From the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
Received for publication, October 30, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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C-type cytochromes are
characterized by having the heme moiety covalently attached via
thioether bonds between the heme vinyl groups and the thiols of
conserved cysteine residues of the polypeptide chain. Previously, we
have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c552 (Daltrop,
O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). In this work we report that thioether bonds can
form spontaneously in vitro between heme and the
apocytochromes c from horse heart and Paracoccus
denitrificans via b-type cytochrome intermediates.
Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each
have an affinity for a hydrophobic ligand. Furthermore, for both
apocytochromes c an intramolecular disulfide can form
between the cysteines of the CXXCH motif that is
characteristic of c-type cytochromes. In vitro
reaction of these apocytochromes c with heme to yield
holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for
cytochrome c maturation in vivo in various organisms.
C-type cytochromes are found in almost all
organisms, being mainly involved in electron transport (1, 2). They
contain a characteristic CXXCH motif, whereby the conserved
cysteine residues are involved in forming a covalent thioether bond
between the thiol functionalities and the vinyl groups of the
prosthetic heme moiety (3). Remarkably, three different systems have
evolved to facilitate cytochrome c formation in
vivo (4, 5). In some eukaryotes, one enzyme, designated heme
lyase, catalyzes the attachment of the heme moiety to the apocytochrome
c (6). These enzymes have a high specificity and do not act
on bacterial apocytochromes c (7). Mammalian organisms make
use of this system as exemplified by horse (Equus caballus)
heart mitochondria.
Many Gram-negative bacteria use a system that involves more than ten
gene products to ensure correct cytochrome c maturation (ccm)1 in the periplasm (5,
8, 9). An example of a cytochrome c that is matured in
vivo using this biosynthesis apparatus is cytochrome
c550 from P. denitrificans (10).
Following our report of the in vitro formation of
holocytochrome c552 from H. thermophilus (11), an important point to be established was
whether the spontaneous attachment of heme to apocytochrome
c is a reaction applicable to other cytochromes c. Because holocytochrome c552 from
H. thermophilus forms exceptionally in the cytoplasm of
Escherichia coli (12), its spontaneous in vitro
formation might not necessarily be anticipated to apply generally to
all c-type cytochromes. The previously reported thioether bond formation in holocytochrome c552 from of
H. thermophilus involved a b-type cytochrome
intermediate (11). Prompted by the previously reported similar ability
of the horse heart apocytochrome to bind heme to yield a species
characteristic of a b-type cytochrome (13), the reaction of
horse heart apocytochrome c with heme has been reinvestigated.
The formation of c-type cytochromes is especially
interesting in light of the different biosynthetic pathways, which are
reviewed extensively elsewhere (4, 5, 9), by which heme is attached to
the apoproteins in vivo. Therefore, a second candidate for studying the reaction of heme with apocytochrome was P. denitrificans cytochrome c550. The two
cytochromes used in this study are matured by two different cytochrome
c maturation systems in vivo. A comparison of the
reactions with respect to the attachment mechanism is anticipated to
have important implications for the molecular basis of heme attachment
in different organisms in vivo.
Protein Production--
Horse heart cytochrome c was
purchased form Sigma, and P. denitrificans c550
was produced as described by Richter et al. (14). Apocytochromes c were prepared analogously to the method of
Fisher et al. (15). After the removal of the Ag+
by addition of dithiothreitol (DTT), the proteins were dialyzed extensively in sodium phosphate buffer (pH 7.0, 20 mM).
Reduced apocytochrome was obtained by dialysis in deoxygenated sodium acetate buffer (pH 5.0, 25 mM).
Protein Characterization--
SDS-PAGE analysis was carried out
using the buffer system described by Laemmli (16), and heme activity
staining was performed according to the method of Goodhew et
al. (17) including non-covalent heme extraction with acidified
acetone. Pre-stained protein marker (New England Biolabs, Beverly,
MA) was used when heme staining was performed. Ellman's reagent
was used according to Riddles et al. (18). Thiol
modification with 4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate (AMS) (Molecular Probes Inc., Leiden, Netherlands) (19) was carried out
by incubating either reduced or oxidized apoprotein (20 µM) in freshly prepared AMS solution (15 mM
AMS, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% SDS) for 45 min at 37 °C with occasional
agitation. Buffer exchange to sodium phosphate buffer (50 mM, pH 7.0) was achieved with Centricon Centrifugal filter units (YM-10; Millipore, Bedford, MA) before analysis of the
incubated proteins. Electrospray ionization mass spectra were recorded
on a Micromass Bio-Q II-ZS triple quadrupole atmospheric pressure mass
spectrometer equipped with an electrospray interface. Samples (10 µl)
were introduced into the electrospray source via a loop injector as a
solution (20 pmol µl Spectroscopic Techniques--
Visible absorption spectra were
recorded on a PerkinElmer Life Sciences Lambda 2 spectrophotometer
using 5 µM protein in 50 mM sodium phosphate
buffer, pH 7.0. The concentrations of holocytochromes were determined
using the extinction coefficients 29.5 mM In Vitro Thioether Bond Formation--
Reconstitution of the
cytochromes was achieved by the addition of apoprotein to (typically
2-5 µM) heme (or mesoheme) in sodium phosphate buffer
(pH 7.0 unless otherwise stated, 50 mM) at 25 °C.
Fe-porphyrins were reduced with disodium dithionite. Apoprotein was
kept reduced by the addition of 5 mM DTT. The presence of oxygen was avoided by thoroughly sparging all solutions with humidified argon. Reactions were carried out in the dark. Free heme and aggregated protein was removed by passing the solution through PD-10 desalting columns (Amersham Biosciences).
Apocytochrome Production and Characterization
Horse Heart Cytochrome c--
Horse heart cytochrome c
was shown to be pure by SDS-PAGE analysis (Fig.
1, lanes a, left hand
panels) and had a mass of 12,359 (calculated 12,360) as
shown by ES-MS analysis. The in vitro produced apocytochrome was shown to be devoid of heme as judged by SDS-PAGE analysis followed by heme staining procedures (Fig. 1, lanes
b, left hand panels), the absence of cytochrome
characteristics in the visible spectra, the disappearance of the
cytochrome c fold as shown by CD analysis (Fig.
2) and ES-MS analysis, which resulted in
a mass of 11,746 (calculated 11,742 and 11,744 for the oxidized and
reduced protein masses, respectively). It is noteworthy that addition
of oxygen to the apoprotein occurred, as judged by the increase of a
mass peak in the ES-MS analysis at 11,760, if the reaction time for the
heme removal was not kept to a maximum of 2 h.
When the apoprotein was dialyzed extensively overnight, the cysteines
within the CXXCH motif (CAQCH for equine cytochrome c) formed a disulfide as shown by Ellman's reagent (18),
which accounted for 0.1 equivalents of thiol per mole of protein
instead of the 2 equivalents that were detected in the starting
material. This disulfide is shown to be internal because the ES-MS
analysis gave the monomeric protein mass and the polypeptide migrates
corresponding to the monomeric weight of the protein in the SDS-PAGE
analysis under non-reducing conditions (Fig. 1, lanes b,
left hand panels). Further evidence to show the presence of
a disulfide was obtained from thiol-labeling studies of reduced and
oxidized apocytochrome with AMS. Oxidized apoprotein was incapable of
reacting with the alkylating agent, whereas the reduced apoprotein was
labeled as shown by SDS-PAGE (Fig.
3A); there was a heavier
protein band for modified polypeptide (Fig. 3A, lane
2). AMS-treated oxidized apoprotein (Fig. 3A,
lane 1) migrated at a weight corresponding to non-reacted
oxidized or reduced apocytochrome (lanes 3 and 4,
respectively). In contrast to the apocytochrome of H. thermophilus cytochrome c552 (11), no
difference was observed between the CD spectra of oxidized and reduced
apocytochrome.
The affinity of the apocytochrome for the hydrophobic ligand ANS was
measured as described under "Experimental Procedures," and the
binding isotherms showed one transition corresponding to a dissociation
constant of 60 (± 20) µM for a single binding site on
oxidized (containing the disulfide) protein. The absence of the
disulfide did not alter the ANS affinity substantially (Kd: 70 (± 20) µM for reduced protein
in the presence of 2 mM DTT). Interestingly, the protein
fluorescence emission maximum was around 355 nm, indicating that the
tryptophan is exposed to the aqueous environment. This observation
suggests that the apoprotein is largely unfolded (25), which is in
agreement with the CD data (Fig. 2). It was shown that the original
holocytochrome neither bound ANS nor gave rise to fluorescence features
of tryptophan residue(s) exposed to a polar environment. The ANS
fluorescence in the presence of apocytochrome had a maximum around 475 nm, with excitation at 380 nm, indicating that the ANS moiety was in a
relatively hydrophobic environment (23). Addition of ANS to either
holoprotein or buffer gave rise to an ANS fluorescence maximum around
515 nm, which is in agreement with ANS being exposed to the aqueous
environment and not bound to a site on the holoprotein (23). The
structure of the protein, which shows that the tryptophan is in an
hydrophobic environment close to the heme (26), is consistent with the
observation of the tryptophan being in a non-polar environment in the
holocytochrome as judged by the protein fluorescence experiments.
It was shown that ANS, which is known to bind to the heme pocket in
apomyoglobin (23), was displaced from the protein upon addition of just
over one equivalent of heme per equivalent of protein. This suggests
that the hydrophobic ligand ANS binds to the same site as heme.
P. denitrificans c550--
This was shown to be pure
(some runs as a polymeric form) by SDS-PAGE analysis (Fig. 1,
lanes a, right hand panels), and ES-MS analysis
resulted in a mass of 15,028 (calculated 15,028). The prepared
apocytochrome was shown to be devoid of heme as determined by using the
same techniques described above for horse heart apocytochrome. SDS-PAGE
analysis (Fig. 1, lanes b, right hand panels) and
CD analysis (Fig. 2) of the obtained apocytochrome are shown. To the
best of our knowledge there has not been a previous report of the
preparation and characterization of the apoform of a mono-heme c-type cytochrome from a mesophilic bacterium. The ES-MS
data showed a mass of 14,409 (calculated 14,410 and 14,412 for oxidized and reduced cysteines, respectively).
Extensive dialysis overnight of the apoprotein resulted in the
formation of a disulfide between the cysteines within the
CXXCH motif (CKACH for P. denitrificans
cytochrome c550) as shown by Ellman's reagent
(18), which accounted for zero equivalents of thiol per mol of protein
compared with two equivalents in the initially produced apoprotein. The
monomeric state of the protein shown by ES-MS and SDS-PAGE analyses
under non-reducing conditions (Fig. 1, lanes b, right
hand panels) show this disulfide to be internal. AMS labeling was
also performed with reduced and oxidized protein. Apoprotein containing
a disulfide was unable to bind any label, whereas reduced apocytochrome
reacted with two AMS moieties as shown by ES-MS and SDS-PAGE analyses
(Fig. 3B). The oxidized protein incubated in AMS solution
had a mass of 14,410, indicating that no alkylation had taken place.
Reduced apocytochrome had a mass peak at 15,420, which is interpreted
as the covalent binding of two AMS molecules (0.5 kDa) and one sodium
adduct. The increased mass also becomes apparent on the SDS-PAGE
analysis (Fig. 3B, lane 2), relative to
unreactive (Fig. 3B, lane 1) and non-reacted
protein (Fig. 3B, lanes 3 and 4).
An ANS dissociation constant of 55 (± 15) µM for
oxidized apoprotein was obtained by fluorescence experiments, proving
that the apoprotein has a pronounced affinity for hydrophobic ligands. The absence of the disulfide did not affect the ANS affinity
(Kd: 55 (± 20) µM). The exposure of
the tryptophan residues and the overall fold of the apocytochrome as
judged by CD spectroscopy (Fig. 2) are analogous to horse heart
apocytochrome and suggest a largely unfolded structure. Holoprotein was
incapable of binding ANS. It was shown that heme could displace ANS
from apocytochrome, indicating the competition for the same binding
site of both hydrophobic molecules.
Reaction of Apocytochromes with Heme
Horse Heart Cytochrome c--
Mixing reduced horse heart
apocytochrome c (no disulfide bond) with ferrous (Fe(II))
heme in the presence of 5 mM DTT at pH 7.0 resulted in an
increase in the absorption around 424 nm relative to that of heme
alone. The same trend was observed around 528 and 559 nm, which are
wavelengths characteristic of the presence of a
reduced b-type cytochrome
(Fig. 4A and Table
I). The pyridine hemochrome spectrum of
this mixture had its
Following nearly quantitative formation of the b-type
cytochrome from the mixture of horse heart apocytochrome c
and heme under reducing conditions, the maximum at 559 nm in the
absorption spectrum shifted to 550 nm with time (Table I). After
48 h, the visible spectrum of protein, following gel-filtration
chromatography to remove any unbound heme and protein aggregates,
showed features both of a b- and c-type
cytochrome in a ratio of ~40:60 (Fig. 4A). After 72 h, the visible spectrum contained broad peaks, but showed absorption
maxima very similar to the original holoprotein (Fig. 4B and
Table I). The pyridine hemochrome spectrum of the reduced protein after
gel-filtration chromatography had a resolved band at 549.5 nm (Table
I), characteristic of saturation of both vinyl groups of the heme (22).
CD spectra of the desalted, oxidized cytochrome c produced
in vitro after 72 h of incubation showed a mixture of
spectral features of holoprotein and apoprotein (Fig. 2). It was
deduced from the ratio of the Soret band relative to the absorption at
280 nm in the absorption spectrum of oxidized protein that 40% of the
apoprotein had formed thioether bonds to heme, and 60% of the protein
was present in the apoform. Heme is assumed to have dissociated from
60% of the initial b-type cytochrome complex and unbound
heme to have been removed upon gel-filtration chromatography. This was
calculated knowing that in the original (oxidized) holoprotein the
ratio of A280/A410 is 1:4. The reconstituted
cytochrome c prepared in the present work had a CD spectrum
that was almost identical to a spectrum that was simulated by adding
the spectra of apocytochrome and original, purchased holocytochrome in
the calculated ratio of 3:2 (Fig. 2). It was shown that CD spectra of
these two components are additive, i.e. the presence of
either form of the protein did not affect the fold or, therefore, the
CD spectrum of the other.
Consequently, it was concluded that 60% of the b-type
cytochrome intermediate was incapable of forming a c-type
cytochrome. Why this is the case is unclear. Possible reasons for this
difference in reactivity may arise from the orientation of the heme
relative to its rotation around the
A noteworthy point is that the CD spectrum of the cytochrome
c generated in vitro had a great similarity with
not only that of the previously reported b-type cytochrome
complex (13), but also an apocytochrome derivative, where the cysteines
had been reacted with a hydrophobic molecule (27). Hence a definite
conclusion whether the full holocytochrome c fold is gained
upon reaction of apocytochrome with heme cannot be reached. Separation
by addition of ammonium sulfate (28) was not achieved to a satisfactory extent with our putative mixture of reconstituted cytochrome
c and unreacted apoprotein.
The observations from the spectral analysis clearly indicate the
formation of a c-type cytochrome, an interpretation that was
substantiated further by heme staining of the proteins on SDS-PAGE gels
(Fig. 1, lanes d, left hand panels), including
treatment of the heme-containing protein with acidified-acetone (17); in these experiments, non-covalently bound heme dissociates from protein but covalently bound heme does not. The data show in
vitro formation of horse heart cytochrome c; the latter
has not been previously reported to form from the b-type intermediate.
An analogue of the b-type cytochrome intermediate formed
upon addition of reduced Fe-mesoporphyrin to reduced apocytochrome c. The product had visible absorption maxima at 549, 519 and
416 nm and did not heme stain on an SDS-PAGE gel (Fig. 1, lanes
c, left hand panels). Mesoporphyrin has ethyl groups in
the positions of the vinyl groups of protoporphyrin and therefore
cannot form thioether bonds with the polypeptide. There was no evidence
for any other type of covalent attachment of mesoporphyrin to apoprotein.
The requirement for both the heme and the apoprotein to be reduced for
holocytochrome c formation was substantiated. Oxidized apoprotein (containing a disulfide), when reacted with ferric heme, did
not give rise to characteristic visible spectra of a cytochrome.
P. denitrificans c550--
Similar to the observation
with horse heart cytochrome c, when reduced P. denitrificans apocytochrome c550 was
reacted with ferrous heme, spectral features of a b-type
cytochrome intermediate were observed (Fig.
5 and Table I). Over a time period of
several hours spectral bands appeared at 552, 523, and 417 nm. A
spectrum of the heme-protein solution after 5 h of incubation
under reductive conditions is shown (Fig. 5). SDS-PAGE analysis of the
reaction product showed that heme was covalently attached to the
protein (Fig. 1, lanes d, right hand panel).
Incubation of ferrous mesoheme with reduced apocytochrome did not
result in covalent attachment (Fig. 1, lanes c, right
hand panel), suggesting that in the reaction with heme thioether
bonds had formed between the protein and the heme vinyl groups. This
interpretation was substantiated by the pyridine hemochrome spectrum of
the reaction product, which had an In this work we show the data obtained from the production of
apocytochromes c from a bacterial and a mammalian organism
and characterize the reaction of the cysteine thiols with the vinyl moieties of heme. Both proteins have very similar properties with regard to their apoforms and their reaction with heme. We show that the
cysteines of the conserved CXXCH motif can form an internal disulfide. This observation raises the question if this oxidation occurs in vivo for the respective proteins in either
organism. For the bacterial system it has been suggested that the
disulfide is an important intermediate during cytochrome c
maturation due to the presence of the disulfide bond forming (Dsb)
proteins in the periplasm, the location of cytochrome c
maturation (2). Furthermore, it has been proposed that proteins (CcmG/H
and DsbD) required for cytochrome c biogenesis are involved
in the reduction of the internal disulfide bond in the apocytochrome
(9). Why this in situ reduction of an apocytochrome has
evolved is unclear, but an internal protection mechanism against
metallation of the coordination site of the reduced apocytochrome might
offer an explanation.
Heme attachment to mitochondrial apocytochrome c occurs in
the intermembrane space (29). The in vivo oxidation state of the cysteines in mitochondrial apocytochromes c has not been
investigated to the best of our knowledge. Whether, as we show here for
the first time in vitro, a disulfide bond occurs during the
in vivo protein maturation process, will depend on both the
reduction potential of its locus and kinetic factors. If formation of a disulfide must be avoided, a thioredoxin-like protein could be required
in the intermembrane space (30). A thioredoxin (Trx3) and its reductase
(Trr2) are known to be present in yeast mitochondria, but
submitochondrial localization to the matrix is suspected rather than to
the intermembrane space (31). These proteins can in any case be deduced
to be dispensable for cytochrome c biogenesis in yeast, as
mutants carrying specific disruptions in the corresponding genes were
able to grow normally under aerobic, respiratory conditions, which
require participation of cytochrome c in the mitochondrial respiratory chain. However, in the case of Arabidopsis
thaliana, it has been recognized, on the basis of the genome
sequence, that there may be a yet to be characterized thioredoxin that
is targeted to the intermembrane space (30). Alternatively, the
formation of holocytochrome c may happen sufficiently fast
that the two thiols of apocytochrome c cannot form a
disulfide during its lifetime in the intermembrane space following
delivery from the reducing environment of the cytoplasm. Such a kinetic
constraint would suffice if the rate of disulfide bond formation in the
intermembrane space were to occur on a similar timescale to that
in vitro (hours as observed in the present work). The
presence of the CcmABCEF components of one cytochrome c
biogenesis pathway, but not of the thioredoxin-like CcmG component, in
plant mitochondria might support the latter view.
The binding of heme to the apoform of mitochondrial cytochrome
c clearly involves some ordering of the polypeptide chain. This is evident from several biophysical studies (13, 28). A
non-covalent complex between heme and apoprotein of H. thermophilus cytochrome c552 generates a protein
structure that is very similar to that of the native protein with
covalently bound heme (32). Although it appears that the structure of
the non-covalent complex between heme and mitochondrial apocytochrome
c does not fully resemble the native, holo structure (13,
28), studies with two monoclonal antibodies that scarcely recognize the
apoprotein, but bind with comparable affinities to both the
non-covalent heme-apoprotein complex and the holoprotein, indicate that
the complex must be similar to the holoprotein in respect of quite
specific structural features at the epitope regions (28). Such
similarities are evidently sufficient to permit the vinyl groups of
heme to be oriented appropriately, in at least a fraction of the
molecules, for reaction with the thiol groups, which have previously
been shown to exhibit nucleophilic reactivity (27). The effect of covalently bound hydrophobic moieties on the folding of cytochrome c has also been discussed (27).
The affinity of the apoproteins for hydrophobic ligands and the ability
of both apoproteins to ligate heme in a b-type cytochrome complex has important implications for the catalytic strategy of the
enzymes involved during cytochrome c maturation. It is unclear from previous studies how the heme lyase functions in mitochondria, except for the proposal that it binds heme via a conserved CPX motif (6). For the bacterial Ccm system, two proteins
have been proposed to function as a heme lyase (33), but the heme
attachment is more complex due to the presence of a heme chaperone,
CcmE (34). However, in view of our data showing that the
b-type cytochrome can spontaneously react to give thioether bond formation yielding holocytochrome c, two key conditions
have to be achieved by the catalytic systems. Firstly, the heme group has to be kept reduced to give optimal reactivity for cysteine-thiol and heme-vinyl group reaction partners. This is in agreement with previous studies (35, 36). And secondly, the orientation of the heme
relative to its rotation around the In the in vitro reaction, the oxidation state of the heme
can be controlled, but the heme delivery is nonspecific with respect to
the stereochemical aspects of the heme orientation. The
stereochemistry for the in vitro produced cytochromes
c is not determined and will be focus of future work.
However, the fact that a fraction of the mitochondrial
b-type cytochrome intermediate remained unreactive might
hint that incorrect orientation of heme does not lead to thioether bond
formation, suggesting the in vitro cytochrome
c formation to be stereoselective. In the case of the
in vitro formation of H. thermophilus
c552 the reaction is stereoselective (11).
An interesting point arises from the observation that neither P. denitrificans c550 (10) nor mitochondrial cytochrome
c, in the absence of a heme lyase (38, 39), can form in the
cytoplasm of E. coli, in contrast to H. thermophilus
c552 (12). These observations might reflect the
stability and the folding state of the apoproteins, but not the
intrinsic difference in their reactivity toward heme, which
is shown from previous (11) and present work to be similar for the
three apocytochromes c. The higher stability and partial
folding (24) of H. thermophilus c552
apocytochrome may lead to a slower rate of degradation in the cytoplasm
of E. coli and a higher heme affinity compared with the two
proteins studied in this work. Therefore, in addition to the aspects
mentioned above, another function of the heme lyase may be to increase
the heme affinity of the apocytochrome c.
In summary, we have shown that two c-type cytochromes, from
a mesophilic bacterium and a mammalian mitochondrion, can form in
vitro from the reaction of apocytochrome and heme. Remarkably, this reaction of horse heart cytochrome has not been reported despite
first having been attempted nearly 30 years ago (15). The reaction
generally seems to proceed from a b-type cytochrome intermediate, provided the potential disulfide between the conserved cysteine residues is avoided and the heme-iron is reduced. Indeed, we
suspect that had Dumont et al. (13) extended their duration, and/or modified the reaction condition for instance by exclusion of
oxygen, of incubation of heme with apocytochrome c, they too might have
seen covalent bond formation to heme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in 1:1 water/acetonitrile, 1%
formic acid) at a flow rate of 10 µl min
1.
1
cm
1 and 30 mM
1
cm
1 at 550 nm in the visible spectrum for reduced equine
and bacterial cytochromes c, respectively (20, 21). For the
apocytochromes (horse heart and P. denitrificans) extinction
coefficients of 10.9 mM
1 cm
1
and 15.3 mM
1 cm
1 (calculated
using the protein sequence) at 280 nm were used, respectively. The
absorption spectra of reduced forms of heme in the presence of
hydroxide and pyridine are characteristic of the type of Fe-porphyrin
present, as well as of any modifications to it (e.g.
covalent attachment to the polypeptide in a c-type cytochrome); such pyridine hemochrome spectra were obtained according to the method of Bartsch (22). Far UV circular dichroism (CD) spectra
were recorded on a Jasco J720 spectropolarimeter. Fluorescence measurements were made using a PerkinElmer Life Sciences 50B
fluorimeter with excitation at 295 nm and emission from 300-400 nm
(slit widths 5 nm). The dissociation constant for ANS
(8-anilino-1-naphthalenesulfonate (Sigma), 1 mM stock in 50 mM sodium phosphate buffer, pH 7.0) was determined by
measuring the quenching of the intrinsic protein fluorescence with
increasing ANS concentrations and the relative fluorescence of free and
bound ANS using standard double-reciprocal and Rosenthal plot analysis.
The displacement of ANS by heme was measured by the decrease in ANS
fluorescence at 480 nm upon addition of aliquots of heme to a mixture
of protein and ANS with excitation at 380 nm (23, 24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (71K):
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Fig. 1.
SDS/15% PAGE of various forms of horse heart
cytochrome c and P. denitrificans cytochrome
c550. A, activity stained for
covalently bound heme followed by Coomassie Blue staining
(B). In vivo-produced holocytochrome
(a), apocytochrome (b), mesoheme-containing
cytochrome (c), and in vitro-produced
holocytochrome (d). M is pre-stained protein
marker corresponding nominally to 16.5, 25, 32.5, and 47.5 kDa from
bottom to top, respectively. 200-400 pmols of
protein were applied to each lane of the gel.
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Fig. 2.
Circular dichroism spectra of various forms
of horse heart cytochrome c (A) and
P. denitrificans c550
(B). Oxidized holocytochrome c ( )
and apocytochrome c (- - -) are presented for proteins
from both organisms. For horse heart cytochrome the reaction product of
apocytochrome and heme is shown (
·
·
)
and compared with the simulated CD spectrum obtained by adding the
spectra of holoprotein and apocytochrome in the ratio 2:3
(·····), which overlaps with the
former spectrum for most of the spectral range. [
]mrw
is mean molar ellipticity per residue. Spectra were recorded using 20 µM protein in 20 mM sodium phosphate buffer,
pH 7.0.
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Fig. 3.
A and B,
SDS/17.5%-PAGE analysis of susceptibility of various forms of
apocytochromes c to modification of cysteine thiols by
AMS. A, corresponds to horse heart apocytochrome
c, and B shows analysis of P. denitrificans apocytochrome c. M' is ultra
low range molecular weight marker (Sigma) with the bands corresponding
to the weights as indicated. Lane 1, oxidized apocytochrome
incubated with AMS labeling solution; lane 2, reduced
apoprotein treated with AMS under identical conditions as the
polypeptide shown in lane 1; lane 3, oxidized
apoprotein; lane 4, reduced apocytochrome. The gels
were stained with Coomassie Blue, and 200-300 pmols of protein were
loaded to each gel.
-band at 556 nm (Table I), indicating that the
heme contained two unreacted vinyl groups (22). These results show that
the apoprotein and heme initially form a species that resembles a
b-type cytochrome, in which the heme is not covalently
attached to the peptide but in which its iron atom is coordinated by
two amino acid side chains from the protein. These results are
consistent with previous studies (13).
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Fig. 4.
A, visible absorption spectra of horse
heart apocytochrome c (5 µM) following mixing
with an equimolar amount of heme under reductive conditions. The
solid line shows a spectrum, corresponding to a
b-type cytochrome, obtained after 10 min after mixing. The
dashed line was obtained after 48 h of incubation.
B, visible spectra of horse heart cytochrome c (5 µM) produced either in vivo (dashed
line) or in vitro (solid line) after 72 h incubation under reductive conditions and subsequent
purification.
Absorption maxima of various forms of cytochrome species
,
meso-axis or the incorrect
fold of the apocytochrome around the prosthetic group.
-band at 552.4 nm, which,
together with the visible spectrum of reduced protein, shows that more
than half of the vinyl groups have been saturated. This data is
interpreted as reflecting a mixture of b- and
c-type cytochrome present in the reaction mixture. The
pyridine hemochrome spectrum of the reaction of mesoheme and apoprotein, which also yields a b-type cytochrome (Table I), remained unchanged from the pyridine hemochrome of mesoheme itself. Again, the reaction of cytochrome c formation from the
b-type intermediate was not quantitative. A similar ratio as
in the horse heart cytochrome c experiments described above
remained as b-type cytochrome. However, the fraction of
apocytochrome c that could form a b-type cytochrome in this
case was very low (5%), suggesting that the heme extraction procedure
had adversely affected the ability of the majority of the apoprotein to
form a hydrophobic pocket. However, it is clear from the data that
reaction of heme and P. denitrificans apocytochrome
c550 can lead to formation of holoprotein with
covalently attached heme in vitro.
View larger version (20K):
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Fig. 5.
Comparison of visible spectra of various
forms of P. denitrificans cytochrome (5 µM). The spectra of the
b-type cytochrome complex between apocytochrome c
and heme obtained after 10 min of incubation under reductive conditions
( ); the reaction product of heme and apocytochrome after 5 h
(·····); the in vivo
produced protein (- - -).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
meso-axis has to be correct
to yield the required stereochemistry at the prochiral
-carbon of
the vinyl substituents of the heme moiety (3). McRee et al.
(37) showed that a recombinant cytochrome c of Thermus thermophilus could be improperly matured in
vivo, but without any catalytic assistance in the cytoplasm of
E. coli, because of both heme inversion and an
intermolecular disulfide bond between cysteine residues, which are
usually involved in heme binding (37). The latter behavior was clearly
reflected in the visible absorption spectrum, which was different from
the reported, properly matured holocytochrome c, and the
pyridine hemochrome spectrum, which was not indicative of two thioether bonds to the heme-vinyl groups (37). For mitochondrial cytochrome c, two thioether bonds have formed in the present work. In
the case of in vitro reaction of heme with P. denitrificans apocytochrome c550 it is not
evident if the reaction yielded a mixture of only b- and
c-type cytochrome complexes or also contained species with only one thioether bond. Due to the presence of DTT during the course
of the reaction, no reaction product containing an intermolecular disulfide bond is obtained in vitro.
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ACKNOWLEDGEMENTS |
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We thank Julie Stevens, Mark Bushell, and James Allen for help and discussions. Christopher Higham and Carsten Richter are gratefully acknowledged for help with the production of P. denitrificans c550.
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FOOTNOTES |
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* This work was supported by Grants C11888 and C13443 from the UK Biotechnology and Biological Sciences Research Council (to S. J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Receiving a University of Oxford scholarship in
association with St. Edmund Hall including a W. R. Miller award.
§ A W. R. Miller fellow of St. Edmund Hall.
¶ To whom correspondence should be addressed. Tel.: 44-1865-275240; Fax: 44-1865-275259; E-mail: stuart.ferguson@bioch.ox.ac.uk.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211124200
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ABBREVIATIONS |
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The abbreviations used are: ccm, cytochrome c maturation; DTT, dithiothreitol; AMS, 4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate; ANS, 8-anilino-1-naphthalenesulfonate; CD, circular dichroism; ES-MS, electrospray mass spectrometry.
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REFERENCES |
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