the hypothesized evolutionary relationship between cytochromes P460 and cytochromes c ' in N. europaea and M. capsulatus [Bergmann, D.J., Zahn, J.A., & DiSpirito, A.A. (2000) Arch. Microbiol . 173 , 29-34], confirm the importance of a heme-crosslink to the spectroscopic properties and catalysis and suggest that the crosslink might form auto-catalytically.