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

Abstract

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 CX(2)CH 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 (CX(2)CK or CX(2)CH). W. succinogenes CcsA2 was found to attach haem to standard CX(2)CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active-site CX(2)CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX(15)CH 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.

Fig. 1

Detection of W. succinogenes c-type cytochromes in cell homogenates and cellular fractions of nitrate-grown cells. 100 μg protein per lane were separated on an SDS polyacrylamide gel (12.5%) and the gel was subjected to haem staining. The size marker (M) comprises pre-stained proteins. NrfA and NrfH have been experimentally identified (Simon et al., 2000, 2001). Assignment of other cytochromes is based on data shown in Table 1. CH, cell homogenate; SF, soluble fraction; MF, membrane fraction; LMWC, low molecular weight cytochromes.

Fig. 2

Production of CCHL isoenzymes from W. succinogenes in E. coli RK103 and their functionality in haem c attachment to a reporter cytochrome with two CX₂CH haem c binding motifs. E. coli strains 21–24 (Table 4) were used. A. Chemiluminescent haem stain detection of B. pertussis CycC in periplasmic extracts (35 μg per lane) of E. coli RK103 producing the indicated CCHL. The cells contained a plasmid that enabled production of the indicated CCHL. The previously observed proteolytic degradation product of CycC (denoted CycC*) was only observed in cells harbouring the H. hepaticus (H.h.) CCHL (Richard-Fogal et al., 2007). B. Immunoblot detection of indicated CCHLs by Western blot analysis using an anti-GST serum. 50 μg of total cell protein were applied to each lane. The detected proteins of around 70 kDa most likely represent proteolytic CCHL fragments, each comprising the GST tag and the CcsB part of the CcsBA fusion protein. C refers to a control experiment using E. coli RK103 cells that contained the empty vectors pRGK330 and pGEX-4T-1 (Feissner et al., 2006). H.h., H. hepaticus.

Fig. 3

Chemiluminescent haem stain detection of B. pertussis CycC variants in periplasmic extracts (30 μg per lane) of E. coli RK103 cells that produced W. succinogenes CcsA2 as sole CCHL (strains 25–30 in Table 4). The two sequences denote the primary structures of the original haem c binding motifs (top, N-terminal motif, bottom, C-terminal motif). Wild-type haem c binding motifs are underlined.

Fig. 4

Heterologous production of catalytically active E. coli NrfA in W. succinogenes. A. Partial physical maps of the genomes of W. succinogenes EcNrfA and W. succinogenes EcNrfA ΔnrfIJ. P_nrf, W. succinogenes cytochrome c nitrite reductase promoter element; SigNrfA, DNA stretch encoding the signal peptide of W. succinogenes NrfA; 2xStrep-Tag, DNA region encoding two consecutive Strep-tags. B. Haem stain analysis of cell homogenates (100 μg protein per lane) from the indicated W. succinogenes strains. C. Visualisation of nitrite reductase activity in cell homogenates of the indicated W. succinogenes strains. Proteins from the soluble cell fraction (50 μg per lane) were subjected to native gel electrophoresis and the gel was stained for activity as described in Experimental procedures. The marker proteins represent monomers, homodimers and homotrimers of bovine serum albumin.

Fig. 5

Heterologous production of C. jejuni NrfA in W. succinogenes. A. Partial physical maps of the genomes of W. succinogenes CjNrfA, W. succinogenes CjNrfA ΔnrfIJ and W. succinogenes CjNrfA H153K. See legend of Fig. 4A for further explanations. B. Haem stain analysis of cell homogenates and cell fractions (100 μg protein per lane) from the indicated W. succinogenes strains. CH, cell homogenate; SF, soluble cell fraction; MF, membrane fraction.