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 lacI, 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.
Bergmann
David J
DJ
Department of Biology, Black Hills State University, Spearfish, SD, USA.
Hooper
Alan B
AB
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D009154:Mutation; D009606:Nitrosomonas / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D003580:Cytochromes; D006418:Heme; C080650:cytochrome P-460