Variation of the axial haem ligands and haem-binding motif as a probe of the Escherichia coli c-type cytochrome maturation (Ccm) system.
Cytochromes c are typically characterized by the covalent attachment of haem to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-Cys-His peptide motif. In many Gram-negative bacteria, the haem is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c552 can be expressed as a stable holocytochrome both in the cytoplasm of Escherichia coli in an apparently uncatalysed reaction and also in the periplasm in a Ccm-mediated reaction. In the present study we show that a Met60-->Ala variant of c552, which does not have the usual distal methionine ligand to the haem iron of the mature cytochrome, can be made in the periplasm by the Ccm system. However, no holocytochrome could be detected when this variant was expressed cytoplasmically. These data highlight differences between the two modes of cytochrome c assembly. In addition, we report investigations of haem attachment to cytochromes altered to have the special Cys-Trp-Ser-Cys-Lys haem-binding motif, and Cys-Trp-Ser-Cys-His and Cys-Trp-Ala-Cys-His analogues, of the active-site haem of nitrite reductase NrfA.
Allen
James W A
JW
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Ferguson
Stuart J
SJ
D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D002352:Carrier Proteins; D003574:Cytochrome c Group; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; C083951:cytochrome c553; D006418:Heme; C022302:cytochrome C-552