title: Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus.
abstract: c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b(562) to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CX(n)CH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: James W A
authors.initials: JW
authors.lastname: Allen
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Sawyer
authors.forename: Michael L
authors.initials: ML
authors.lastname: Ginger
authors.forename: Paul D
authors.initials: PD
authors.lastname: Barker
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006418:Heme; D045304:Cytochromes c; C017850:cytochrome b562, E coli; D003545:Cysteine