Journal of Bacteriology, January 2003, p . 175-183, Vol . 185, No . 1

Biochemical and Mutational Characterization of the Heme Chaperone CcmE Reveals a Heme Binding Site

Elisabeth Enggist, Michael J . Schneider, Henk Schulz, and Linda Thöny-Meyer^*

Departement Biologie, Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland

Received 8 August 2002/ Accepted 7 October 2002

Remarkably, CcmE binds heme covalently at the histidine H130, as demonstrated by Edman degradation, ion spray, and tandem mass spectrometry of the CcmE-derived tryptic heme peptide (18) . However, the nature of the heme-histidine bond is not known yet . It has also been shown previously that H130 is required for heme binding of CcmE and also for cytochrome c maturation (18) . Therefore, the covalent attachment of heme to H130 is not an artifact but represents a true intermediate of the heme delivery pathway (20) . Heme binding to CcmE has a strict requirement only for CcmC, but it is most efficient in the presence of CcmABCD (17) . CcmC was shown to interact directly with both heme and CcmE (14) .

It was the aim of this work to characterize CcmE biochemically and genetically . The soluble domain of CcmE was purified in a His-tagged form and analyzed spectroscopically . Different methods were applied to achieve information about the heme binding site, the nature of the covalent heme-histidine bond, and the mechanism of the transfer of heme to c-type cytochromes . A sequence alignment of 27 bacterial and plant CcmE homologues revealed 26 residues with >90% sequence identity . From these, 11 mostly invariant charged, polar, or hydrophobic amino acids were selected and changed to alanine by site-directed mutagenesis . In addition to these 11 mutants, another three less-conserved residues were investigated . In a first step, single amino acids were exchanged to seek for CcmE point mutants expected to be deficient in either heme binding or heme release . With the exception of H130, no other residue was found to be required strictly for CcmE function . H130 was also mutated to a cysteine to mimic the situation in c-type cytochromes, where a covalent heme-cysteine bond is formed . Wild-type and H130C mutant CcmE were overproduced and purified, and their heme binding characteristics were compared . The results are discussed in the context of the structure of apo-CcmE that has been solved recently by nuclear magnetic resonance (6), and a model of heme binding is presented .

 
Construction of plasmids. E . coli DH5 was used as the host for cloning . All used primers were purchased from Microsynth (Balgach, Switzerland) . They are listed in Table 2 .

 
Plasmid pEC302 was constructed by QuikChange site-directed mutagenesis (Stratagene) with pEC415 as the template . For the likewise construction of pEC469, pEC472, pEC479, pEC480, pEC481, pEC482, pEC485, pEC491, pEC492, pEC493, and pEC496, the template pEC458 was used . pEC472 served as the template for constructing pEC488 and pEC495 . pEC489 resulted from a one-step PCR mutagenesis with pEC458 as the template . The 947-bp BglII/SalI fragment was ligated into BglII/SalI-digested pEC482 . The product of a similar PCR with different primers ligated into pEC481 yielded pEC494 . For pEC498, pEC472 served as the template . The 869-bp NheI/SalI fragment was ligated into NheI/SalI-digested pEC472 . For the construction of pEC499, the 377-bp NheI/SalI fragment from the PCR with pEC476 as the template was cloned into NheI/SalI-digested pEC476, which was the result of a two-step PCR . A 105-bp fragment was amplified with primers ccmEC and ccmEE132Af with pEC458 as the template . This fragment was used as a primer together with ccmEN2, again with pEC458 as the template . The resulting 535-bp fragment was digested with BamHI/EcoRV and ligated into BamHI/EcoRV-digested pBR322 . Cloning of the 707-bp NruI fragment from pEC419 into NruI-digested pEC458 yielded pEC467, and the ligation of the 585-bp NruI fragment of pEC472 into NruI-digested pEC481 resulted in pEC497 .

For the construction of pEC101 expressing ccmABCD, a 1,189-bp AflII/SspI fragment containing ccm'BCD was ligated into AflII/FspI-digested pEC86 with ccmAB' . All mutations of PCR-derived plasmids were confirmed by DNA sequencing with an ABI Prism 310 genetic analyzer (Perkin Elmer) .

Cell fractionation. Periplasmic fractions of 500 ml of anaerobically grown cultures were isolated by treatment with polymyxin B sulfate (Sigma) . The cells were harvested by centrifugation at 4,000 x g, washed in cold 50 mM Tris-HCl, pH 8.0, and resuspended in cold extraction buffer (1 mg of polymyxin B sulfate/ml, 20 mM Tris-HCl, 500 mM NaCl, 10 mM EDTA [pH 8.0]) (1.5 ml/g of wet cells) . The suspension was stirred for 1 h at 4°C and centrifuged at 40,000 x g for 20 min at 4°C . The supernatant contained the periplasmic fraction .

Membrane fractions of 250 ml of aerobically grown cultures were prepared as follows . The cells were harvested by centrifugation at 4,000 x g, washed in cold 50 mM Tris-HCl (pH 8.0), resuspended in 3 ml of cold 50 mM Tris-HCl (pH 8.0) containing 5 µg of DNase I/ml, and passed twice through a French pressure cell at 110 MPa . Cell debris were separated by centrifugation at 40,000 x g for 20 min at 4°C . The supernatant was subjected to ultracentrifugation at 150,000 x g for 1 h . The membrane fraction was washed once with 1 ml of cold 50 mM Tris-HCl, pH 8.0, and resuspended in 200 µl of the same buffer .

Purification of His-tagged soluble CcmE. The E . coli strain EC06 carrying pEC415 overproduced soluble apo-CcmE-H₆ (hexahistidine tagged) . One liter of LB was inoculated with 10 ml of culture grown overnight . The pellet obtained 12 h after induction with 0.1% arabinose was washed once with 50 mM sodium phosphate, pH 7.2, and resuspended in 6 ml of polymyxin buffer (150 mM NaCl, 1 mM EDTA, 50 mM sodium phosphate [pH 7.2], and 2 mg of polymyxin B [Fluka]/ml) . The solution was stirred gently for 1 h at 4°C . After centrifugation, the pellet was resuspended in the same amount of polymyxin B buffer for a repeated extraction of periplasmic proteins . The two supernatants containing the periplasmic proteins were precipitated with ammonium sulfate that was added slowly to 60% saturation . After centrifugation, the pellet was dialyzed twice against 2 liters of 300 mM NaCl and 50 mM sodium phosphate, pH 7.2 . Purification of CcmE-H₆ was performed by affinity chromatography on nickel-nitrilotriacetic acid (NTA)-agarose (Qiagen) with 3 column volumes of a linear gradient from 0 to 250 mM imidazole in 300 mM NaCl and 50 mM sodium phosphate, pH 7.2 . Apo-CcmE-H₆ was present in fractions eluted with 100 to 200 mM imidazole . After dialyzing twice against 2 liters of 300 mM NaCl and 50 mM sodium phosphate, pH 7.2, soluble CcmE-H₆ was concentrated with Centricon-10 (Millipore) up to 60 mg/ml .

When plasmid pEC101 containing ccmABCD was overexpressed in cells containing pEC415, a mixture of soluble holo- and apo-CcmE-H₆ was obtained and purified by the same procedure . To isolate holo-CcmE-H₆, the affinity-purified proteins were subjected to hydrophobic interaction chromatography (phenyl Sepharose 6 fast flow, high sub; Amersham Biosciences) . The sample was brought to 1 M ammonium sulfate, 300 mM sodium chloride, and 50 mM sodium phosphate (pH 7.2) and eluted with a linear gradient of 3 column volumes from 1 to 0 M ammonium sulfate . Both components eluted shortly after reaching 0 M ammonium sulfate in two peaks, and apo-CcmE-H₆ eluted first . The purity of the samples was checked by reverse-phase chromatography on a LiChrospher 100 RP-18 high-performance liquid chromatography (HPLC) cartridge (Hewlett-Packard) with a linear acetonitrile gradient in 0.1% trifluoroacetic acid . The protein was monitored at 215 and 398 nm . Samples with an A₄₀₀/A₂₈₀ ratio of >2.7 were found to contain only holo-CcmE . To remove degradation products, which had lost some C-terminal amino acids, including the His-tag, a second purification by nickel-NTA-agarose was performed, in which only full-length protein was present .

Biochemical methods. Protein concentrations were determined by using the Bradford assay (Bio-Rad) . Heme staining of proteins separated by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis was carried out with o-dianisidine (Sigma) as the substrate as described previously (19) . Immunoblot analysis was performed with a CcmE-specific antiserum directed against the synthetic peptide ¹²⁹KHDENYTPPEVEKAME¹⁴⁴ (18) . Signals were detected by using goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Bio-Rad) as a secondary antibody and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1^3,7]decan}-4-yl)phenyl phosphate (Roche Diagnostics) as the substrate .

Purified soluble CcmE-H₆ was digested with 1% (wt/wt) trypsin (sequencing grade; Roche Diagnostics) overnight at 37°C . After centrifugation, the sample was prepared for HPLC by filtering it through a 0.22-µm-pore size filter . The peptides were separated on a Zorbax 300SB-C18 reverse-phase column (Hewlett-Packard) with a linear acetonitrile gradient (20 to 30%) in 0.1% (vol/vol) trifluoroacetic acid . Peptides were monitored at 215 nm, and heme was monitored at 398 nm . The peak fractions containing the heme-binding peptides were collected and lyophilized .

Protein precipitation with trichloroacetic acid was performed with a 100% (wt/vol) solution, which was added to a final concentration of 10% . Samples were kept on ice for 15 min and then centrifuged at maximal speed . The pellet was washed quickly with 2 M Tris-HCl, pH 8.0, and resuspended in 3x SDS loading dye .

Spectroscopic methods. Optical spectra were recorded on a Hitachi model U-3300 spectrophotometer . Samples were reduced by adding a few grains of sodium dithionite and oxidized by adding K₃Fe(CN)₆ to a final concentration of 0.05 mM . For the CO difference spectrum, the reduced sample was bubbled for 2 min with CO and measured immediately . Pyridine hemochrome spectra were used for quantification of heme with an extinction coefficient at 551 nm of 29.1 mM^-1 cm^-1 (7) .

For mass spectroscopy, the peptides were resuspended in a saturated solution of sinapinic acid in H₂O-acetonitrile (2:1) and 0.1% trifluoroacetic acid . One microliter was deposited directly onto the mass spectrometer target . After drying, the sample was washed again with 1 µl of H₂O-acetonitrile (2:1) and 0.1% trifluoroacetic acid . Mass spectroscopy was performed on a Perseptive Biosystem Voyager Elite matrix-assisted laser desorption ionization-time of flight (mass spectrometry) with reflector and delayed extraction .

The constructed plasmids were cotransformed with a plasmid carrying ccmC into strain EC06 (ccmA-H) . In this minimal system, wild-type CcmE can bind heme, but no c-type cytochromes are formed due to the lack of the other ccm genes (17) . The mutants were tested for their ability to form holo-CcmE by heme staining of membrane proteins (Fig . 1A) . Different quantities of holo-CcmE were detected in the mutants (Fig . 1A, lanes 3 to 9) . No single mutant showed a complete deficiency of heme binding as it is found for mutant H130A (18) (Fig . 1A, lane 16) . The presence of the CcmE polypeptide in the membrane was assessed by Western blot analysis . All mutants produced wild-type or only slightly reduced levels of CcmE (Fig . 1B, lanes 3 to 9) .

 
To check if these mutants were affected in heme delivery to cytochrome c, a ccmE in-frame deletion strain was complemented with a plasmid carrying either wild-type or mutant ccmE alleles . Coexpression of the heterologous Bradyrhizobium japonicum gene cycA under anaerobic growth conditions with nitrite as the terminal electron acceptor allowed us to analyze the formation of two soluble periplasmic c-type cytochromes, the foreign B . japonicum cytochrome c₅₅₀ (2), and the endogenous NapB diheme cytochrome c (12) . Heme staining of the periplasmic fractions showed that all CcmE versions with point mutations of charged residues were able to complement the ccmE strain for cytochrome c biogenesis (Fig . 2, lanes 1 and 3 to 9) .

 
We next attempted to introduce more drastic changes by constructing the double and triple mutants K129A/D131A, D131A/E132A, K129A/D131A/E132A, and R104A/K129A . All tested combinations yielded CcmE versions that retained some ability to bind heme (Fig . 1A, lanes 10 to 13) and to produce c-type cytochromes (Fig . 2, lanes 10 to 13) . However, multiple changes also led to a decrease of the CcmE signal (Fig . 1B, lanes 10 to 13), perhaps affecting the stability of the protein . In conclusion, we have not found evidence for acid- or base-catalyzed heme binding or release by removing specific charged residues .

Point mutations of conserved, noncharged amino acids in CcmE. Binding of the hydrophobic heme molecule to CcmE is expected to involve hydrophobic interactions between cofactor and polypeptide . Thus, some of the best conserved noncharged and hydrophobic amino acids were changed to alanine: the aromatic residues F37, Y95, F103, and Y134, the hydrophobic L127, and the polar S70 . In addition, the triple mutant F103A/R104A/E105A was constructed in order to introduce a drastic change in the conserved motif ⁹⁹LPDLFREG¹⁰⁶ . Expression of these CcmE versions together with CcmC resulted in the heme staining bands presented in Fig . 1A (lanes 17 to 23) . Mutants F37A and L127A were only slightly impaired in heme binding, whereas the other mutants produced low levels of holo-CcmE . The polypeptide levels of mutants S70A and Y95A were also strongly affected as detected by Western blot analysis (Fig . 1B, lanes 18 and 19) . However, the corresponding mutant proteins were able to transfer heme as well as the wild type was (Fig . 2, lanes 18 and 19) . Most likely these mutations affected the stability, but not the function, of CcmE . The decreased amount of mutated CcmE is thus not limiting with respect to cytochrome c maturation .

Mutant F103A contained the lowest levels of heme of all of the single mutants, but it also showed reduced protein levels and was affected in cytochrome c maturation . The additional mutation of R104 and E105 did not alter this phenotype, which excludes a cooperative behavior of these residues . L127A and Y134A showed a decreased heme binding capacity but wild-type levels of CcmE polypeptide . Both mutants were impaired in heme transfer to cytochrome c . In summary, hydrophobic residues play a major role in the binding of heme to CcmE . Likewise, changes of hydrophobic residues F37, F103, L127, and Y134 led to the most significant loss of cytochrome c formation .

Quantification of c-type cytochromes by absorption spectroscopy. To quantify the concentration of soluble c-type cytochromes in the different strains, the reduced minus the oxidized absorption spectra of the periplasmic fractions were recorded . The difference of the absorption at 550 and 536 nm is proportional to the amount of c-type cytochromes . The value 100% was assigned to the wild type, and the relative levels of cytochromes formed by the mutants were calculated accordingly . All measurements were performed with at least two independent experiments . The values obtained from the spectra corresponded well to the intensities of the heme staining bands (Fig . 2) .

The CcmE H130C mutant can bind heme but does not form c-type cytochromes. H130 is the only residue of CcmE which is essential for heme binding . In mutant H130A, the lack of heme binding (Fig . 1, lane 16) abolishes cytochrome c biosynthesis (Fig . 2, lane 16), suggesting that heme binding is an essential step during the maturation pathway . H130 binds heme covalently, but the nature of this bond is not known (18) . We speculate that the binding of the histidine side chain occurs at one of the two heme vinyl groups, which is somewhat reminiscent of the heme-cysteine bond of c-type cytochromes . If the formation of this bond is based mainly on a correct positioning of heme and the polypeptide, one might expect that replacement of H130 by a cysteine can result in the formation of a thioether bond, like in cytochrome c . Thus, we constructed an H130C version of CcmE and analyzed heme binding and heme transfer . In fact, a faint heme staining band was present in the membranes of the H130C mutant (Fig . 3A), whereas the level of expressed protein was comparable to that of the wild type (Fig . 3B) . However, no c-type cytochromes were detected (Fig . 3C) .

 
Purification and characterization of soluble CcmE-H₆. To further characterize the CcmE heme chaperone, large-scale purification of the protein was necessary . The membrane anchor of CcmE was replaced by a cleavable signal sequence to produce soluble, periplasmic CcmE (18) . In addition, a hexahistidine tag was added to the C terminus . The resulting soluble CcmE-H₆ protein was purified in three forms: as apo-CcmE without heme, as holo-CcmE with heme, and as a mixture of these two forms . When ccmE was expressed in a ccmA-H background, only apo-CcmE was produced because the covalent attachment of heme requires the presence of CcmC (17) . When plasmid pEC101 carrying ccmABCD was coexpressed, a mixture of apo- and holo-CcmE in a ratio of about 10:1 was obtained . The proteins were purified by affinity chromatography on nickel-NTA-agarose (Fig . 4), yielding 20 mg of pure protein per liter of cells . Further separation of apo- and holo-CcmE was achieved by hydrophobic interaction chromatography . This step repeatedly produced degradation products . Four fragments with masses of 13,036, 12,851, 12,591, and 12,391 m/z were detected by matrix-assisted laser desorption ionization mass spectroscopy . They corresponded to the following degradation products (theoretical masses are given in parentheses): apo-CcmE^30-146 (13,037 m/z), apo-CcmE^30-144 (12,852 m/z), apo-CcmE^30-142 (12,591 m/z), and apo-CcmE^30-140 (12,392 m/z), respectively . For three of them, the heme-binding forms were also found: 13,655 m/z for holo-CcmE^30-146 (13,654 m/z), 13,468 m/z for holo-CcmE^30-144 (12,468 m/z), and 13,208 m/z for holo-CcmE^30-142 (13,208 m/z) . These fragments are lacking their C-terminal His tags . In order to eliminate the degradation products, the fractions containing only holo-CcmE, as detected by reverse-phase HPLC, were subjected a second time to nickel-NTA affinity chromatography . The eluted, nondegraded holo-CcmE was found to be stable for several weeks at 4°C if kept in 300 mM NaCl and 50 mM sodium phosphate (pH 7.2) . Lower salt concentrations (100 mM NaCl) caused rapid degradation of the protein .

 
Visible absorption spectra of holo-CcmE-H₆ and the holo-CcmE-apo-CcmE mixture were identical . Characteristics of the spectra from the mixture are presented in Fig . 5 . Maxima for the difference spectrum (Fig . 5C) of the reduced (Fig . 5B) minus oxidized (Fig . 5A) holo-CcmE correspond well with the previously published ones of membrane bound holo-CcmE (18) . The bands of the reduced sample (Fig . 5B) at 556 nm and of the pyridine hemochrome (Fig . 5E) at 551 nm are in agreement with saturation of a heme vinyl by a covalent modification . Similar bands have been found in the holo-CcmE preparation of Daltrop et al . (5) . The minimum at 555 nm in the reduced CO complex (Fig . 5D) suggests that the heme iron is available for axial ligation with CO . The maximum at 620 nm in the oxidized spectrum (Fig . 5A) indicates that iron is present in the high-spin state (3) .

 
CcmE H130C requires CcmABCD for heme binding. To facilitate purification of CcmE H130C, the soluble, His-tagged version of this mutant was constructed . As for the wild type, heme binding of this mutant was observed only in the presence of coexpressed ccmABCD (Fig . 6A) . While the H130C mutant protein was expressed equally well as the wild-type protein (Fig . 6B), only a small fraction bound heme .

 
To address the nature of the newly formed heme-cysteine bond, the soluble holo-CcmE-H₆ H130C protein was purified . Optical spectra of the purified H130C mutant protein showed the same characteristics as wild-type CcmE (Fig . 5F); however, at least 200 times less of the holo-CcmE form was present .

Tryptic peptides of CcmE bind heme covalently. Purified wild-type CcmE and CcmE H130C were digested with trypsin . From wild-type CcmE, two heme-binding peptides were isolated by reverse-phase HPLC and analyzed by mass spectroscopy . The average mass of 2,074.1 m/z of the first peptide corresponded exactly to the sum of that of the expected 12mer peptide ¹³⁰HDENYTPPEVEK¹⁴¹ (1,457.5 m/z) plus heme (616.5 m/z) . The second isolated peptide had a mass of 2,614.5 m/z coming from heme plus the 17mer ¹²⁵EVLAKHDENYTPPEVEK¹⁴¹ (1,998.2 m/z) resulting from a partial digest . We thus speculate the reaction of heme with the histidine to be an addition, whereby one of the heme vinyl side chains reacts with a nitrogen of the histidine imidazole ring . A substitution would lead to the loss of two hydrogen atoms .

After the tryptic digest of CcmE H130C and separation by reverse-phase HPLC, a peptide absorbing at 398 nm was isolated . The identified mass of 2,040.3 m/z corresponds to the dodecamer peptide ¹³⁰CDENYTPPEVEK¹⁴¹ (1,423.5 m/z) plus heme . This indicates that heme is bound covalently to the mutated cysteine 130 .

Heme binding to and release from CcmE is catalyzed in vivo by the membrane proteins CcmC and CcmF, respectively, which have been demonstrated to interact directly with CcmE (13, 14) . D86, the amino acid with the strongest effect on cytochrome c formation upon mutation among the charged residues is located at the surface of CcmE, distant from the putative heme binding pocket . D86 may be responsible for protein-protein interactions, either with CcmC or CcmF . Both proteins contain a number of conserved basic residues in periplasmic domains which are candidates for interaction with D86 of CcmE: R55 and R128 for CcmC, and R84, R149, R269, K273, R295, R311, K564, and R565 for CcmF .

H130 is the only amino acid that is absolutely required for CcmE function . CcmE H130A neither binds heme nor supports production of c-type cytochromes (18) . When H130 was mutated to a cysteine, residual heme binding to CcmE was detected, but it was more than 200-fold decreased . The resulting H130C protein showed an absorption spectrum indistinguishable from that of wild-type CcmE . In c-type cytochromes, cysteine reacts with heme by forming a thioether bond . As for the heme-histidine bond formed in the wild type, the heme-cysteine bond of the mutant CcmE was formed in vivo only in the presence of other proteins of the Ccm complex . CcmC has been shown to be necessary and sufficient to transfer heme to CcmE (19) . It either catalyzes the formation of the covalent bond or presents heme in the correct spatial orientation to CcmE . The H130C mutant was blocked in cytochrome c synthesis . Apparently, the transfer of heme that is bound to a cysteine cannot occur due to the stability of the thioether bond .

We suspect that, in the wild-type CcmE, heme binding to H130 occurs at one of the two vinyl groups for the following reasons . (i) Mass spectra of the heme peptides showed that no protons are lost when heme is bound, indicating an addition rather than a substitution . (ii) The optical spectrum of holo-CcmE has an maximum at 556 nm under conditions where the spectrum of apo-CcmE plus heme has a maximum at 559 nm (6) . The shift of the band to a shorter wavelength in the holo form is in agreement with the presence of a covalent bond removing one of the two heme vinyl groups (11) . (iii) Our idea to mimic the covalent attachment of heme by the formation of a thioether bond like that found in c-type cytochromes in an H130C mutant proved to be feasible, although heme binding was very inefficient . In a parallel study, Daltrop et al . found that soluble CcmE-H₆ can bind ferric heme in vitro . Upon reduction to the ferrous form, heme was bound covalently to the polypeptide when protoheme was used but not when mesoheme lacking the vinyl groups was used (5) . These data support the idea that the heme-histidine bond is the product of an addition to the vinyl double bond .

With the protocol for purifying soluble apo-CcmE and holo-CcmE, it should now be possible to elucidate the most interesting features of CcmE: the nature of the heme-histidine bond and the mechanism by which this bond is formed and subsequently broken to transfer heme . We speculate that CcmE selects one of the two heme vinyl groups to form a covalent bond, thus implementing a stereospecifically correct and efficient delivery of heme to c-type cytochromes .

This work was supported by grants from the Swiss National Foundation for Scientific Research and from the ETH .

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