title: The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus.
abstract: c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: James W A
authors.initials: JW
authors.lastname: Allen
authors.forename: Nicholas
authors.initials: N
authors.lastname: Leach
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D003573:Cytochrome b Group / Q000235:genetics* / Q000378:metabolism*; D045304:Cytochromes c / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006639:Histidine; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding
chemical_list: D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006418:Heme; D006639:Histidine; D045304:Cytochromes c; C017850:cytochrome b562, E coli; D001120:Arginine; D008239:Lysine