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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  and  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  subclass, these genes are ccmA to -H (3, 18), whereas in B. japonicum, a member of the  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 (ccmE_Bj) 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 (CcmE_Ec) is able to bind heme in the presence of CcmABCD (15). The E. coli ccm 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 CcmE_Ec and CcmE_Bj 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 CcmE_Bj was changed to alanine, a heme-staining band was not detected (Fig. 1A, lane 4). To analyze whether the mutant CcmE_Bj (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 CcmE_Ec and CcmE_Bj bound heme (Fig. 1A, lanes 5 and 6). Thus, mutation H122A in CcmE_Bj led to a stable membrane-bound protein that was no longer able to bind heme. This finding implies that, like H130 in CcmE_Ec, H122 is the heme binding residue in CcmE_Bj. In fact, the histidine lies within the segment LAKHDE, which is absolutely conserved in all CcmE sequences known so far.

View larger version (43K): [in this window] [in a new window]   FIG. 1.   Ability of CcmEBj to bind heme and to transfer it to apocytochrome c. (A) The ccm 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 ccmE 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 CcmE_Bj 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 (ccmE), 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. CcmE_Bj and CcmE_Ec were able to complement the ccmE 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 CcmE_Bj was used. The CcmE_Bj H122A mutant was unable to complement a ccmE 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 CcmE_Bj 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 cycY (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 ccmE_Bj 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 CcmC_Bj has a similar function when expressed in E. coli. ccmC_Bj was amplified by PCR and cloned into the expression vector pISC-2 (Table 1). The E. coli ccm 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 CcmC_Bj and CcmC_Ec were able to insert heme into CcmE_Ec (Fig. 2A, lanes 2 and 3). A Western blot probed with anti-CcmE_Ec serum revealed similar amounts of CcmE_Ec polypeptide in all membrane protein fractions (Fig. 2B). Yet CcmC_Bj was significantly less active in holo-CcmE formation than CcmC_Ec (Fig. 2A, compare lanes 2 and 3).

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Ability of ccmCBj to attach heme to E. coli CcmE and to complement an E. coli ccmC mutant for formation of holo-cytochrome c. (A) The ccm 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 ccmC 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 (ccmC), 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. CcmC_Bj was able to complement a ccmC mutant to synthesize holo-cytochrome c (Fig. 2C, lane 2). However, much smaller amounts of cytochrome c were produced than with CcmC_Ec (Fig. 2C, lane 3). CcmC_Bj shows an activity similar to, but strongly reduced from, that of CcmC_Ec, 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.