The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

Abstract

A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels ofBradyrhizobium japonicum holocytochromec 550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.

Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

“…CcmG is a 20-kDa protein with an N-terminal membrane anchor and faces the periplasm with its hydrophilic C-terminal domain containing the active site ( Fig. 2B) [89]. Around the C-X-X-C motif several additional residues are conserved between CcmG and the reductases Trx1 and DsbD, suggesting that CcmG also assumes the thioredoxin fold ( Fig.…”

“…In E. coli, it has been shown that ccmG mutants having one or both cysteine residues of the active site replaced by serine produce strongly decreased levels of holocytochrome c. Furthermore, cytochrome c maturation could be restored when CcmG active-site mutants were grown in the presence of reducing compounds such as cysteine or 2-mercaptoethanesulfonic acid. Remarkably, a ccmG in-frame deletion mutant was irreversibly defective [89,94,98]. In R. capsulatus, the active-site mutants were tested under conditions of photosynthetic growth, for which c-type cytochromes are absolutely essential, and shown to be lethal under these conditions [92].…”

“…In R. capsulatus, the active-site mutants were tested under conditions of photosynthetic growth, for which c-type cytochromes are absolutely essential, and shown to be lethal under these conditions [92]. It was concluded that CcmG is especially required for the reducing pathway of cytochrome c maturation [89].…”

“…Cytochrome c maturation has been shown to require various thiol:disul¢de oxidoreductases [41,61,72,88,89,122]. E. coli is a facultative anaerobe that does not naturally synthesise c-type cytochromes during aerobic growth.…”

“…When the apoprotein is translocated to the periplasm, it is most likely to be in the reduced state, and haem addition should in principle be possible. The fact that extra reductants such as CcmG and CcmH are required for cytochrome c maturation [89,90] raises the question of whether this requirement is due to a rapid, non-speci¢c oxidation of the cysteines by DsbA. Does the CcmG/CcmH system re-reduce oxidised apocytochrome c, or does it prevent oxidation of newly translocated apocytochrome by DsbA ?…”