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J Bacteriol. 2004 June; 186(12): 4030–4033.
PMCID: PMC419958

The Acidic Nature of the CcmG Redox-Active Center Is Important for Cytochrome c Maturation in Escherichia coli


Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABCDEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.

Type c cytochromes are heme-binding proteins that function as electron transfer proteins in photosynthetic and/or respiratory chains. Unlike other classes of cytochromes, c-type cytochromes bind heme covalently (28). This binding requires the formation of two thioether bonds between the vinyl groups of heme and two cysteines in the conserved Cys-X-X-Cys-His motif of the apocytochrome. Three different posttranslational systems have evolved to facilitate cytochrome c formation in vivo (15). The most complicated of these systems, system I, is found in many gram-negative bacteria. For example, at least eight genes (ccmABCDEFGH) are essential for cytochrome c maturation in Escherichia coli (30). This is in stark contrast to the system of vertebrates and invertebrate mitochondria (system III), in which a single enzyme (cytochrome c heme lyase) facilitates the process (27). For system II, found in gram-positive bacteria, cyanobacteria, and chloroplasts, four accessory proteins have so far been identified (16).

One of the critical steps in the biogenesis of cytochrome c in E. coli is the ligation of heme to apocytochrome c. Because ligation occurs in the oxidizing environment of the periplasm, the heme-binding cysteines are likely to be oxidized by disulfide oxidases in this compartment. This idea is supported by the finding that E. coli strains deficient in the periplasmic protein oxidants DsbA and DsbB do not synthesize cytochrome c (22). Furthermore, recent evidence has shown that the Cys-X-X-Cys-His motif in apocytochrome c is capable of forming a disulfide bond and that this disulfide prevents the formation of mature cytochrome c in vitro (5). However, DsbA may not be involved directly in the maturation of cytochromes c (1, 6).

Several findings point to CcmG facilitating the reduction of apocytochrome c in vivo before heme attachment. Probably the most convincing evidence is that CcmG homologues contain a conserved Cys-X-X-Cys motif that is redox active (10) and that is required for its role in cytochrome c maturation in vivo (8). The Cys-X-X-Cys motif is characteristic of thioredoxin-like (TRX-like) proteins, of which thioredoxin (TRX) is the archetype. TRX maintains a reducing environment in the cytoplasm by reverting disulfide bonds to dithiols in cytoplasmic proteins (12). However, the redox activity of CcmG is different from that of TRX and TRX-like proteins in that it is highly specific and limited to cytochrome c maturation (10, 23). Furthermore, CcmG, unlike other TRX-like proteins, is not a catalyst of the insulin reduction assay (10). In addition, CcmG is reduced by the transmembrane electron transfer protein DsbD, supporting the notion that it plays a reducing role in cytochrome c maturation (14).

The crystal structure of a CcmG homologue from Bradyrhizobium japonicum was recently determined (7) and revealed the presence of a core TRX fold with several distinguishing features. We have previously shown that one of these features, an insert in the TRX fold, is required for CcmG function (7). Another distinguishing feature is the acidic nature of the CcmG redox-active center compared with those of other TRX-like proteins (7). Three conserved acidic residues (Asp97, Glu98, and Glu158 in B. japonicum CcmG) contribute to the negative charge. Here, we investigated the role of these acidic residues and other conserved features of CcmG in cytochrome c maturation by testing the ability of E. coli CcmG mutants to complement cytochrome c maturation in a ΔccmG E. coli strain.

Asp107 and Asp129 in the central insert are not required for cytochrome c maturation.

Asp107 and Asp129 of E. coli CcmG (Fig. (Fig.1)1) are surface exposed and highly conserved in CcmG homologues. Furthermore, both are part of the central insert in CcmG, which is required for cytochrome c maturation (7). To investigate a possible role for Asp107 and Asp129, each was mutated to alanine and the resulting ccmG mutant was used to transform a ΔccmG E. coli strain containing a plasmid directing apocytochrome c to the periplasm (25). Cells expressing CcmGAsp107Ala or CcmGAsp129Ala were grown under anaerobic conditions to induce the expression of chromosomal cytochrome c maturation genes (ccmABCDEFH). Periplasmic proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and holocytochrome c was detected by heme staining of the electrophoresed proteins (26). Surprisingly, despite their prime positions and conservation in CcmG homologues, neither Asp107 nor Asp129 was required for CcmG function. Rather, both CcmGAsp107Ala (Fig. (Fig.2A,2A, lane 1) and CcmGAsp129Ala (Fig. (Fig.2A,2A, lane 2) were able to complement cytochrome c maturation to wild-type levels in a ΔccmG E. coli strain.

FIG. 1.
CcmG structure and sequence. (a) Sequence alignment of E. coli and B. japonicum CcmGs. Residues mutated in E. coli CcmG are marked by asterisks. The residues deleted to create CcmGΔLeu24-Thr66 are italicized, and the residues deleted to create ...
FIG. 2.
Characterization of CcmG mutants. (A) Heme stain of cytochromes produced by CcmG single mutants (30 μg of periplasmic protein). Lane 1, CcmGAsp107Ala; lane 2, CcmGAsp129Ala; lane 3, CcmGGlu86Ala; lane 4, CcmGGlu145Ala; lane 5, CcmGAsp162Ala; lane ...

The acidic nature of the redox-active center plays a role in cytochrome c maturation.

The redox-active center of CcmG is relatively acidic (7) compared to other TRX-like oxidoreductases, including TRX (13), DsbA (20), DsbC (21), and the closest structural relative to CcmG, TlpA (2). Three acidic residues in CcmG, of which two are exposed to solvent (Asp97 and Glu158 in B. japonicum CcmG) and one is partly buried (Glu98 in B. japonicum CcmG), contribute to the acidic redox-active center. Glu98 and Glu158 are conserved in all CcmG homologues identified to date. Asp97 is conserved in three CcmG homologues (B. japonicum, Rhizobium leguminosarum, and Haemophilus influenzae homologue 2), and a nearby residue is often acidic in three other CcmG homologues (Asp162 in E. coli, Asp157 in Pseudomonas fluorescens, and Asp160 in H. influenzae homologue 1). These three acidic residues are all within 5 to 8 Å of the redox-active center (Fig. (Fig.1b).1b). The finding that these three residues are conserved in CcmG but not in other TRX-like proteins suggests that the acidic nature of the CcmG redox-active center may be important for cytochrome c maturation. A functional role for these three residues in E. coli CcmG (Glu86, Glu145, and Asp162) (Fig. (Fig.1)1) was investigated by testing the ability of the respective single CcmG mutants (Glu86Ala, Glu145Ala, and Asp162Ala) to complement cytochrome c maturation in a ΔccmG E. coli strain. However, all three single mutants complemented cytochrome c to wild-type levels in the ΔccmG E. coli strain (Fig. (Fig.2A,2A, lanes 3 to 5).

To further investigate a functional role for the three acidic residues near the redox-active center of CcmG, a CcmG double mutant (CcmGGlu86Ala/Glu145Ala) and a CcmG triple mutant (CcmGGlu86Ala/Glu145Ala/Asp162Ala) were made and characterized. Two of the three acidic residues involved are conserved in all CcmG homologues identified to date. These two residues were simultaneously mutated to alanines to create a CcmG double mutant. Cells expressing CcmGGlu86Ala/Glu145Ala produced lower levels of holocytochrome c (Fig. (Fig.2B,2B, lane 1) than cells expressing wild-type CcmG (Fig. (Fig.2B,2B, lane 3). This result supports the proposal that the acidic nature of the redox-active center in CcmG is required for the specific function of CcmG in cytochrome c maturation. A triple mutant was also constructed by mutating to alanine each of the three acidic residues near the redox-active center in E. coli CcmG. Cells expressing the triple mutant (CcmGGlu86Ala/Glu145Ala/Asp162Ala) (Fig. (Fig.2B,2B, lane 2) produced lower levels of cytochrome c than cells expressing wild-type CcmG (Fig. (Fig.2B,2B, lane 3).

Quantification of c-type cytochromes produced from cells carrying wild-type CcmG or the double-mutant or triple-mutant version was performed by absorption difference spectroscopy of periplasmic fractions (Fig. (Fig.3).3). The double mutant and the triple mutant produced 44 and 39%, respectively, of the type c cytochromes produced by the wild type (100%).

FIG. 3.
Absorption difference spectra of c-type cytochromes. A ΔccmG mutant expressing apocytochrome c was complemented with a plasmid expressing either wild-type CcmG (A), the double mutant CcmGGlu86Ala/Glu145Ala (B), or the triple mutant CcmGGlu86Ala/Glu145Ala/Asp162Ala ...

The N-terminal β-hairpin-like structure is associated with CcmG stability.

The structure of CcmG includes an addition of ~30 residues to the TRX fold at the N terminus (7) that forms a β-hairpin like structure. The role of this addition to the TRX fold is unknown. In order to investigate a possible functional role, a ccmG deletion mutant was designed to link the membrane anchor in E. coli CcmG (Met1 to Trp22) (10) directly to the first strand of the TRX fold in CcmG by removing residues 24 to 66 (CcmGΔLeu24-Thr66) (Fig. (Fig.1).1). This variant was unable to complement cytochrome c maturation in the ΔccmG E. coli strain (Fig. (Fig.2C,2C, upper panel, lane 1). The stability of CcmGΔLeu24-Thr66 was investigated by Western blotting of total cell protein (with an anti-His tag antibody), which failed to detect protein (Fig. (Fig.2C,2C, lower panel, lane 1), suggesting that the β-hairpin may play a role in stabilizing the protein. A second deletion that removed a shorter section of the N terminal region, leaving additional residues to connect the membrane anchor with the TRX fold of CcmG, was constructed. This variant of (CcmGΔAsp31-Gln67) included seven more residues after the membrane anchor than the first deletion (CcmGΔLeu24-Thr66) (Fig. (Fig.1).1). However CcmGΔAsp31-Gln67, like CcmGΔLeu24-Thr66, did not complement cytochrome c maturation in ΔccmG E. coli (Fig. (Fig.2C,2C, upper panel, lane 2) and also did not produce a stable product (Fig. (Fig.2C,2C, lower panel, lane 2). Taken together, these results suggest that the β-hairpin-like structure at the N terminus of CcmG is required for stability. Perhaps this region is important for interacting with other Ccm proteins, an idea that is consistent with the proposal that Ccm proteins may associate at the membrane, forming a cytochrome c maturation complex (29).


ΔccmG E. coli strains complemented with either CcmGGlu86Ala/Glu145Ala or CcmGGlu86Ala/Glu145Ala/Asp162Ala produced similar levels of cytochrome c. This result suggests that Asp162 is not as important for function as Glu86 or Glu145. This idea is consistent with the fact that Asp162 is not as highly conserved as Glu86 or Glu145. An alternative interpretation of these results is that the mutation of any two of the three acidic residues near the redox-active center is sufficient to create a mutant phenotype.

The double mutant (CcmGGlu86Ala/Glu145Ala) produced lower levels of cytochrome c than wild-type CcmG did, indicating that the acidic residues are involved in CcmG function in c-type cytochrome maturation. Glu86 (Glu98 in B. japonicum CcmG) is located in a position in the TRX fold similar to that of Asp26 of E. coli TRX (13). Asp26 in TRX has been implicated in deprotonating the second cysteine in the Cys-X-X-Cys motif via a nearby water molecule (4, 17). Interestingly, B. japonicum TlpA, a periplasmic TRX-like protein that is required for the maturation of aa3-type cytochromes (2, 19), contains an acidic residue (Glu78) that aligns with the equivalent residue in CcmG. Therefore, Glu86 in CcmG and Glu78 in TlpA are in a prime position to fulfill a role in catalysis similar to that of Asp26 in TRX.

The other highly conserved acidic residue, Glu145 (Glu158 in B. japonicum CcmG), follows the cis-Pro residue in the fingerprint motif of CcmGs (residues 139-Gly-Val-X-Gly-Ala/Val-cis-Pro-Glu-145). A cis-Pro in this position is conserved in TRX-like oxidoreductases and exposes the main chain oxygen of the preceding residue for interaction with other residues. The structurally equivalent regions in TRX (24) and DsbA (3) have been implicated in substrate binding. Based on these findings, Glu145 may also be involved in binding CcmG substrates. Possible substrates may be the electron donor DsbD or the electron acceptor apocytochrome c, though the latter interaction could be mediated by another redox-active Ccm protein, CcmH (9). The equivalent residue in TRX and TlpA is not acidic, suggesting that these proteins interact with different substrates.

The pKa of the N-terminal cysteine at the redox-active center is used as a means of comparing biochemical and redox activities of TRX-like redox proteins. The pKa of the thiol for E. coli CcmG (DsbE) is reported to be 6.8 (18), and that of the thiol for B. japonicum CcmG is expected to be similar, since the two proteins have similar reductant functions. By comparison, the pKa of 5.0 is much lower for Mycobacterium tuberculosis DsbE (11). Although structurally very similar to CcmG, the oxidant properties of M. tuberculosis DsbE indicate that it is not involved in cytochrome c biogenesis (11). Our results support this notion, since M. tuberculosis DsbE lacks two of the three conserved acidic residues identified here as important for cytochrome c biogenesis. Furthermore, the fingerprint region indicative of CcmG function is not conserved in M. tuberculosis DsbE (Asn-Val-X-Trp-Gln-cis-Pro-Ala) (11). In this context, not only is the acidic residue Glu145 replaced by Ala but the highly conserved hydrophobic residue that precedes cis-Pro in almost all TRX-like proteins is hydrophilic in M. tuberculosis DsbE. Hydrophilic residues at this position are also found in disulfide isomerases such as DsbC and DsbG, suggesting the possibility that M. tuberculosis DsbE may have isomerizing activity.


This research was supported by grants from the Australian Research Council (J.L.M.), the Swiss National Foundation (L.T.-M.), and the ETH (L.T.-M.). J.L.M. is an ARC Senior Research Fellow. M.A.E. is the recipient of a University of Queensland Postgraduate Research Scholarship and Graduate School Research Travel Award.


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