Journal of Bacteriology, June 2004, p . 4030-4033, Vol . 186, No . 12

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

Melissa A . Edeling,^1, Umesh Ahuja,² Begoña Heras,¹ Linda Thöny-Meyer,² and Jennifer L . Martin^1*

Institute for Molecular Bioscience and Special Research Centre for Functional and Applied Genomics, The University of Queensland, Brisbane, Queensland 4072, Australia,¹ Institute for Microbiology, ETH Zürich, CH-8092 Zürich, Switzerland²

Received 20 November 2003/ Accepted 19 February 2004

 
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 ccmG 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 . 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 ccmG E . coli strain containing a plasmid directing apocytochrome c to the periplasm (25) . Cells expressing CcmG_Asp107Ala or CcmG_Asp129Ala 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 CcmG_Asp107Ala (Fig . 2A, lane 1) and CcmG_Asp129Ala (Fig . 2A, lane 2) were able to complement cytochrome c maturation to wild-type levels in a ccmG E . coli strain .

 
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 . 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 . 1) was investigated by testing the ability of the respective single CcmG mutants (Glu86Ala, Glu145Ala, and Asp162Ala) to complement cytochrome c maturation in a ccmG E . coli strain . However, all three single mutants complemented cytochrome c to wild-type levels in the ccmG E . coli strain (Fig . 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 (CcmG_{Glu86Ala/Glu145Ala}) and a CcmG triple mutant (CcmG_{Glu86Ala/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 CcmG_{Glu86Ala/Glu145Ala} produced lower levels of holocytochrome c (Fig . 2B, lane 1) than cells expressing wild-type CcmG (Fig . 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 (CcmG_{Glu86Ala/Glu145Ala/Asp162Ala}) (Fig . 2B, lane 2) produced lower levels of cytochrome c than cells expressing wild-type CcmG (Fig. 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 . 3) . The double mutant and the triple mutant produced 44 and 39%, respectively, of the type c cytochromes produced by the wild type (100%) .

 
The N-terminal ß-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 ß-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 (CcmG_Leu24-Thr66) (Fig . 1) . This variant was unable to complement cytochrome c maturation in the ccmG E . coli strain (Fig . 2C, upper panel, lane 1) . The stability of CcmG_Leu24-Thr66 was investigated by Western blotting of total cell protein (with an anti-His tag antibody), which failed to detect protein (Fig. 2C, lower panel, lane 1), suggesting that the ß-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 (CcmG_Asp31-Gln67) included seven more residues after the membrane anchor than the first deletion (CcmG_Leu24-Thr66) (Fig . 1) . However CcmG_Asp31-Gln67, like CcmG_Leu24-Thr66, did not complement cytochrome c maturation in ccmG E . coli (Fig . 2C, upper panel, lane 2) and also did not produce a stable product (Fig . 2C, lower panel, lane 2) . Taken together, these results suggest that the ß-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) .

Conclusions. ccmG E . coli strains complemented with either CcmG_{Glu86Ala/Glu145Ala} or CcmG_{Glu86Ala/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 (CcmG_{Glu86Ala/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 pK_a 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 pK_a 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 pK_a 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 .

 
* Corresponding author . Mailing address: Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia . Phone: 61 7 3346 2016 . Fax: 61 7 3346 2101 . E-mail: j.martin@imb.uq.edu.au.

Present address: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge CB2 2XY, United Kingdom .

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