Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1

• Published in: Molecular microbiology 2010-01, Vol.75 (1), p.122-137
• Main Authors: Kern, Melanie, Eisel, Florian, Scheithauer, Juliane, Kranz, Robert G, Simon, Jörg
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Campylobacter jejuni - enzymology; Campylobacter jejuni - genetics; E coli; Enzymes; Escherichia coli - enzymology; Escherichia coli - genetics; Genomics; Heme - metabolism; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Lyases - genetics; Lyases - metabolism; Nitrates; Proteomics; Substrate Specificity; Wolinella - enzymology
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2009.06965.x

Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX₂CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c-type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active-site HBMs (CX₂CK or CX₂CH). W. succinogenes CcsA2 was found to attach haem to standard CX₂CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active-site CX₂CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX₁₅CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterized structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome, a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c-type cytochromes.