Interaction of Heme with Variants of the Heme Chaperone CcmE Carrying Active Site Mutations and a Cleavable N-terminal His Tag*
Julie M. Stevens
,
Oliver Daltrop
,
Christopher W. Higham and
Stuart J. Ferguson ¶
From the
Department of Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU, United Kingdom
Received for publication, December 18, 2002
, and in revised form, March 21, 2003.
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ABSTRACT
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Cytochrome c maturation in the periplasms of many bacteria
requires the heme chaperone CcmE, which binds heme covalently both in
vivo and in vitro via a histidine residue before transferring
the heme to apocytochromes c. To investigate the mechanism and
specificity of heme attachment to CcmE, we have mutated the conserved
histidine 130 of a soluble C-terminally His-tagged version of CcmE
(CcmEsol-C-His6) from Escherichia coli to
alanine or cysteine. Remarkably, covalent bond formation with heme occurs with
the protein carrying the cysteine mutation, and the process occurs both in
vivo and in vitro. The yield of holo-H130C
CcmEsol-C-His6 produced in vivo is low compared
with the wild type. In vitro heme attachment occurs only under
reducing conditions. We demonstrate the involvement of one of the heme vinyl
groups and a side chain at residue 130 in the bond formation by showing that
in vitro attachment does not occur either with the heme analogue
mesoheme or when alanine is present at residue 130. These results have
implications for the mechanism of heme attachment to the histidine of CcmE.
In vitro, CcmEsol lacking a His tag binds
8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and
via a covalent bond from the histidine side chain, similarly to the tagged
proteins, thus countering a recent proposal that the His tag causes the heme
binding. However, the His tag does appear to enhance the rate of in
vitro covalent heme binding and to affect the heme ligation in the ferric
b-type cytochrome form.
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INTRODUCTION
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c-type cytochromes are important ubiquitous proteins that bind
heme covalently via two thioether bonds between the cysteines in a conserved
CXXCH motif in the protein and the vinyl groups of heme. In many
Gram-negative bacteria, formation of these covalent bonds occurs in the
periplasm in a multistep process involving the so-called Ccm
(cytochrome c maturation) proteins
AH, all of which are essential
(1,
2). The apocytochromes are
synthesized in the cytoplasm and transported to the periplasm via the Sec
pathway (3). The mechanism by
which heme, which is also synthesized cytoplasmically, is transported to the
periplasm is as yet uncertain
(4,
5,
6). However, it has been shown
that the membrane-bound protein CcmC is required for presentation of heme to
the membrane-anchored periplasmic protein CcmE
(7). CcmE has been identified
as the heme chaperone and binds heme covalently via a conserved histidine
residue (His130 in Escherichia coli) before transferring
the heme to apocytochromes (8).
Recently, structures of the CcmE apoproteins from two different bacterial
species have been reported (9,
10), which shows that the
heme-binding histidine is exposed on the protein surface but provides no clue
as to the unusual properties of this residue. CcmE has been shown to be part
of a complex with CcmF in vivo
(11), which together with CcmH
forms what is proposed to be a bacterial heme lyase. CcmH interacts with CcmG,
which is part of the system that provides reductant to the periplasm,
specifically for reducing the cysteines in the CXXCH motif of the
apocytochromes (12).
The nature of the novel bond between the histidine of CcmE and the heme
remains unknown, although it has been shown that one of the vinyl groups of
heme is involved (13). CcmE
has been identified in a number of bacteria as well as in Arabidopsis
and presumably other plant mitochondria
(14). In vitro
studies of a soluble version of CcmE (CcmE') have shown that the
covalent attachment occurs under reducing conditions and that release of the
heme to apocytochromes will only occur if heme is in its ferrous state
(13). To probe further the
covalent attachment of heme in vitro or in vivo, we have
altered by site-directed mutagenesis the histidine 130 that has been shown
previously to be the site of heme attachment in vivo
(8). Replacement of this
histidine by cysteine or alanine was expected to generate proteins with either
a potentially reactive or an unreactive side chain at position 130. The
CcmE' protein used in previous in vitro studies
(13) had a His tag at the C
terminus. Very recently it has been suggested that such a His tag may
significantly influence the binding of heme to CcmE' in vitro
to the extent that protein from Shewanella putrefaciens lacking a tag
is reported to be unable to bind heme in vitro
(9). Any possibility that a His
tag might direct heme to form a nonphysiological covalent bond to other than
residue 130 would also be addressed by the present mutagenesis studies.
Further test of whether a His tag affects the binding of heme has been made by
comparing the tagged and untagged proteins with histidine at 130. Furthermore,
the mechanistic implications of our studies are discussed.
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EXPERIMENTAL PROCEDURES
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Plasmid Construction and MutagenesisE. coli strain
DH5
was used for cloning, and JM109(DE3) was used for protein
expression. The expression vector for CcmEsol-C-His6
(CcmE') was constructed as described
(13) and produces the soluble
periplasmic region of the protein from Ser32 with a cleavable
pelB signal sequence for periplasmic targeting of the protein. Amino
acid substitutions in this expression vector were performed using the ExSite
PCR-based site-directed mutagenesis method (Stratagene) using the primers
pET22bH130AF and -R for the H130A mutant and pET22bH130CF and -R for the H130C
mutant (as listed in Table I).
The vector for the cytoplasmic expression of the thrombin-cleavable His-tagged
version of the protein (N-His6-CcmEsol) was constructed
by PCR amplification of the gene from the plasmid pEC86 (which was kindly
provided by L. Thöny-Meyer) using the primers pET15bF and -R, which
include XhoI and BamHI restriction sites, respectively
(Table I). The PCR products
were cloned into the vector pET15-b (Novagen) using these restriction sites,
producing the plasmid pE151. Mutations were made in this plasmid using the
QuikChange method (Stratagene). The mutations made were H130A using the
primers pET15bH130AF and -R, H130C using the primers pET15bH130CF and -R, as
well as mutation of the His and Met (both to Ala) that remain on the protein
following thrombin cleavage, using the primers pET15bHMAAF and -R
(Table I). All of the resulting
plasmids were sequenced to confirm that only the desired mutations had been
incorporated.
Protein Expression and PurificationFor expression of the
holoforms of the periplasmic proteins, the expression vectors pE221, pE222,
and pE223 were co-transformed with the plasmid pEC86
(15), which expresses the Ccm
proteins AH. Co-expression of these proteins is essential for
production of wild-type holo-CcmE'
(13). High levels of the
apoproteins were expressed in the periplasm in the absence of pEC86. E.
coli cultures were grown as described previously, and the proteins were
purified using Ni2+-chelating Sepharose columns equilibrated with
50 mM Tris-HCl, pH 7.4, as described
(13). The cytoplasmically
expressed proteins (from the plasmids pE151, pE152, pE153, and pE154) were
purified in the same way, except that the cells were sonicated on ice three
times for 30 s to prepare the cell extracts, and the buffer contained 300
mM NaCl throughout. Thrombin cleavage of
N-His6-CcmEsol and mutants thereof was performed using a
thrombin CleanCleave Kit (Sigma) according to the manufacturer's instructions.
Uncleaved protein was removed by re-applying the reaction mixture to the
Ni2+-Sepharose column. Western blots were performed using a
peroxidase conjugate of a monoclonal anti-polyhistidine antibody (Sigma) to
confirm that the His tags had been completely cleaved.
Protein CharacterizationDiscontinuous SDS-PAGE (15 or 17.5%
acrylamide) (16) was used to
analyze the proteins, and staining for covalently bound heme was performed
according to the method of Goodhew et al.
(17), following acidified
acetone extraction to remove noncovalently bound heme. Visible absorption
spectra were recorded on a Perkin-Elmer Lambda 2 spectrophotometer using
between 2 and 5 µM heme-protein samples in either 50
mM sodium phosphate buffer, pH 7.0, or 50 mM Tris-HCl
buffer, pH 7.4, 300 mM NaCl. Pyridine hemochrome spectra were
obtained according to the method of Bartsch
(18) using 5 µM
protein in 19% (v/v) pyridine and 0.15 M NaOH. Electrospray
ionization mass spectrometry
(ES-MS)1 was performed
using a Micromass Bio-Q II-ZS triple quadrupole atmospheric pressure mass
spectrometer. 10-µl protein samples in 1:1 water:acetonitrile, 1% formic
acid at a concentration of 20 pmol/µl were injected into the electrospray
source at a flow rate of 10 µl/min.
Heme AdditionHemin (Sigma) or mesoheme (Frontier Science)
(1 mM in Me2SO) was added to apoprotein solutions in 50
mM sodium phosphate buffer, pH 7.0. Quantitative loading of the
proteins with heme was achieved by incubating 1 equivalent of protein with 1.1
equivalents of heme at room temperature. Desalting columns were used to remove
excess heme from the protein solutions. Disodium dithionite (Sigma) was used
to reduce heme. The fluorescence measurements were made using a Perkin Elmer
LS 50B fluorimeter, and the dissociation constants for heme and ANS were
determined as described
(13).
Covalent Heme AttachmentDisodium dithionite and
dithiothreitol (5 mM) were added to solutions (in 50 mM
sodium phosphate buffer, pH 7.0) containing protein (50 µM) and
heme (10 µM; added from 1 mM stocks in
Me2SO), which were incubated at room temperature. The solutions
were deoxygenated by thoroughly sparging with humidified argon. The reactions
were carried out in the dark.
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RESULTS
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Characterization of CcmEsol Lacking a His
TagRecently, it was suggested that the His tag on a soluble CcmE
construct, described in our previous work
(13), causes artifactual
noncovalent binding of heme and that the heme binding described is
nonphysiological (9). To
address this point, we have made a construct of CcmE that can be studied with
and without the His tag, using a protease cleavage site between the His tag
and the soluble CcmE domain. The protein with the cleavable His tag
(N-His6-CcmEsol) was analyzed by ES-MS and had a mass of
16,641 Da (theoretical mass, 16,641 Da); the protein ran as a single band
during SDS-PAGE analysis (Fig.
1, lane 1). After thrombin treatment and removal of
uncleaved protein, the cleaved protein (CcmEsol) had a mass of
14,890 Da (theoretical mass, 14,890). The difference in the mass of the
protein could also be seen by SDS-PAGE analysis
(Fig. 1, lane 4). The
absence of the His tag was also confirmed by Western blotting.

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FIG. 1. SDS-PAGE (17.5%) analysis of various forms of
N-His6-CcmEsol and CcmEsol. A,
Coomassie Blue stain. B, activity stained for bound heme. Lanes
1, apoform of N-His6-CcmEsol. Lanes 2,
heme-N-His6-CcmEsol obtained after reaction of
N-His6-CcmEsol with reduced heme for 14 h. Lanes
3, protein obtained after incubation of protein sample shown in lane
2 with thrombin. Lanes 4, apoform of CcmEsol obtained
following treatment of N-His6-CcmEsol with thrombin.
Lanes 5, CcmEsol reacted with reduced heme for 40 h.
M is prestained molecular mass marker corresponding to 25 and 16.5
kDa from top to bottom.
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After the addition of ferric heme to apoprotein lacking a His tag, followed
by reduction by disodium dithionite, no differences in the visible spectra,
characteristic of a low spin state, were observed compared with the His
tag-containing protein (Table
II). There was a subsequent disappearance of these spectral
characteristics upon reduction, as we have observed for the wild-type protein
with the C-terminal His tag
(13). Interestingly, in the
visible spectrum of ferric heme and CcmEsol, a species was obtained
that is similar to that observed upon the addition of ferric heme to horse
heart apocytochrome c
(19), showing features of a
high spin heme-protein species. Thus, the spectrum of the oxidized
heme-protein complex obtained without a His tag (a broad Soret band around 400
nm) was different from those for the proteins with the His tag at either the C
or N terminus, both of which have a Soret band at 413 nm. These data suggest
that the His tag can affect the coordination chemistry of the ferric
heme-CcmEsol complex leading to a high spin/low spin equilibrium.
However, in light of the known effects of polyhistidine on the coordination
characteristics of heme, this is arguably not surprising
(20). This contrasts with the
spectrum of the reduced heme-CcmEsol complex not being altered by
the absence of the His tag (Table
II).
In addition, further analysis of the protein without the His tag showed
that it has a similar ANS affinity to the protein with the tag and the same
maximum emission wavelength at 480 nm (results not shown). Also, it was
possible to displace bound ANS from CcmEsol by the addition of
heme, as we have shown for the tagged protein
(13). The reaction of ferrous
heme with N-His6-CcmEsol led to a protein with
covalently bound heme that had characteristics indistinguishable from
CcmEsol-C-His6
(13)
(Fig. 1, lane 2, and
data not shown). After thrombin cleavage of the His tag, heme was shown to be
covalently bound to CcmEsol as shown in
Fig. 1 (lane 3).
Reaction of CcmEsol with heme in the absence of the His tag led to
the same qualitative result (Fig.
1, lane 5), but the half-life of reaction was increased
by at least 10-fold relative to versions of the protein with His tags at
either end.
Following thrombin cleavage of the protein expressed from pET15-b, two
potential extra heme-ligating residues remain on the N terminus of the
protein, namely histidine and methionine. To avoid any potential artifactual
heme ligation by the untagged protein obtained by thrombin treatment of the
N-terminal His-tagged protein, these residues were mutated to alanines. The
proteins with and without the His tag were pure as judged by SDS-PAGE analysis
(data not shown) and had the expected masses as indicated by ES-MS analysis.
Upon the addition of ferric heme to apoprotein without the His tag, the
spectrum changed compared with the spectrum of free ferric heme
(Fig. 2). The heme-protein
complex appeared to be high spin in the ferric state and switched to low spin
in the reduced state upon the addition of disodium dithionite
(Fig. 2). Therefore, the
presence of an extra methionine and histidine, derived from the linker region,
had no observable effect on interactions with heme.

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FIG. 2. Absorption spectra of the b-type cytochrome formed after the
addition of heme to apo-CcmEsol, showing the oxidized spectrum
(dashed line), the reduced spectrum obtained immediately after the
addition of disodium dithionite (solid line), and the spectrum of
free heme (dotted line). The absorption spectra were recorded by
using 5 µM protein in 50 mM Tris-HCl buffer, pH 7.4,
300 mM NaCl.
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Characterization of the Mutants H130A and H130CBoth the
H130A and H130C variants of CcmEsol-C-His6 were
expressed well as their apoforms in the E. coli periplasm. The masses
of the proteins were confirmed by ES-MS, which also showed that the
periplasmic targeting sequences had been completely cleaved. The observed
masses were 15,447 Da for the H130A mutant (theoretical mass, 15,450 Da) and
15,483 Da for the H130C mutant (theoretical mass, 15,482 Da). Both proteins
were also expressed in E. coli with co-expression of the other Ccm
proteins, and the covalent attachment of heme to the proteins was examined by
SDS-PAGE analysis followed by heme staining
(Fig. 3).
Fig. 3A shows that the
proteins were purified to homogeneity (lanes 2 and 4 for
H130A and H130C, respectively). As expected, the H130A mutant did not appear
to bind heme covalently in vivo as judged by heme staining of the
SDS-PAGE gel (Fig. 3B,
lane 2), which is in agreement with previous experiments
(8). The H130C mutant, however,
was found to heme stain when expressed with the other Ccm proteins
(Fig. 3B, lane
6), indicating that the protein was recognized to some extent by the Ccm
system for heme delivery and attachment. To detect the stain from covalently
bound heme, the gel had to be overloaded such that a broad band was seen. It
was found that the apo-H130C protein formed intermolecular disulfide bonds
in vitro when dialyzed extensively against oxygenated 50
mM sodium phosphate buffer, pH 7.0, as shown in
Fig. 3A (lane
5). This observation was supported by analysis with Ellman's reagent,
which showed 1 and 0.2 equivalents of free thiol for reduced and oxidized
protein, respectively. A significant proportion of the protein ran at a
molecular mass of
30 kDa, corresponding to a covalently linked dimer,
which was also identified by ES-MS analysis. In vivo-produced H130C
holo-CcmEsol-C-His6 is produced at a very low level,
where less than 0.5% of the protein is in the holo-form containing covalently
bound heme, as determined from the relative ratio of the Soret band to the
absorption at 280 nm.

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FIG. 3. SDS-PAGE (15%) analysis of various forms of
CcmEsol-C-His6. A, Coomassie Blue stain.
B, activity stained for covalently bound heme. M, molecular
mass markers (16.5, 25, 32.5, and 47.5 kDa (only in A) from
bottom to top). Lanes 1, wild-type
holo-CcmEsol-C-His6 produced in vivo. Lanes 2,
H130A mutant produced in the presence of pEC86. Lanes 3, H130A
reacted with reduced heme in vitro for 14 h. Lanes 4, the
apoform of H130C isolated from the periplasm of E. coli. Lanes 5, the
extensively dialyzed apo-H130C. Lanes 6, the holo-H130C produced
in vivo in the presence of pEC86. 300 pmol and 1.5 nmol of protein
were loaded in A and B, respectively. The protein was
overloaded so that the heme-staining band could be clearly observed. Lanes
7, the apoform of H130C reacted with oxidized heme in vitro for
14 h. Lanes 8, apo-H130C reacted with reduced heme for 14 h.
Lanes 9, the reaction product of incubation apo-H130C and reduced
mesoheme in vitro for 14 h. 100200 pmol of protein were loaded
into each lane, unless otherwise stated.
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In Vitro Heme Binding to the H130A and H130C
ApoproteinsUpon the addition of ferric heme to the mutant
CcmEsol-C-His6, both H130A and H130C formed noncovalent
b-type cytochrome complexes, as determined by visible spectroscopy.
The complexes formed within the mixing time and appeared to be stable in this
form for several hours. The absorbance maxima for the complexes of both
proteins with ferrous heme and mesoheme as well as the pyridine hemochrome
spectra are shown in Table II.
Mesoheme is a heme analogue that has ethyl substituents in the normal
positions of the vinyl groups. The visible spectrum of the complex of H130C
with ferric heme shows the characteristics of a b-type cytochrome,
with an
-band at 560 nm following reduction with dithionite and
immediate recording of the spectrum. Interestingly, it was not possible to
record an accurate visible spectrum of the dithionite-reduced H130A mutant
b-type complex because dissociation of heme from the protein was too
rapid. Heme dissociation upon reduction was also observed with the wild-type
heme-protein complex (13). The
pyridine hemochrome spectra of the b-type complexes of both mutants
with heme have absorbance maxima at 556 nm, which is characteristic of
unsaturated vinyl groups and shows that covalent attachment does not occur
under these conditions.
The dissociation constants (Kd) of the mutant
proteins for ferric heme were measured by fluorescence spectroscopy as
described for the wild-type protein
(13). The dissociation
constants of the high affinity binding sites were compared, because these are
likely to be the physiologically relevant sites. The
Kd of the H130A mutant was found to be 0.72
(± 0.16) µM, which shows that it has a slightly lower
affinity for heme than the wild-type protein (Kd,
0.2 µM (13)).
This result is not unexpected because the loss of the histidine side chain is
likely to have changed the conformation of the heme-binding site in the
protein. The Kd of the H130C mutant was found to
be 0.48 ± 0.08 µM, also higher than the wild type. Both
mutant proteins, however, have retained a significant affinity for heme. It
should be noted, however, that the model of heme binding presented for this
protein suggests that a conformational change occurs in the flexible
C-terminal region upon heme binding
(10).
Covalent Attachment of Heme to H130CUpon reduction by
disodium dithionite of the C-terminal His-tagged b-type H130C variant
protein, with a 5-fold excess of protein over heme, the absorbance spectrum
shifted toward a cytochrome spectrum corresponding to a covalent bond between
heme and protein. The
-band shifted from 560 to 556 nm over several
hours, and the
-band and Soret band also shifted accordingly with time
(Table II). After desalting the
reaction mixture, the spectrum of H130C produced in this way was very similar
to the spectrum of the in vivo-produced H130C protein
(Table II). The pyridine
hemochrome spectrum of the in vitro produced holo-form of the protein
yielded a maximum around 553 nm, which is consistent with the presence of a
single free vinyl group, as has been observed for single cysteine variants of
c-type cytochromes
(21). Interestingly, the
in vivo produced holo-form of this mutant had broad absorption maxima
in the reduced and pyridine hemochrome spectra. This observation suggests that
as a consequence of the substitution of histidine 130 with a cysteine residue,
heme attachment is not completely selective and that the formation of
incorrect side products can occur. The oxidation state of heme and the
cysteine thiol in vivo might not be as tightly controlled during the
periplasmic attachment of heme to the protein compared with the reducing
conditions of the in vitro experiments.
To prove that the spectroscopic data were indicative of in vivo
and in vitro covalent bond formation between heme and the H130C
mutant protein, SDS-PAGE analysis followed by heme staining was performed.
Fig. 3 (lane 8) shows
the reaction of the b-type complex of heme and H130C with dithionite
after 14 h. The fact that the protein stains for covalently bound heme
(Fig. 3B, lane
8), as does the in vivo produced holo-H130C in lane 6, indicates
that in vitro and in vivo covalent attachment of heme to the
protein had occurred. The controls for this experiment are shown in lanes
3, 7, and 9. These are H130A incubated with ferric heme followed
by reduction with dithionite, H130C incubated with ferric heme, and the
addition of ferric mesoheme to H130C protein followed by reduction with
dithionite and incubation for 14 h, respectively. These controls show that
covalent attachment of heme to the CcmEsol protein samples did not
occur under these conditions, because no heme staining could be observed for
CcmEsol. The results establish that one of the vinyl groups of heme
is involved in formation of the covalent bond, because attachment was not
observed with mesoheme. The results also show that the bond forms with the
cysteine residue of the protein, because it is not observed with the alanine
mutant. Therefore, it is shown that covalent heme binding can only occur with
a reactive side chain of amino acid 130. As was observed for the wild-type
protein (13), these results
also highlight the requirement for reduction of the heme in the covalent bond
formation, because no heme staining was observed with the oxidized sample.
To remove any ambiguity regarding the effect of the His tag on the covalent
heme attachment to the H130C variant and the inability of the H130A mutant to
bind heme covalently in vitro, proteins with these active site
mutations were also made with a N-terminal cleavable His tag. The purified
proteins were shown to be pure by SDS-PAGE analysis as shown in
Fig. 4 (lanes 1 and
2, for H130A and H130C, respectively). Upon the addition of heme
under reductive conditions followed by removal of the His tag by thrombin
cleavage, the H130C mutant was found to contain covalently bound heme as
determined by SDS-PAGE analysis followed by heme staining
(Fig. 4, lane 4) and
visible spectroscopy (Fig. 5).
The visible spectrum compares very well with the in vivo produced
H130C heme-containing protein. The H130A mutant failed to stain for covalently
bound heme (Fig. 4, lane
3) and did not show any characteristics of a heme-containing protein in
the visible spectrum (data not shown).

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FIG. 4. SDS-PAGE (17.5%) analysis of heme attachment to H130A and H130C
CcmEsol-C-His6. A, activity stained for
covalently bound heme. B, Coomassie Blue stained. M shows
molecular mass markers (6.5 (faint), 16.5, 25, and 32.5 kDa from
bottom to top). Lanes 1, apo-H130A
CcmEsol-C-His6. Lanes 2, apo-H130C
CcmEsol-C-His6. In the Coomassie-stained gel it is clear
that some dimerization has occurred via disulfide bonds between the cysteines
at position 130. Lanes 3, H130A CcmEsol-C-His6
reacted with reduced heme for 14 h followed by thrombin cleavage of the His
tag. Lanes 4, H130C CcmEsol-C-His6 reacted with
reduced heme for 14 h followed by cleavage of the His tag.
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FIG. 5. Absorption spectra of holo-H130C CcmEsol produced in
vitro, showing the oxidized spectrum (solid line) and the reduced
spectrum obtained immediately after the addition of disodium dithionite
(dashed line). 4 µM protein samples in 50
mM Tris-HCl buffer, pH 7.4, 300 mM NaCl were used.
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DISCUSSION
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Effect of the His TagThe failure of heme to bind covalently
to the H130A mutant of CcmEsol establishes that the covalent
binding of heme observed with the wild-type H130 and H130C proteins involves
the specific participation of the imidazole or thiol groups of histidine or
cysteine at this residue position. It is reasonable to assume that the initial
noncovalent binding of ferric heme to any of the three proteins, i.e.
H130, H130C, and H130A, positions the heme appropriately for the subsequent
uncatalyzed covalent bond formation with the former two proteins. This is an
important result in the context that the His tag might itself contribute to
the noncovalent binding site for heme. Recently, Arnesano et al.
(9) have argued that untagged
CcmE from S. putrefaciens does not bind heme and that a His tag can
be responsible for introducing a heme-binding site into this and other
proteins. In the present work we show that the untagged CcmEsol
protein from E. coli retains a heme-binding site at which a covalent
bond forms between His 130 and a vinyl group in vitro under the same
reductive conditions as described before
(13). In this respect our
results are at variance with those of Arnesano et al.
(9). However, we have noticed
that the presence of the His tag affects the visible absorption spectrum of
the initial ferric noncovalent CcmEsol-heme complexes, consistent
with the heme iron having a histidine ligand provided by one of the residues
of the tag. In this sense the His tag appears to facilitate the in
vitro covalent incorporation of heme into CcmEsol. A His tag
at either the N terminus (present work) or at the C terminus
(13) appears to act similarly
in this respect. However, in the present work we show that the heme is bound
covalently to CcmEsol after the addition of ferric heme to
CcmEsol-C-His6 followed by incubation under reductive
conditions and thrombin cleavage. Furthermore, CcmEsol, which has
had the N-terminal His tag removed by thrombin treatment, can also bind heme
both covalently and noncovalently. Thus, we conclude that the presence of a
His tag at either end of the protein does not alter the nature of the covalent
bond formation. Any facilitation of the interaction of heme with
CcmEsol by the His tag may be a coincidental partial mimicking of
the histidine-rich region on the CcmC protein that is generally agreed to
participate in the binding of heme to CcmE in vivo
(5).
It is difficult to explain the differences with the results with the
protein from Shewanella that imply that CcmE alone cannot bind heme,
a view that is argued to be supported by inspection of the structure of the
apoprotein which does not have a classic hydrophobic pocket for binding heme
(9). On the other hand, Enggist
et al. (10), having
determined essentially the same structure for the apo-CcmE from E.
coli as Arnesano et al.
(9), have modeled a
heme-binding site onto a hydrophobic patch on the surface of the protein. We
assume that this patch provides the previously described ANS-binding site
(13), which the present work
establishes is also present in CcmEsol that lacks a His tag.
Judging from the fluorescence emission maximum at 480 nm, this ANS site is not
as hydrophobic as can be found in some proteins, consistent with ANS binding
in the relatively exposed heme-binding site advocated by Enggist et
al. (10). Our previous
observations that heme can displace ANS from CcmEsol support this
view. Indeed it would be surprising if the presence of a His tag did promote
the binding of ANS, given that this probe has a preference for hydrophobic
sites and that histidine is a hydrophilic amino acid. However, it cannot be
excluded that a C-terminal His tag helps stabilize the C terminus of the
protein. The effect of the His tag on the kinetic acceleration of the covalent
bond formation between heme and CcmEsol in vitro suggests
that either the supplied ligand field and stabilization of the noncovalent
heme-protein complex by the His tag is vital or that the His tag may even
provide an acid-base catalytic effect on the heme-His130 bond
formation.
Heme Binding to H130C CcmEsolOur previous in
vitro studies have shown that covalent bond formation can occur
spontaneously between the histidine of the heme chaperone
CcmEsol-C-His6 and ferrous heme
(13). Upon the addition of
ferric heme, the apoprotein binds heme noncovalently with a high affinity, and
the covalent bond forms only when the heme is reduced. To investigate this
process further, we have performed similar studies on this protein with the
heme-binding histidine mutated to a cysteine. Surprisingly, we found that the
covalent bond still formed, both in vitro and in vivo,
although in the latter case to a lesser extent compared with the wild-type
protein. The unusual similarity between the heme binding process occurring
with the wild type and the H130C mutant leads to interesting mechanistic
proposals. During revision of the present manuscript, an in vivo
study was published in agreement with our findings showing that a His-tagged
H130C mutant of CcmE can similarly (as judged by several criteria) bind heme
covalently on this cysteine, albeit to a low level
(22).
Implications for the Roles of Other Ccm ProteinsThe results
presented here also provide some insight into the role of the protein CcmC,
which has been shown to bind heme and to present CcmE with heme in the
periplasm (5,
7). The fact that the
covalently attached form of the H130C mutant is produced in vivo in
the presence of the other Ccm proteins to a considerably lesser extent than
the wild-type protein, in contrast to the comparable yields in vitro,
suggests that CcmC is not effective in catalyzing the attachment of the heme
to the cysteine compared with the attachment to histidine. This lower
efficiency may indicate that enzymatic action specifically requires the
heme-binding histidine residue on the apo-CcmE protein. Ligation effects to
the heme iron within the ligand field in the proximity of the heme chaperone
might render the vinyl group more reactive to histidine rather than cysteine
residues. The observation that the H130C mutant can readily form covalent
dimers via a disulfide bridge between the cysteines at position 130 suggests
that residue 130 is relatively accessible in CcmE, which is in agreement with
the published structure for this protein
(10). Because the heme is
bound to the histidine residue in this position of the wild-type protein, it
is expected that the heme is also accessible, which would allow the
apocytochrome c to take up the heme from the heme chaperone.
Coordination by a functional group from either the Ccm proteins or the
solution leading to a weak ligand field would also enhance the heme transfer
upon ligation by the apocytochrome c inducing a strong ligand
field.
Mechanistic ImplicationsThe successful attachment of the
heme to cysteine at residue 130, both in vivo and in vitro,
provides some mechanistic insight into heme attachment to CcmE. The in
vitro reaction appears to resemble the uncatalyzed formation from
polypeptide and heme of a c-type cytochrome, or its derivatives
carrying just one cysteine in the heme-binding motif that was recently
reported (21). The pyridine
hemochrome spectra reported here suggest that CcmEsol H130C is
similar to the known c-type cytochromes with a single thioether bond.
At this stage we have been unable to determine the axial ligands for either
the oxidized or reduced heme-protein. In vitro formation of thioether
bonds of a c-type cytochrome presumably requires protonation of the
-carbon of the vinyl groups of heme and formation of the thioether bond
between the cysteine residues and the
-carbon of the vinyl groups of
heme (23). A mechanism for
this process is envisaged to involve attack of the thiol moiety of the
cysteine residues on the
-carbon. For thioether bond formation to
occur, it was shown experimentally that ferrous heme is required
(23). In our work, we have
shown that the addition of either the histidine
(13) or the cysteine residue
to the heme requires ferrous heme. Given the similarities of the in
vitro heme binding of either histidine or cysteine variant of
CcmEsol, it is likely that they occur by a similar mechanism.
Therefore, the mechanism of histidine-heme attachment is suggested to be
analogous to the thioether bond formation in c-type cytochromes. The
histidine residue of wild-type CcmE could add onto the
-carbon of one
of the vinyl groups. Recently a covalent histidine-heme bond of this nature
has been reported for an unusual form of hemoglobin, which is formed in
vitro under reductive conditions
(24). If the heme attachment
to CcmE occurs with a specific vinyl group and heme can bind stereoselectively
to CcmE in one orientation, relative to the
,
meso axis of the
heme moiety, stereospecificity of the heme transfer reaction to apocytochrome
c would be achieved. The proposed process of heme binding and heme
release from CcmE is summarized in Fig.
6. However, a radical mechanism as proposed for in vitro
thioether bond formation between heme and cysteine
(25) cannot be excluded on the
basis of the current experimental data. The release of the heme from the heme
chaperone has similarities with a synthetic reaction yielding dipyridyl
sulfides whereby a quaternary pyridinium moiety acts as a leaving group upon
nucleophilic attack of a thiol functionality
(26).

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|
FIG. 6. The proposed chemical process of heme binding to CcmE and heme transfer
to apocytochrome c. P denotes the protein moiety of the
apocytochrome c, and R abbreviates a leaving group stable as
a cation species. In vitro this is presumably a proton.
Alternatively, the nucleophilic attack from the apocytochrome is conducted by
a thiolate group. The identity of the imidazole N atom participating
in CcmE function is not known; N has been chosen for
illustration.
|
|
Additional studies will be required to further describe the processes
involved during heme transfer, especially with respect to which vinyl moiety
reacts with CcmE and, therefore, the exact nature of the histidine-heme bond.
It will also be interesting to find out how the proposed disulfide
intermediate with the cysteine thiols of the apocytochrome c during
cytochrome c maturation
(27) will affect the heme
transfer reaction. However, this work contributes to the understanding of the
molecular basis of a central step in cytochrome c biogenesis.
 |
FOOTNOTES
|
---|
* This work was supported by Grants C11888
[GenBank]
and C13443
[GenBank]
from the Biotechnology
and Biological Sciences Research Council (to S. J. F.) and other funding from
the BBSRC (to J. M. S. and C. W. H.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. 
These authors contributed equally to this work. 
Recipient of a University of Oxford scholarship in association with St.
Edmund Hall, Oxford. 
¶
To whom correspondence should be addressed. Tel.: 44-1865-275240; Fax:
44-1865-275259; E-mail:
stuart.ferguson{at}bioch.ox.ac.uk.
1 The abbreviations used are: ES-MS, electrospray ionization mass
spectrometry; ANS, 8-anilino-1-naphthalenesulfonate. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Kevin M. Smith for comments on the reaction mechanism and Paul
Barker for helpful discussion and acknowledge Matthew Ellington, Richard
Zajicek, and James Allen for assistance and advice.
 |
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