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Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.

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.

Cytochrome c′ of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome c555. This cytochrome is spectrally similar to other cytochromes c′ but is larger (16,000 Da) and has a lower midpoint potential (–205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c′ from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c′, only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c′ from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c′-type cytochrome which developed a covalent cross-link between a lysine residue and the c′-heme. [ABSTRACT FROM AUTHOR]

Presents the isolation of cytochrome C5530, the polypeptide and structural gene for a high-molecular-mass c-type cytochrome from the methanotroph Methylococcus capsulatus Bath. Purification of the cytochrome c5530; Heme and metal components of the cytochrome; Spectral properties.

Provides information on a study which examined the gene encoding cytochrome P460 from Methylococcus capsulatus Bath and compared the deduced amino acid sequence to that of cytochrome P460 from Nitrosomonas europaea. Materials and methods; Results from the transcriptional start site mapping; Discussion.

Investigates the organization of genes for three proteins in ammonia oxidation in Nitrosomonas europaea. Cloning and sequencing for two tetraheme c cytochromes; Amino acid sequence of heme-containing proteolytic peptides; Sequence of the gene for cytochrome C554; Sequence of ORF2.