THE CCME PROTEIN FROM ESCHERICHIA-COLI IS A HEME-BINDING PROTEIN

We previously reported that a 17.5-kDa haem-binding polypeptide accumu lates in Escherichia coli K-12 mutants defective in an essential gene for cytochrome c assembly, ccmF, and speculated that this polypeptide is either CcmE or CcmG. The haem-containing polypeptide, which is asso ciated with the cytoplasmic membrane, has now been identified by N-ter minal sequencing to be CcmE. The haem-dependent peroxidase activity of CcmE is clearly visible not only in a ccmF mutant, but also in ccmG a nd ccmH mutants, implying that CcmE functions either before or in the same step as CcmF, CcmG and CcmH in cytochrome c maturation. A trxA mu tant, like the dipZ mutant, was unable to assemble c-type cytochromes or catalyse formate-dependent nitrite reduction: both activities were restored in the trxA and dipZ, but not ccmG, mutants by the reducing a gent, 2-mercaptoethanesulphonic acid. Our data suggest that haem trans ferred across the cytoplasmic membrane by the CcmABCD complex becomes associated with CcmE, possibly by a labile covalent bond, before it is transferred to the cytochrome c apoproteins by the periplasmic haem l yase encoded by ccmF and ccmH. We further propose that CcmG is essenti al to reduce the disulphide bonds formed in cytochrome c apoproteins b y DsbA, before haem is attached by the haem lyase. Electrons for disul phide bond reduction are supplied from thioredoxin in the cytoplasm vi a DipZ in the membrane, but can be replaced by the chemical reductant, 2-mercaptoethanesulphonic acid. According to this model, CcmG is the last protein in the reducing pathway which interacts stereospecificall y with the apoprotein. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.