title: The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrome c.
abstract: Cytochromes c are typically characterized by the covalent attachment of heme 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 heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c(552), which has a normal CXXCH heme-binding motif, and variants with AXXCH, CXXAH, and AXXAH motifs, can be expressed as stable holocytochromes in the cytoplasm of Escherichia coli. By targeting these proteins to the periplasm using a signal peptide, with or without co-expression of the Ccm proteins, we have assessed the ability of the Ccm system to attach heme to proteins with no, one, or two cysteine residues in the heme-binding motif. Only the wild-type protein, with two cysteines, was effectively processed and thus accumulated in the periplasm as a holocytochrome. This is strong evidence for disulfide bond formation involving the two cysteine residues of apocytochrome c as an intermediate in Ccm-type Gram-negative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: James W A
authors.initials: JW
authors.lastname: Allen
authors.forename: Esther J
authors.initials: EJ
authors.lastname: Tomlinson
authors.forename: Lin
authors.initials: L
authors.lastname: Hong
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D020816:Amino Acid Motifs; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c; D004926:Escherichia coli / Q000201:enzymology*; D006418:Heme / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D003574:Cytochrome c Group; D006418:Heme; D045304:Cytochromes c; D003545:Cysteine