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 an aerobic growth with trimethylamine-N-oxide, nitrite or low concentrations o f nitrate as the terminal electron acceptor. A thioredoxin-like protein, Cc mG, 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 att achment by CcmF and CcmH. We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming protei ns. DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic a cid. 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 othe r proteins essential for cytochrome c assembly. The oxidized form of CcmG a ccumulated not only in trxA or dipZ mutants defective in the transfer of el ectrons from the cytoplasm for disulphide isomerization and reduction react ions in the periplasm. but also in ccmF and ccmH mutants. The requirement o f both CcmF and CcmH for the reduction of the disulphide bond in CcmG indic ates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer From the membrane-associated DipZ (also k nown 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 .