Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus

James W.A. Allen, Elizabeth B. Sawyer, Michael L. Ginger, Paul D. Barker, Stuart J. Ferguson

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 b562 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 b562 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 CXnCH 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.

Original languageEnglish
Pages (from-to)177-184
Number of pages8
JournalBiochemical Journal
Volume419
Issue number1
DOIs
Publication statusPublished - 1 Apr 2009
Externally publishedYes

Fingerprint

Cytochrome c Group
Substrate Specificity
Cytochromes c
Heme
Escherichia coli
Substrates
Sulfides
Cytochromes
Proteins
Periplasm
Escherichia coli Proteins
Post Translational Protein Processing
Sulfur
Sulfhydryl Compounds
Disulfides
Cysteine
Iron
Genes
Genome
Oxygen

Cite this

Allen, James W.A. ; Sawyer, Elizabeth B. ; Ginger, Michael L. ; Barker, Paul D. ; Ferguson, Stuart J. / Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus. In: Biochemical Journal. 2009 ; Vol. 419, No. 1. pp. 177-184.
@article{7eda7bb34cf9459caadfa41f41de5ae8,
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 b562 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 b562 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 CXnCH 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.",
keywords = "Biogenesis, C-type cytochrome, Cytochrome b, Cytochrome c maturation, Haem-binding motif, Post-translational modification",
author = "Allen, {James W.A.} and Sawyer, {Elizabeth B.} and Ginger, {Michael L.} and Barker, {Paul D.} and Ferguson, {Stuart J.}",
year = "2009",
month = "4",
day = "1",
doi = "10.1042/BJ20081999",
language = "English",
volume = "419",
pages = "177--184",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus. / Allen, James W.A.; Sawyer, Elizabeth B.; Ginger, Michael L.; Barker, Paul D.; Ferguson, Stuart J.

In: Biochemical Journal, Vol. 419, No. 1, 01.04.2009, p. 177-184.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus

AU - Allen, James W.A.

AU - Sawyer, Elizabeth B.

AU - Ginger, Michael L.

AU - Barker, Paul D.

AU - Ferguson, Stuart J.

PY - 2009/4/1

Y1 - 2009/4/1

N2 - 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 b562 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 b562 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 CXnCH 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.

AB - 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 b562 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 b562 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 CXnCH 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.

KW - Biogenesis

KW - C-type cytochrome

KW - Cytochrome b

KW - Cytochrome c maturation

KW - Haem-binding motif

KW - Post-translational modification

UR - http://www.scopus.com/inward/record.url?scp=62749109417&partnerID=8YFLogxK

U2 - 10.1042/BJ20081999

DO - 10.1042/BJ20081999

M3 - Article

VL - 419

SP - 177

EP - 184

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -