ALTERATION OF HEME-ATTACHMENT AND SIGNAL-CLEAVAGE SITES FOR PARACOCCUS-DENITRIFICANS CYTOCHROME C(550) PROBES PATHWAY OF C-TYPE CYTOCHROME BIOGENESIS IN ESCHERICHIA-COLI

Paracoccus denitrificans cytochrome c(550) is expressed as a periplasm ic hole-protein in Escherichia coli; amino acid substitutions of cyste ine residues in the haem-binding motif (Cys-X-X-Cys-His), either toget her or singly, prevented covalent attachment of haem but not polypepti de translocation into the periplasm. When the three alanine residues a t positions -3 to -1 in the native signal-cleavage site were deleted, or alanine at -1 was changed to glutamine, signal cleavage was at alte rnative sites (after only ten residues in the latter case), but haem a ttachment still occurred, When the same three alanines were changed to Asp-Glu-Asp, a membrane-associated ape product that had retained the complete signal sequence was detected, These and other results present ed here indicate that (i) haem attachment is not required for the apo- cytochrome c(550) export to the periplasm; (ii) haem cannot attach to apocytochrome c(550) when attached to the cytoplasmic membrane, sugges ting that signal-sequence cleavage precedes periplasmic haem attachmen t, which can occur at as few as six residues from the mature N-terminu s; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate, The gene for Saccharomyc es cerevisiae mitochondrial iso-l-cytochrome c was expressed as a hole -protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome c(550), indicating that the E. c oli cellular apparatus for the c-type cytochrome biogenesis has a broa d substrate specificity.