title: Avoidance of the cytochrome c biogenesis system by periplasmic CXXCH motifs.
abstract: The CXXCH motif is usually recognized in the bacterial periplasm as a haem attachment site in apocytochromes c. There is evidence that the Escherichia coli Ccm (cytochrome c maturation) system recognizes little more than the CXXCH sequence. A limited number of periplasmic proteins have this motif and yet are not c-type cytochromes. To explore how unwanted haem attachment to CXXCH might be avoided, and to determine whether haem attachment to the surface of a non-cytochrome protein would be possible, we converted the active-site CXXCK motif of a thioredoxin-like protein into CXXCH, the C-terminal domain of the transmembrane oxidoreductase DsbD (cDsbD). The E. coli Ccm system was found to catalyse haem attachment to a very small percentage of the resultant protein ( approximately 0.2%). We argue that cDsbD folds sufficiently rapidly that only a small fraction fails to avoid the Ccm system, in contrast with bona fide c-type cytochromes that only adopt their tertiary structure following haem attachment. We also demonstrate covalent haem attachment at a low level in vivo to the periplasmic disulfide isomerase DsbC, which contains a native CXXCH motif. These observations provide insight into substrate recognition by the Ccm system and expand our understanding of the requirements for covalent haem attachment to proteins. The possible evolutionary relationship between thioredoxins and c-type cytochromes is discussed.
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.
authors.forename: Despoina A I
authors.initials: DA
authors.lastname: Mavridou
authors.forename: Martin
authors.initials: M
authors.lastname: Braun
authors.forename: Linda
authors.initials: L
authors.lastname: Thöny-Meyer
authors.forename: Julie M
authors.initials: JM
authors.lastname: Stevens
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D045304:Cytochromes c / Q000096:biosynthesis* / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D006418:Heme; D045304:Cytochromes c; D010088:Oxidoreductases; C094848:DsbD electron transport protein, E coli; D019704:Protein Disulfide-Isomerases; C526649:dsbC protein, E coli