title: Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III.
abstract: The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.
authors.affiliation: Department of Biology, Washington University, St. Louis, MO 63130, USA.
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Richard-Fogal
authors.forename: Brian
authors.initials: B
authors.lastname: San Francisco
authors.forename: Elaine R
authors.initials: ER
authors.lastname: Frawley
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D045304:Cytochromes c / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017353:Gene Deletion; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D006801:Humans; D008190:Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D033903:Periplasmic Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001059:Apoproteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments; D033903:Periplasmic Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D006418:Heme; D045304:Cytochromes c; D010088:Oxidoreductases; C094848:DsbD electron transport protein, E coli; D008190:Lyases; C031581:cytochrome C synthetase; D019704:Protein Disulfide-Isomerases; C526649:dsbC protein, E coli