Abstract
Biogenesis of c-type cytochromes in α- and γ-proteobacteria requires the function of a set of orthologous genes (ccm genes) that encode specific maturation factors. The Escherichia coli CcmE protein is a periplasmic heme chaperone. The membrane protein CcmC is required for loading CcmE with heme. By expressing CcmE (CycJ) from Bradyrhizobium japonicum in E. coli we demonstrated that heme is bound covalently to this protein at a strictly conserved histidine residue. The B. japonicum homologue can transfer heme to apocytochrome c in E. coli, suggesting that it functions as a heme chaperone. CcmC (CycZ) from B. japonicum expressed in E. coli was capable of inserting heme into CcmE.
In c-type cytochromes, the heme cofactor is attached covalently via two thioether bonds to a conserved CXXCH sequence motif within the apoprotein. Bacterial c-type cytochromes are localized on the periplasmic side of the cytoplasmic membrane. In Bradyrhizobium japonicum, the root nodule endosymbiont of the soybean plant, the c-type cytochromes of the bc1 complex and of the cbb3-type oxidase are essential for symbiotic nitrogen fixation (8, 9, 20).
In members of the α and γ subclasses of proteobacteria, a set of genes has been identified that is essential for cytochrome c maturation (4, 6, 17, 22). In Escherichia coli, a member of the α subclass, these genes are ccmA to -H (3, 18), whereas in B. japonicum, a member of the γ subclass, these genes have been named cyc, emphasizing their involvement in the formation of cytochrome c (10, 12, 13, 17).
In E. coli, CcmE was found to be an intermediate in the heme delivery pathway specific for c-type cytochromes. It binds heme transiently in the periplasm at a single histidine residue and transfers it to apocytochrome c, thereby acting as a periplasmic heme chaperone (15). In addition, it was found that CcmC is essential and sufficient to insert heme into CcmE (14). The biochemical characterization of the cytochrome c maturation factors in B. japonicum is difficult, due to the lack of nonpolar ccm mutants, the low abundance of these proteins, and the lack of a system for overexpression. For example, a heme binding form of B. japonicum CcmE (CycJ) (13) is not detectable in wild-type cells. So far, only the function of the B. japonicum CcmG (CycY) protein as a periplasmic thiol-disulfide oxidoreductase has been elucidated after overproduction and purification of the protein in E. coli (2). Here, we report on the characterization of the B. japonicum CcmE and CcmC proteins in the cytochrome c maturation pathway in a situation where they replace the corresponding E. coli orthologues.
We analyzed the ability of B. japonicum CcmE to bind heme when it was expressed in E. coli. The B. japonicum ccmE gene (ccmEBj) was amplified by PCR and cloned into the arabinose-inducible expression vector pISC-2 (19), resulting in pRJ3294 (Table 1). The E. coli CcmE protein (CcmEEc) is able to bind heme in the presence of CcmABCD (15). The E. coli Δccm mutant EC06, expressing the E. coli ccmABCD genes from plasmid pEC101 (Table 1), was cotransformed with plasmids expressing different ccmE derivatives (Table 1). Membrane proteins were isolated and stained for covalently bound heme. Both CcmEEc and CcmEBj were able to bind heme (Fig. 1A, lanes 2 and 3, respectively). They both sometimes migrate as doublets and form dimers that are not resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The reason for this behavior is not known. When the conserved H122 in CcmEBj was changed to alanine, a heme-staining band was not detected (Fig. 1A, lane 4). To analyze whether the mutant CcmEBj (H122A) forms a stable polypeptide in the membrane, a hexahistidine tag was fused to the N termini of the CcmE proteins. These fusion proteins could be detected in membrane protein fractions using a monoclonal antipentahistidine antibody (Qiagen, Hilden, Germany) (Fig. 1B, lanes 5 to 7). The hexahistidine tag did not interfere with the activity of the proteins, as both His-tagged CcmEEc and CcmEBj bound heme (Fig. 1A, lanes 5 and 6). Thus, mutation H122A in CcmEBj led to a stable membrane-bound protein that was no longer able to bind heme. This finding implies that, like H130 in CcmEEc, H122 is the heme binding residue in CcmEBj. In fact, the histidine lies within the segment LAKHDE, which is absolutely conserved in all CcmE sequences known so far.
TABLE 1.
Strains and plasmids used in this work
Strain or plasmid | Relevant characteristics | Reference |
---|---|---|
Strains | ||
E. coli | ||
MC1061 | hsdR mcrB araD139 Δ(araABC-leu)7679 ΔlacX74 galU galK rpsL thi | 5 |
EC06 | ΔccmA-H derivative of MC1061; Kmr | 18 |
EC28 | ΔccmC derivative of MC1061 | 21 |
EC65 | ΔccmE derivative of MC1061 | 15 |
B. japonicum | ||
Bj110spc4 | Spcr (referred to as wild type) | 11 |
Bj2746 | Spcr Kmr ΔcycY::aph | 2 |
Plasmids | ||
pEC101 | ccmABCDEc cloned into pACYC184; Cmr | 15 |
pEC410 | ccmEEc cloned into pACYC184; Cmr | 14 |
pEC411 | H6-ccmEEc cloned into pISC-3; Apr | 15 |
pEC412 | ccmEEc cloned into pISC-2; Apr | 15 |
pEC432 | ccmCEc cloned into pISC-2; Apr | This work |
pRJ3291 | cycABj cloned into pISC-2; Kmr | 14 |
pRJ3292 | ccmCBj cloned into pISC-2; Apr | This work |
pRJ3294 | ccmEBj cloned into pISC-2; Apr | This work |
pRJ3295 | H6-ccmEBj cloned into pISC-3; Apr | This work |
pRJ3297 | ccmEBj (H122A) cloned into pISC-2; Apr | This work |
pRJ3298 | H6-ccmEBj (H122A) cloned into pISC-3; Apr | This work |
pRJ3454 | cycHJBj cloned into pRKPol2; Tcr | 12 |
FIG. 1.
Ability of CcmEBj to bind heme and to transfer it to apocytochrome c. (A) The Δccm mutant expressing ccmABCD constitutively from plasmid pEC101 was cotransformed with plasmids expressing different ccmE derivatives. Membrane proteins (50 μg) were separated by SDS–15% PAGE and stained for covalently bound heme. (B) Western blot of samples (20 μg) identical to those shown in panel A probed with anti-penta-His immunoglobulins. CcmE can form dimers that are detectable in both heme stain and Western blot analysis, as shown previously (15). (C) The ΔccmE mutant was cotransformed with plasmids expressing B. japonicum or E. coli ccmE derivatives and with pRJ3291, encoding cytochrome c550 (Cyt c550). Cells were grown anaerobically in the presence of nitrite. Trichloroacetic acid-precipitated periplasmic proteins (100 μg) (16) were stained for covalently bound heme. Plasmids carrying the respective genes were as follows: pISC-2 (lane 1), pEC412 (lane 2), pRJ3294 (lane 3), pRJ3295 (lane 4), pEC411 (lane 5), pRJ3297 (lane 6), and pRJ3298 (lane 7). Molecular masses of marker proteins (in kilodaltons) are indicated on the left.
Next, we analyzed whether CcmEBj is able to transfer heme to E. coli and to B. japonicum c-type cytochromes. The periplasmic B. japonicum cytochrome c550, encoded by cycA, can be expressed in E. coli from plasmid pRJ3291 with the addition of arabinose (14). E. coli strain EC65, containing an in-frame deletion in ccmE (ΔccmE), was transformed with pRJ3291 and with plasmids expressing either E. coli or B. japonicum ccmE (cycJ). The cells were grown anaerobically in the presence of nitrite as an electron acceptor to ensure expression of the ccm operon and the structural genes napBC, which encode the c-type cytochromes of the periplasmic nitrate reductase (7). After induction of cycA expression, holocytochrome c formation was analyzed by heme staining of periplasmic proteins. CcmEBj and CcmEEc were able to complement the ΔccmE phenotype and synthesized both overproduced cytochrome c550 and NapB (Fig. 1C, lanes 2 and 3). However, the level of native cytochrome c was reduced when CcmEBj was used. The CcmEBj H122A mutant was unable to complement a ΔccmE mutant (Fig. 1C, lane 4). The hexahistidine tag did not interfere with the activity of the proteins (Fig. 1C, lanes 5, 6, and 7). A mutation in the conserved histidine of the CcmE orthologue CycJ from Rhodobacter capsulatus has also been shown to block cytochrome c maturation (1). We propose that it is a general characteristic of CcmE to bind heme at the conserved histidine residue and subsequently to transfer it to apocytochrome c.
To analyze whether CcmEBj is also a heme binding protein in B. japonicum, plasmid pRJ3454 expressing cycH (ccmE) from the cycH promoter (12) was conjugated into a B. japonicum wild-type strain and into the ΔcycY (ccmG) mutant Bj2746. In Bj2746 no c-type cytochromes are formed (2), which let us assume that the amount of holo-CcmE in the membranes of this strain could be increased due to inhibition of heme release from CcmE. B. japonicum cells were grown aerobically, and membrane proteins were analyzed for covalently bound heme. In membrane fractions of wild-type B. japonicum containing pRJ3454, two c-type cytochromes with apparent molecular masses of 28 kDa (cytochrome c1) and 20 kDa (CycM) were present (data not shown). However, no holo-CcmE could be detected, either in the wild type or in the mutant strain Bj2746 containing pRJ3454 (data not shown). Expression of ccmEBj from its natural promoter on the low-copy-number plasmid pRJ3454 was apparently not sufficient for the detection of holo-CcmE. In E. coli, holo-CcmE can be seen only when ccmE is overexpressed (15). In a study with the CcmE orthologue, CycJ, of R. capsulatus the heme binding form also could not be obtained (1), again most likely because it accumulates to detectable levels only after a significant overproduction.
In E. coli, expression of ccmC is necessary and sufficient to incorporate heme covalently into CcmE. CcmC (CycZ) is an integral membrane protein. A strictly conserved tryptophan-rich motif and flanking histidines are involved in its function, perhaps by interaction with heme (16). We tested whether the B. japonicum homologue CcmCBj has a similar function when expressed in E. coli. ccmCBj was amplified by PCR and cloned into the expression vector pISC-2 (Table 1). The E. coli Δccm mutant EC06, expressing E. coli ccmE from plasmid pEC410, was cotransformed with plasmids expressing ccmC of either B. japonicum or E. coli. Membrane proteins were isolated and stained for covalently bound heme. Both CcmCBj and CcmCEc were able to insert heme into CcmEEc (Fig. 2A, lanes 2 and 3). A Western blot probed with anti-CcmEEc serum revealed similar amounts of CcmEEc polypeptide in all membrane protein fractions (Fig. 2B). Yet CcmCBj was significantly less active in holo-CcmE formation than CcmCEc (Fig. 2A, compare lanes 2 and 3).
FIG. 2.
Ability of ccmCBj to attach heme to E. coli CcmE and to complement an E. coli ΔccmC mutant for formation of holo-cytochrome c. (A) The Δccm mutant EC06 was cotransformed with plasmids expressing B. japonicum (Bj) and E. coli (Ec) ccmC and with a plasmid expressing ccmEEc. Cells were grown aerobically in Luria-Bertani medium, and protein expression was induced with 0.1% arabinose. Membrane protein fractions (100 μg) were separated by SDS–15% PAGE and stained for covalently bound heme. (B) Western blot of membrane fractions (20 μg) identical to those shown in panel A probed with anti-CcmE serum. (C) The ΔccmC mutant was cotransformed with plasmids expressing B. japonicum or E. coli ccmC, with a plasmid expressing ccmEEc, and with pRJ3291, encoding cytochrome c550 (Cyt c550). Cells were grown anaerobically in the presence of nitrite, and protein expression was induced with 0.1% arabinose. Trichloroacetic acid-precipitated periplasmic proteins (100 μg) were stained for covalently bound heme. Plasmids carrying the respective genes were as follows: pISC-2 (lane 1), pRJ3292 (lane 2), and pEC432 (lane 3).
E. coli strain EC28, containing an in-frame deletion in ccmC (ΔccmC), was transformed with pRJ3291, pEC410, and plasmids expressing ccmC of either B. japonicum or E. coli. The cells were grown anaerobically in the presence of nitrite as an electron acceptor. Holo-cytochrome c formation was analyzed by heme staining of periplasmic proteins. CcmCBj was able to complement a ΔccmC mutant to synthesize holo-cytochrome c (Fig. 2C, lane 2). However, much smaller amounts of cytochrome c were produced than with CcmCEc (Fig. 2C, lane 3). CcmCBj shows an activity similar to, but strongly reduced from, that of CcmCEc, being necessary and sufficient for incorporation of heme into the heme chaperone CcmE and, as a consequence, for holo-cytochrome c formation.
In conclusion, we state that both ccmC and ccmE of B. japonicum can complement the respective E. coli mutants, albeit not at wild-type levels. It is therefore likely that in B. japonicum and also in other organisms CcmC incorporates heme covalently into CcmE, which acts as a periplasmic heme chaperone: heme is bound at a conserved histidine residue of CcmE and is then transferred to apocytochrome c. Our current model of CcmC- and CcmE-mediated heme delivery during cytochrome c biogenesis involves protein-protein interactions between CcmC, CcmE, and the small membrane protein CcmD (16). Although the amino acid identities between the CcmE, CcmC, and CcmD proteins of E. coli and B. japonicum are only 45, 49, and 25%, respectively, the interaction of Ccm proteins derived from different species was apparently sufficient to support cytochrome c maturation.
Acknowledgments
We thank N. Spielewoy for fruitful discussions and John A. DeMoss for critical reading of the manuscript.
This work was supported by grants from the Swiss National Foundation for Scientific Research and from the ETH.
REFERENCES
- 1.Deshmukh M, Brasseur G, Daldal F. Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes. Mol Microbiol. 2000;35:123–138. doi: 10.1046/j.1365-2958.2000.01683.x. [DOI] [PubMed] [Google Scholar]
- 2.Fabianek R A, Huber-Wunderlich M, Glockshuber R, Künzler P, Hennecke H, Thöny-Meyer L. Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes. J Biol Chem. 1997;272:4467–4473. doi: 10.1074/jbc.272.7.4467. [DOI] [PubMed] [Google Scholar]
- 3.Grove J, Tanapongpipat S, Thomas G, Griffiths L, Crooke H, Cole J. Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. Mol Microbiol. 1996;19:467–481. doi: 10.1046/j.1365-2958.1996.383914.x. [DOI] [PubMed] [Google Scholar]
- 4.Kranz R, Lill R, Goldman B, Bonnard G, Merchant S. Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol Microbiol. 1998;29:383–396. doi: 10.1046/j.1365-2958.1998.00869.x. [DOI] [PubMed] [Google Scholar]
- 5.Meissner P S, Sisk W P, Bergman M L. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA. 1987;84:4171–4175. doi: 10.1073/pnas.84.12.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Page M D, Sambongi Y, Ferguson S J. Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria. Trends Biochem Sci. 1998;23:103–108. doi: 10.1016/s0968-0004(98)01173-6. [DOI] [PubMed] [Google Scholar]
- 7.Potter L C, Cole J A. Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12. Biochem J. 1999;344:69–76. doi: 10.1042/0264-6021:3440069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Preisig O, Anthamatten D, Hennecke H. Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen fixing endosymbiosis. Proc Natl Acad Sci USA. 1993;90:3309–3313. doi: 10.1073/pnas.90.8.3309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Preisig O, Zufferey R, Thöny-Meyer L, Appleby C A, Hennecke H. A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. J Bacteriol. 1996;178:1532–1538. doi: 10.1128/jb.178.6.1532-1538.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Ramseier T M, Winteler H V, Hennecke H. Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes. J Biol Chem. 1991;266:7793–7803. [PubMed] [Google Scholar]
- 11.Regensburger B, Hennecke H. RNA polymerase from Bradyrhizobium japonicum. Arch Microbiol. 1983;135:103–109. doi: 10.1007/BF00408017. [DOI] [PubMed] [Google Scholar]
- 12.Ritz D, Bott M, Hennecke H. Formation of several bacterial c-type cytochromes requires a novel membrane-anchored protein that faces the periplasm. Mol Microbiol. 1993;9:729–740. doi: 10.1111/j.1365-2958.1993.tb01733.x. [DOI] [PubMed] [Google Scholar]
- 13.Ritz D, Thöny-Meyer L, Hennecke H. The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum. Mol Gen Genet. 1995;247:27–38. doi: 10.1007/BF00425818. [DOI] [PubMed] [Google Scholar]
- 14.Schulz H, Fabianek R A, Pellicioli E C, Hennecke H, Thöny-Meyer L. Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB. Proc Natl Acad Sci USA. 1999;96:6462–6467. doi: 10.1073/pnas.96.11.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Schulz H, Hennecke H, Thöny-Meyer L. Prototype of a heme chaperone essential for cytochrome c maturation. Science. 1998;281:1197–1200. doi: 10.1126/science.281.5380.1197. [DOI] [PubMed] [Google Scholar]
- 16.Schulz H, Pellicioli E, Thöny-Meyer L. New insights into the role of CcmC, CcmD, and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC. Mol Microbiol. 2000;37:1379–1388. doi: 10.1046/j.1365-2958.2000.02083.x. [DOI] [PubMed] [Google Scholar]
- 17.Thöny-Meyer L. Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev. 1997;61:337–376. doi: 10.1128/mmbr.61.3.337-376.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Thöny-Meyer L, Fischer F, Künzler P, Ritz D, Hennecke H. Escherichia coli genes required for cytochrome c maturation. J Bacteriol. 1995;177:4321–4326. doi: 10.1128/jb.177.15.4321-4326.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Thöny-Meyer L, Künzler P, Hennecke H. Requirements for maturation of Bradyrhizobium japonicum cytochrome c550 in Escherichia coli. Eur J Biochem. 1996;235:754–761. doi: 10.1111/j.1432-1033.1996.00754.x. [DOI] [PubMed] [Google Scholar]
- 20.Thöny-Meyer L, Stax D, Hennecke H. An unusual gene cluster for the cytochrome bc1 complex in Bradyrhizobium japonicum and its requirement for effective root nodule symbiosis. Cell. 1989;57:683–697. doi: 10.1016/0092-8674(89)90137-2. [DOI] [PubMed] [Google Scholar]
- 21.Throne-Holst M, Thöny-Meyer L, Hederstedt L. Escherichia coli in-frame deletion mutants can produce periplasmic cytochrome b, but not cytochrome c. FEBS Lett. 1997;410:351–355. doi: 10.1016/s0014-5793(97)00656-x. [DOI] [PubMed] [Google Scholar]
- 22.Xie Z, Merchant S. A novel pathway for cytochromes c biogenesis in chloroplasts. Biochim Biophys Acta. 1998;1365:309–318. doi: 10.1016/s0005-2728(98)00085-1. [DOI] [PubMed] [Google Scholar]