Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome c550 probes pathway of c-type cytochrome biogenesis in Escherichia coli

Authors

  • Yoshihiro Sambongi,

    1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
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    • ISIR, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567, Japan

  • Raphael Stoll,

    1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
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    • Physiologisch-chemisches lnstitut der Universitat, Hoppe-Seyler Strasse 4, D-72076 Tubingen, Germany.

  • Stuart J. Ferguson

    Corresponding author
    1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
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Summary

Paracoccus denitrificans cytochrome c550 is expressed as a periplasmic holo-protein in Escherichia coli; amino acid substitutions of cysteine residues in the haembinding motif (Cys-X-X-Cys-His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions -3 to -1 in the native signal-cleavage site were deleted, or alanine at -1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp-Glu-Asp, a membrane-associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo-cytochrome c550 export to the periplasm; (ii) haem cannot attach to apocytochrome c550 when attached to the cytoplasmic membrane, suggesting that signal-sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N-terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso-1-cytochrome c was expressed as a holo-protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome c550, indicating that the E. coli cellular apparatus for the c-type cytochrome biogenesis has a broad substrate specificity.

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