title: The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.
abstract: Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches haem covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of holocytochromes c by CcmFH and CcmG, a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH-only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I, with a cleaved signal sequence and two covalent bonds to haem. We engineered increased cytochrome c production by CcmFGH-only, with yields approaching those from the full system I. Three conserved histidines in CcmF (TM-His1, TM-His2 and P-His1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH. Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis.
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO, 63130, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: San Francisco
authors.forename: Molly C
authors.initials: MC
authors.lastname: Sutherland
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D045304:Cytochromes c / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008190:Lyases / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D006418:Heme; D006639:Histidine; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase; D003545:Cysteine