The Escherichia coli CcmG protein fulfils a specific role in cytochrome c assembly.
In Escherichia coli K-12, c-type cytochromes are synthesized only during anaerobic growth with trimethylamine-N-oxide, nitrite or low concentrations of nitrate as the terminal electron acceptor. A thioredoxin-like protein, CcmG, is one of 12 proteins required for their assembly in the periplasm. Its postulated function is to reduce disulphide bonds formed between correctly paired cysteine residues in the cytochrome c apoproteins prior to haem attachment by CcmF and CcmH. We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming proteins, DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic acid. An anti-CcmG polyclonal antibody was used in Western-blot analysis to probe the redox state of CcmG in mutants defective in the synthesis of other proteins essential for cytochrome c assembly. The oxidized form of CcmG accumulated not only in trxA or dipZ mutants defective in the transfer of electrons from the cytoplasm for disulphide isomerization and reduction reactions in the periplasm, but also in ccmF and ccmH mutants. The requirement of both CcmF and CcmH for the reduction of the disulphide bond in CcmG indicates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer from the membrane-associated DipZ (also known as DsbD) to CcmH. The data support a model proposed by others in which CcmG catalyses one of the last reactions specific to cytochrome c assembly.
Reid
E
E
School of Biosciences, University of Birmingham, Birmingham B15 2TT, U.K.
Cole
J
J
Eaves
D J
DJ
D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003574:Cytochrome c Group / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000276:immunology / Q000502:physiology*; D033903:Periplasmic Proteins; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D003574:Cytochrome c Group; D017931:DNA Primers; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C077913:periplasmic protein disulfide oxidoreductase