From the Department of Molecular Biology, the Scripps
Research Institute, La Jolla, California 92037, § Protein
Chemistry Laboratory and Department of Biochemistry and Molecular
Biology, University of New Mexico, Albuquerque, New Mexico 87131-5221, the ¶ Department of Chemistry, University of Rochester, Rochester,
New York 14627, and the
Department of Biology, University of
California at San Diego, La Jolla, California 92093
Received for publication, September 14, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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Cytochrome rC557 is an
improperly matured, dimeric cytochrome c obtained from
expression of the "signal peptide-lacking" Thermus thermophilus cycA gene in the cytoplasm of Escherichia
coli. It is characterized by its Q00 (or Cytochrome c biosynthesis in Gram-negative, prokaryotic
cells occurs from the cycA structural gene that normally
encodes a preapoprotein having a short N-terminal signal peptide to
direct the protein to the periplasm. The process occurs largely in the periplasm, where its completion requires a host of cytochrome c maturation factors
(Ccm)1 (see Refs. 1-4 for a
review). Interestingly, two reports describe expression in
Escherichia coli of truncated cycA genes (those lacking code for the signal peptide) from thermophilic organisms, Hydrogenobacter thermophilus (5) and T. thermophilus (6), to obtain significant amounts of cytochrome
c-like material. Because truncated cycA genes
from mesophilic organisms are generally expressed quite inefficiently
in E. coli (see Ref. 6 for a review), it was suggested that
thermophilic apoproteins fold properly in the cytoplasm, thereby
permitting spontaneous heme insertion and thioether formation (7). In
the case of the truncated cycA gene from Thermus,
it was evident that the resulting product was actually a complex
mixture of cytochrome c-like material, which, when examined carefully by a number of spectral techniques, was found to differ significantly from the native protein (Ref. 6 and see below).
From spontaneous maturation of "signal peptide-deficient"
Thermus CycA in the cytoplasm of E. coli, two
major fractions are obtained, both of which are flawed. The majority
product, cytochrome rC552, is an ensemble of
molecules, some of which have the chemical potential, in a yet unknown
form, to spontaneously convert to a novel cytochrome pigment, p572,
which, in its reduced form, has optical absorption bands at ~430 and
572 nm (cf. Ref. 8 for a brief description of p572). The
minority product, cytochrome rC557, representing
~30% of the cytochrome, is a dimer having its Q00 band
(also called the Here we describe the nature of cytochrome rC557
and offer speculation on the mechanism of its in vivo
formation. This protein was originally thought to be an improperly
folded cytochrome c in which both cysteine thiols had
reacted with the porphyrin, histidine rather than methionine was bound
in the sixth coordination position, and the dimer was stabilized by
noncovalent interactions. At the time, this idea accounted for the
unusual spectral features of the as-isolated protein, its normal heme C
pyridine hemochrome spectrum and the absence of both Cys-11 and Cys-14
during standard N-terminal sequencing (6). The current results support
a different model.
Bacterial Strains and Plasmids--
E. coli strain
DH10B from Life Technologies, Inc. (genotype: F- mcrA
Molecular Genetics--
Procedures were carried out generally as
described by Sambrook et al. (11). Oligonucleotides were
purchased from Life Technologies, Inc. The polymerase chain reaction
was performed using ready-to-go polymerase chain reaction beads
(Amersham Pharmacia Biotech). The polymerase chain reaction products
were purified using Qiagen purification kits to remove the mineral oil
and excess primer. The QiaExII kit was used for isolating restriction
fragments from agarose gels. DNA sequencing was done at the UCSD
Molecular Pathology Shared Resource in the UCSD Cancer Center.
Construction of the cycA Gene Encoding Cys-11 to
Ala-11--
Mutation of the cycA gene to encode for alanine
rather than cysteine at position 11 (mature, native numbering) was
carried out using the Stratagene (San Diego CA)
QuikChangeTM site-directed mutagenesis kit. This procedure
requires two polymerase chain reaction primers, both having the desired
mutation. Plasmids pRC552 and pRSC552 were isolated from E. coli DH10B. The sense primer is
5'-GATCTACGCCCAGGCCGCGGGGTGCC-3', and the antisense primer is 5'-GGCACCCCGCGGCCTGGGCGTAGATC-3' (the
Cys-11 to Ala-11 mutation is underlined). The manufacturer's
instructions were followed to introduce the mutation into both
plasmids, and the mutation was confirmed by DNA sequencing. We denote
these plasmids pRC552(C11A) and pRSC552(C11A).
Protein Expression and Purification--
E. coli
cells were grown, and cytochrome rC557 was
isolated as described by Keightley et al. (6), except that
residual rC552 was removed by treatment with
hydroxyapatite.2 As predicted
from the work of Sambongi et al. (12), cytochrome rsC552(C11A) was not synthesized in E. coli cells bearing plasmids pRSC552(C11A) and pEC86, probably
because of obligatory formation of a Cys-11-Cys-14 disulfide bond
during cytochrome synthesis (cf. Ref. 12). However,
cytochrome rC552 (C11A) was synthesized in good
quantity by E. coli cells bearing the plasmid pRC552(C11A), presumably by a spontaneous route that occurs in the cytoplasm. The
resulting cytochrome rC552 (C11A) was purified
by the same procedure as described for rsC552
(6). Optical absorption spectra of cytochrome solutions were recorded
using a SLM/AMINCO model DB3500 spectrophotometer. The standard,
recombinant cytochrome rsC552 has a reduced
minus oxidized extinction coefficient, Chemical Methods--
N-terminal amino acid sequencing was
carried out at the Protein Chemistry Laboratory (Department of
Biochemistry and Molecular Biology, University of New Mexico) as
described (15). Reduction and alkylation of the cysteine residues in
rC557 was carried out by dissolving 8 nmol of
the protein into 100 µl of 6 M guanidine hydrochloride
containing 10 µmol of dithiothreitol in Tris buffer, pH 8.5 (16).
This solution was incubated at 37 °C for 20 h under a blanket
of nitrogen. The protein thus reduced was reacted with 20 µmol of
iodoacetamide for 20 min at room temperature, after which the pH of the
solution was adjusted to pH 2-3 with trifluoroacetic acid, and the
protein was recovered by passage through a Vydac C-4 reversed phase
column. As a positive control, 8 nmol of
Hydrazine hydrate was used to selectively reduce heme vinyl to ethyl
groups as described by Fischer and Gibian (18). The cytochromes were
treated as follows. 100 µl of ~20 µM cytochrome c, 400 µl of 95% hydrazine hydrate was diluted to 900 µl with 100 mM Tris-HCl buffer at pH 8. This solution was
heated at 90 °C for 5 min and cooled to room temperature before its
optical absorption spectrum was recorded; hydrazine also reduces the
iron of the heme.
High Resolution NMR Spectra--
Protein samples for
1H NMR spectroscopy consisted of 1.0-2.0 mM
protein in 100 mM potassium phosphate, pH 7.0, in 90%
H2O, 10% D2O or in 99% D2O.
Samples were oxidized with a 3-fold molar excess of potassium
ferricyanide and concentrated using a Centricon-10 device prior to data
collection. 1H NMR spectra were collected on a 500-MHz
Varian INOVA instrument. One-dimensional NMR spectra were collected
with recycle times of 250 ms, 350 ms, or 2 s, a sweep width of
40,000 or 50,000 Hz, and presaturation to suppress the solvent resonance.
X-ray Crystallography--
Crystals of recombinant
Thermus cytochrome rC557 were
obtained against 42% MPEG 5K (w/w), 0.1 M
imidazole-malate buffer, pH 6.3, 0-50 mM NaCl, using ~12
mg/ml protein suspended in 25 mM Tris/HCl, pH 8.0, and a
drop size of 1 µl of protein, 1 µl of mother liquor. The crystals
belong to space group P21212, with cell
dimensions 98.72, 69.05, and 36.58 Å. Diffraction data to 3.0-Å
resolution were collected at two wavelengths, at the white line peak
(1.7389 Å) and at a remote high wavelength (1.4586 Å) at the Stanford
Synchrotron Radiation Laboratory, beamline 9.2. The data were processed
using MOSFLM (19), scaled, and further reduced using the CCP4 suite of
programs (20) (see Table I). Anomalous Patterson maps featured
two prominent peaks, attributable to two heme groups in the asymmetric
unit. Experimental phases were calculated using Xheavy (21) and
solvent-flattened and 2-fold averaged using the automask averaging
procedure of Ref. 22. Two copies of the previously determined
cytochrome c552 (PDB accession 1C52) were
positioned in the experimental electron density maps. A bijvoet
difference Fourier map made using the 1.7389-Å Bijvoet differences and
the experimental phases showed two large peaks at the positions of the
heme. The two copies were then subjected to rigid body minimization,
rebuilt using Xfit/XtalView (21), and further refined using CNS (23)
(cf. Table I). Atomic coordinates have been deposited with the Protein Data Bank (1FOC).
Chemical and Optical Absorption Studies--
Cytochrome
rC557's activity as an electron transfer
partner with cytochrome ba3 (6) was reported
earlier to be ~8% of native Thermus cytochrome
c552, but subsequent comparisons in this system indicate activity ranging from 0 to 5% (data not shown). Because the
current purification procedure2 removes contaminating
rC552, dimeric rC557 is
judged to be inactive, although some low level, nonspecific electron
transfer may occur.
Fig. 1A compares the optical
absorption and Fig. 1B compares the second derivative
absorption spectra of cytochromes rsC552 (top), rC552 (C11A)
(middle), and rC557
(bottom). Cytochrome rsC552 serves
here as the primary standard, because it was previously shown to
possess chemical, spectral, and structural properties virtually
identical to those of native Thermus cytochrome
c552 (8). Additionally, its reduced pyridine
hemochrome spectrum has the Q00 band at 551 nm, typical of
heme C, and is not affected by heating with hydrazine, indicating the
expected absence of vinyl groups (18). The Cys-11 to Ala mutant
cytochrome rC552(C11A) has not been described
previously.3 Its
Q00 band appears at 553 nm, as may be expected from a
monothioether c-type cytochrome (24-27), and it also
exhibits a red-shifted, reduced pyridine hemochrome spectrum having a
maximum at 553 nm. Upon heating with hydrazine, the Q00
band of rC552(C11A) is shifted to 551 nm,
indicating the presence of a free vinyl group. Both observations are
consistent with the fact that this mutant form of the cytochrome can
possess only one heme thioether linkage. Spectra of cytochrome
rC557 are the lower traces
in Fig. 1, A and B. It was reported previously
(6) that the Q00 peak of its pyridine hemochrome is at 551 nm (see "Discussion"). Upon hydrazine treatment, Q00 of
rC557 is shifted to 550 nm, indicating the
presence of at least one free vinyl group on each heme. Note in Fig.
1B that each of the recombinant cytochromes c
exhibits splitting of its Q00 band at room
temperature.4
The chemical evidence indicating a free vinyl group in
rC557 prompted us to search for a cysteine
residue having a free thiol group. Accordingly, cytochrome
rC557 was dissolved into 6 M
guanidine-HCl, treated with an excess of dithiothreitol under anaerobic
conditions, alkylated with iodoacetamide, and subjected to quantitative
amino acid analysis (see "Experimental Procedures"). In two
separate experiments, 0.8 and 1.04 mol of carboxymethyl-cysteine were
found per mol of heme C.
High Resolution NMR Studies--
NMR spectroscopy is a sensitive
tool with which to probe the environment of the heme in cytochromes.
For example, due to the presence of the S = 1/2,
low spin ferric ion, oxidized cytochromes c show a rich
spectrum of 1H NMR resonances shifted both downfield and
upfield of the diamagnetic region. Strongly upfield shifted
1H NMR resonances may also be observed in the
S = 0, low spin ferrous cytochromes, as a result of
ring current effects. Fig. 2 shows the
upfield shifted 1H NMR spectra of reduced
rsC552 (native structure),
rC552(C11A) and rC557.
Resonances in the region
While already indicated in the optical spectrum of
rC557, the paramagnetically shifted
1H NMR spectrum of oxidized rC557
reveals that the heme environment in this cytochrome is distinctly
different from that in native protein. Downfield shifted 1H
NMR spectra of oxidized rsC552,
rC552(C11A) and rC557 are
shown in Fig. 3. The heme methyl
resonances in these proteins (see Fig. 4
for numbering) were identified by analysis of one- and two-dimensional NOESY spectra, and were assigned for some of the protein
forms.5 The monomeric
proteins, rsC552 and
rC552(C11A), have downfield-shifted heme
3-CH3 and 8-CH3 resonances with shifts of
33-35 ppm, characteristic of c-type cytochromes with
His-Met ligands oriented approximately as shown in Fig. 4 (31, 32). As
expected, rsC552 (Fig. 3A) and
rC552(C11A) (Fig. 3B) also have
5-CH3 and 1-CH3 resonances between 10 and 20 ppm, with the shift of 5-CH3 greater than that of
1-CH3. In contrast, rC557 displays a
significantly perturbed 1H NMR spectrum (Fig.
3C), with heme methyl shifts of 39.4, 34.0, and 17.3 ppm
(the fourth methyl resonance has not been identified). Although
significantly broadened under these recording conditions, the presence
of only three well resolved, hyperfine-shifted three-intensity peaks
indicates that the two hemes in the rC557 dimer
are in similar environments. Efforts to assign these three-intensity
peaks, however, failed to yield reliable
information.6 The
difficulties in assigning the NMR resonances of
rC557 are attributed to conformational
heterogeneity exhibited by the nonnatively folded protein in solution
(see "Discussion").
Crystallographic Studies--
We determined the structure of
cytochrome rC557 to a resolution of 3.0 Å using
multiwavelength anomalous dispersion. The dimer of cytochrome
c protomers is formed by a disulfide bond between the Cys-11
residue of each cytochrome c. The overall structure is shown
in Fig. 5, which is a view down an
approximate 2-fold axis that relates the two halves of the dimer. This
is a noncrystallographic symmetry axis and does not apply to the entire
molecule because the structure in the region of the disulfide bond is
significantly distorted. Fig. 6 provides
a stereo view of electron density around one of the hemes that shows
the thioether linkage between the heme and Cys-14 and continuous
density connecting Cys-11 on one molecule with Cys-11 on the other
molecule of the dimer.
Close examination of the electron density accounting for the heme
cofactor provides evidence that the heme is inverted about its
Additional evidence for incorrect insertion of the heme in
rC557 can be found in global changes in the
molecule. Fig. 8 shows a partial view of cytochrome
rsC552 (red) overlaid on cytochrome rC557 (yellow). Cytochromes
rC557 and rsC552 were
aligned by fitting C
The mean B factor is 75 with a root mean square deviation of
28, which is consistent with the low resolution limit of the diffraction from the crystal and may be compared with the 1.2-Å structure of Than et al., where the mean B is 11.2 and has a
root mean square deviation of 12 (33). Subjective evaluation of the electron density maps indicates that the electron density is weak or
significantly smeared in the sequence regions 10-13, 20-23, 30-39,
74-82, and 94-118; in addition, there is no density for residues 92 and 93, and the C terminus is disordered. In the regions 74-82 and
94-118, the local B value is significantly above the global
average, whereas in the 1.2-Å structure, these regions do not have
significantly different B factors; examination of the
interface between the protomers offers some explanation for this (see below).
In addition to the disulfide, the interface between the two protomers
is made up of several hydrophobic residues: Ala-10, Cys-11 (as a
disulfide), Ala-12, Cys-14 (as a thioether), Ile-22, Ala-25, Phe-26,
Val-68, and Phe-72; the hemes are also in van der Waals contact. It is
estimated that ~10% of each protomer's surface is occluded, with
the area of the interface being 1330 Å2. The surface area
was computed using a 1.4-Å radius probe.
Returning to the higher than average B value in specific
regions of the molecule, the distortion in the 74-82 region most likely results from unfavorable interaction between Gln-74 across the
dimer interface with Cys-11 and Ala-12, while the distortion in the
94-118 region probably results from unfavorable interaction of Asp-77
across the interface with Ala-105. It is noteworthy that these regions
of higher than average B value are part of the C-terminal
"thermo helices" region (33) that is wrapped around the inner,
canonical cytochrome c structure. Residues 30-39 form a
helix that packs on one side of the heme, and smearing of its density
probably results from an unfavorable interaction of the helical unit
with the flipped, displaced heme.
Our observations describe in considerable detail one of the dead
end situations available to a cytochrome c molecule during unassisted assembly in the cytosol of E. coli. Knowledge of
how the spontaneous synthesis can go wrong may shed light on why the bewilderingly complex cytochrome c maturation process has evolved.
How Does Cytochrome rC557 Form?--
Studies with
noncovalently attached hemoproteins such as myoglobins (34),
hemoglobins (35), cytochrome b5 (36-38), and E. coli cytochrome b562 (39) revealed
what is called rotational isomerization of the heme. La Mar and
coworkers (37, 40) have obtained information about the relative
stability of the A and B isomers of recombinant rabbit liver cytochrome
b5 and their rate of interconversion. Thus, when
the apo form of cytochrome b5 is reconstituted
with heme, the A and B isomers form in ~1:1 ratio, and over a period
of hundreds of minutes, depending on pH, the A to B ratio increases to
~8:1. Recently, Banci et al. (38) and Arnesano et al. (41)
determined the solution structures of the oxidized and reduced forms of
the A and B conformers of recombinant rat liver cytochrome
b5. In these examples, the free energy
differences between the A and B forms are very small, on the order of 1 kBT.
A major distinction between heme binding in cytochromes
b5 and cytochromes c is that the
"hydrophobic end" of the heme penetrates the protein and the
propionate side chains are exposed to solvent in the b-type
cytochromes, whereas the opposite holds for heme binding in the
c-type cytochromes (see Fig. 4). For Thermus
cytochrome c552, in which the heme is present in
the A conformation, the carboxyl group of propionate 7 forms a salt
bridge with the side chain of Arg-125, while the carboxyl group of
propionate 6 is in hydrogen bonding distance to main chain NH and/or
oxygen of residues Gln-55, Gly-56, Asn-66, and Gly-67. Although the
geometry of these hydrogen bonds is poor in the current structural
model, this may partially reflect the relatively low resolution of the data and the B factors in the range 58-98 in these regions.
Because the "hydrophilic" portion of the heme is indistinguishable
in the two conformations (see Fig. 4), any energetic difference between the A and B isomers must result from different interactions between the
protein and porphyrin substituents at positions 1-4. These are likely
to be small, making it reasonable to suggest that, prior to thioether
bond formation, the protein accommodates A and B conformations of the
heme with similar affinity. Trapping the B conformation probably occurs
upon formation of the Cys-14/2-vinyl thioether bond, while Cys-11
subsequently forms a disulfide link with another
pre-rC557 molecule. Because these reactions are
occurring without intervention of cytochrome c maturation
factors, the final A to B ratio of ~3 most likely depends on relative
rates of thioether formation in the noncovalent A and B conformations.
Structural Perturbations Caused by Heme Inversion and Disulfide
Formation--
By whatever mechanism, once the heme is fixed in the B
conformation and the disulfide bond has formed, the molecule differs in
minor but significant ways from the native structure. Small structural
differences are distributed throughout the molecule, while the larger
ones are found in those regions affected by interactions across the
dimer interface, as noted from the comparison of local B
factors. Although we are unable to distinguish changes due to accommodation of the inverted heme within the heme pocket and those
induced by protein-protein interactions across the dimer interface, it
is likely that the protomer, as it is captured in the
rC557 dimer, is unable to achieve the global
free energy minimum represented by the native structure. One may
imagine this as a perturbation of the energy landscape near the energy
minimum of the folding funnel
(42).7 Our NMR data are
consistent with this interpretation (cf. Footnote 6).
Novel Spectral Features of Cytochrome rC557--
Now
that we know the structure of cytochrome rC557,
it is possible to provide some rationalization of its unique optical
absorption and hyperfine shifted 1H NMR spectra. The energy
and shape of the Q00 band contains information about the
structure and environment of the heme. This band is actually composed
of two normally degenerate xy polarized transitions, which
occur at different energies only when perturbations on the heme
significantly lower its normal D2h symmetry (43, 44). The
average energy of the Q00 transitions depends on the nature of the heme substituents, to a lesser extent on the axial ligands of
the iron, and to a much lesser extent on the dielectric environment. The energy separating the two Q00 transitions depends on a
variety of factors including distortion of the heme from planarity and the dispositions of charges around the heme pocket (cf.
Rasnik et al.8 for
additional references). Of interest here are the vinyl groups of the
heme and their ability to form thioether linkages with the protein. A
general rule of thumb in b- and c-type
cytochromes is that the presence of one vinyl group shifts the
absorption ~5 nm to the red, while two free vinyl groups shift the
absorption an additional ~5 nm (cf. Ref. 24). Accordingly,
Q00 of a typical b-type cytochrome containing
Fe(II)-protoporphyrin IX occurs around 560 nm, at ~555 in a
monothioether cytochrome c, and close to 550 nm in a
dithioether cytochrome c. In naturally occurring (24) and
synthetic monothioether cytochromes c (26, 46), the
Q00 bands occur over the range 553-561 nm. Because the
Q00 band of cytochrome rC557 lies
within this range, why was the possibility of a monothioether
arrangement not suggested earlier? The principal reason was that
Q00 of its pyridine hemochromogen of
rC557 occurs at 551 nm (6), which is identical
to that of rsC552 and is diagnostic of a
dithioether hemochromogen. We speculate here that under the strongly
basic and reducing conditions required to form the pyridine
hemochromogen, the disulfide is reduced to yield a thiolate group that
reacts with the free vinyl to form dithioether heme. Thus, it is likely
that the one free vinyl group in "as-isolated" rC557 accounts for the bulk of the observed red
shift of its Q00 band.
Heme methyl 1H NMR shifts of c-type cytochromes
with ligands oriented as shown in the A conformation (Fig. 4) show
little variation across species, even for proteins with significantly
different folds (32). Thus, the differences between the pattern of
hyperfine shifts of rC557 compared with the
monomeric proteins indicates a significant change in heme electronic
structure. One possible explanation for this could be a change in the
heme axial ligands. However, as noted above, structurally similar
His-Met ligation occurs in both valence states of
rC557. Another possible explanation for the
perturbation of the rC557 NMR spectrum could be
the breakage of the thioether bond to the heme. However,
rC552 (C11A) necessarily has one vinyl in place
of a thioether, and its heme methyl shift pattern is similar to that of
rsC552 Other modifications of the porphyrin,
such as oxidation, also can be eliminated by examination of the optical
absorption spectrum of rC557 and its pyridine
hemochrome. The presence of a second, nearby heme group in
rC557 is not expected to significantly change
chemical shifts through dipolar effects because the shortest distance
between the iron of one heme and a proton on the other is >10 Å (47)
This leaves the possibility of the heme being oriented differently in
the rC557 protein compared with the wild-type
monomer. Rotation of the heme about its
According to recent theories explaining the paramagnetically shifted
1H NMR resonances, the magnitude of the shift of each
methyl group resonance is determined by the orientations of the filled
p-orbital (or sp3-orbital) on the
sulfur atom of the liganding methionine and the
As a substrate of cytochrome ba3, cytochrome
rC557 is essentially devoid of activity. The
likely reason for this is that the surface around the exposed heme
edge, which is most likely the binding interface with cytochrome
ba3, is largely occluded in dimer interface of
rC557. Cursory comparison of this surface with the analogous region of horse heart cytochrome c reveals
that this face of Thermus cytochrome
c552 is largely neutral, as already noted in
Ref. 33, whereas that of the horse heart cytochrome c has
several exposed positive residues in this same area. The latter
explains why horse heart cytochrome c is only a weakly active substrate for ba3 (45, 49).
-)
optical absorption band at 557 nm in the reduced form (Keightley,
J. A., Sanders, D., Todaro, T. R., Pastuszyn, A., and Fee,
J. A. (1998) J. Biol. Chem. 273, 12006-12016).
We report results of a broad ranging, biochemical and spectral
characterization of this protein that reveals the presence of a free
vinyl group on the porphyrin and a disulfide bond between the
protomers and supports His-Met ligation in both valence states of the
iron. A 3-Å resolution x-ray structure shows that, in comparison with
the native protein, the heme moiety is rotated 180° about its
,
-axis; cysteine 14 has formed a thioether bond with the 2-vinyl
of pyrrole ring I instead of the 4-vinyl of pyrrole ring II, as occurs
in the native protein; and a cysteine 11 from each protomer has formed
an intermolecular disulfide bond. Numerous, minor perturbations exist
within the structure of rC557 in comparison
with that of native protein, which result from heme inversion and
protein-protein interactions across the dimer interface. The unusual
spectral properties of rC557 are rationalized
in terms of this structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-band) at 557 nm (6). It has recently been shown
that these "errors" in c552 maturation can be avoided by coexpressing a mature, chimeric cycA gene
along with the ccmABCDEFGH genes on a separate plasmid. The
resulting recombinant cytochrome c552, denoted
rsC552, obtained in this manner has been
examined in functional and structural detail and is identical to native
cytochrome c552 except for having lost two amino
acid residues, Gln-Ala, from its N terminus (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(mrr- hsdRMS-
mcrBC) j80dlacZ
M15
lacX74
deoR recA1 endA1 araD139
(ara, leu)7697 galU
galK l- rpsL nupG) (9) was used for the
ligation and sequencing steps. E. coli strain BL21(DE3) from
Novagen (Madison, WI) (genotype: F- ompT
hsdSb (rB-
mB
) gal dcm (DE3)) (10) was used
for expression of the cytochrome c552 genes.
Plasmid pCYC552, carrying the truncated Thermus cycA gene,
was described by Keightley et al. (6), and plasmid pRSC552, bearing a chimeric cycA gene composed of code for an
N-terminal signal sequence and the mature cytochrome
c552, was described by Fee et al.
(8).
552 = 14.26 mM
1 cm
1
(8). Second derivative optical absorption spectra were obtained using
the vector manipulation program, IGOR (WaveMetrics, Lake Oswego, OR).
Pyridine hemochromes were prepared and quantified by the methods of
Berry and Trumpower (13). Electrospray mass spectrometry was carried
out at the Scripps Research Institute of Mass Spectrometry Facility (La
Jolla, CA) using a PerkinElmer Life Sciences SCIEX API III mass
analyzer with the orifice potential set at 100 V (14). Mass spectra
were obtained from ion spectra using the PerkinElmer Life Sciences
program for Macintosh, BioMultiView. Electron transfer activity with
cytochrome ba3 was carried out as described in
Ref. 8.
-lactalbumin were processed
in parallel. Amino acid analysis was performed using the PicoTag method
(17).
Crystallography data
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Visible absorption (A) and
negative second derivative (B) spectra of recombinant
T. thermophilus cytochromes
rsC552 (top),
rC552(C11A) (middle), and
rC557
(bottom).
2 to
3 ppm are diagnostic for
Fe(II)-S(Met) coordination in the reduced form of cytochromes, where
the single three-intensity peak near
3 ppm is attributed to
the axial Met
-CH3 (28, 29). Moreover, a weak optical absorption band attributed to a Met(S)-to-Fe(III) charge transfer occurs at ~690 nm (29, 30) in the oxidized form of the protein (data
not shown). These observations support His-Met axial ligation for
rC557 in both valence states and obviate the
previous suggestion of bis-histidine ligation (6).
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Fig. 2.
Upfield-shifted 1H NMR resonances
for reduced T. thermophilus cytochromes
rsC552 (A),
rC552(C11A) (B), and
rC557 (C). The
three-proton intensity peaks at 2.7 to
2.9 ppm indicate
methionine-sulfur coordination to Fe(II).
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Fig. 3.
Downfield-shifted 1H NMR
resonances for oxidized T. thermophilus cytochromes
rsC552 (A),
rC552(C11A) (B), and
rC557 (C). Assigned
heme methyls are indicated. Asterisks in B
indicate a minor component proposed to have an inverted heme.
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Fig. 4.
Schematic representation of heme orientation
and coordination geometry in rsC552
(A) and rC557
(B), looking down the Met-iron-His axis. The
axial Met side chain is indicated by balls and
sticks, and the axial His ring plane is indicated with a
heavy line.
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Fig. 5.
Stereo view of the overall fold of
recombinant T. thermophilus cytochrome
rC557, showing the approximate
noncrystallographic 2-fold axis relationship between the two
protomers in the asymmetric unit. The polypeptide chain is colored
from blue at the N terminus to red at the C
terminus. The heme, ligand residues Met-69 and His-15, and the S
atom of Cys-11 (yellow) are depicted as
ball and stick. The disulfide bridge between
Cys-11 on the two protomers is shown as a solid
line between the two sulfur atoms (yellow
balls) in the center of the
figure.
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Fig. 6.
Stereo view of a portion of the final
A weighted electron density map
obtained from crystals of recombinant T. thermophilus
cytochrome rC557 contoured at 1
(blue) and 5
(red). This side-on view of the heme in one
of the protomers shows the two axially liganding amino acids Met-69 and
His-15, the thio ether linkage of Cys-14 to the heme (see Fig.
4B), the chain tracing from Cys-14 to Cys-11, and the
electron density arising from the disulfide bond to the Cys-11 of the
adjacent protomer (in gray). Sulfur atoms are in
light blue, and nitrogen atoms are in
dark blue. Note the close proximity of the
disulfide sulfur atom to the heme.
-
axis (see Fig. 4). Flipping the heme in such a manner also changes the
relative positions of the methyl and vinyl groups on the I and II rings
of the heme. Further evidence for this comes from the fact that the
density for Cys-14 clearly shows it linking to what would have been the
methyl position if the heme were not flipped, which is chemically
unreasonable. Flipped over, however, the bond forms to the new vinyl
position. This can be seen in Fig. 7,
which provides a stereo view of heme electron density in
rC557 into which an inverted heme is fitted
(yellow model) and overlaid with the model of the
heme as it is found in cytochrome rsC552
(red model). Compared with
rsC552, the heme in rC557
is shifted "up" about 1 Å toward the propionates in Fig.
8. Cys-14 has formed a thioether linkage
in both molecules, while the C
of Cys-11 has moved
~4.5 Å from its position in rsC552; note
S
shown as the isolated yellow ball in
Fig. 7. The fit of the inverted heme (yellow
model) into the electron density from
rC557 is good, whereas both the Cys-11 and
Cys-14 thioether linkages observed in rsC552
fall outside the rC557 electron density
(red). By rotating the heme about its
-
axis, the free
vinyl group of heme ring II in rC557 fits nicely
into the rC557 electron density. Further, the
thioether linkage between the vinyl group on ring II and Cys-14 in
rsC552 (red) clearly falls outside
the electron density of rC557. These
observations indicate that the heme is indeed rotated 180° between
rC557 and rsC552
structures. Overlays of the heme axial ligand regions of
rC557 with the corresponding parts of rsC552 reveal that neither the bond lengths nor
the orientations of the imidazole ring and
CH2-S-CH3 planes of the histidine and methionine ligands, respectively, differ in the two molecules (not
shown). Thus, the chirality of methionine ligation is preserved in the
two molecules and corresponds to that found in mitochondrial cytochromes c (cf. Ref. 30).
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Fig. 7.
Stereo view of a portion of the final
A weighted electron density map
(blue) obtained from recombinant T. thermophilus cytochrome
rC557. The inverted
rC557 heme in yellow is fitted
into its electron density and compared with the heme position and
orientation in cytochrome rsC552 in
red. This view shows that the rC557
heme is moved slightly away from its position in
rsC552, that the Cys-14 in
rC557 suffers a relatively small distortion in
making the thioether bond with 2-vinyl and that Cys-11 moves away from
the heme in rC557 (see also Fig. 8). The view is
down the Met(S)-iron-N(His) axis as shown in Fig. 4. The electron
density is contoured at 1.3
.
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Fig. 8.
Stereo view of the backbone tracings
(C ) and hemes of
rsC552 (Protein Data Bank accession 1DT1)
in red and monomeric rC557 in
yellow; Met-69 and His-15 residues are also
shown. Sulfur atoms of Cys-11, Cys-14, and Met-69 are
light blue balls; the nitrogen atoms
of the His-15 rings are dark blue
balls; and the propionate oxygen atoms are
solid red balls. The two sulfur atoms
on the right of the rC557 model
correspond to the disulfide bridge between the Cys-11 residues from the
two protomers. The two molecules were superimposed using the best fit
of C
atoms between residues 60 and 131 (see
"Results"). The black arrow points to
a cleft between the domains "above" and "below" the hemes that
is opened slightly in rC557.
atoms in the C-terminal two-thirds
of the molecule. The two molecules are well matched in the region of
residues 60-131, root mean square difference of 0.39 Å, whereas in
the region of residues 4-59, the structures deviate significantly with
a root mean square difference of 1.13 Å. It can be seen that the
position of the sulfur atom of the axial ligand Met-69 is similar in
the two structures but that the heme iron in
rC557 has moved about 1 Å to the
left in Fig. 8, appearing to have pivoted about the sulfur
atom of Met-69. This movement is accompanied by a slight opening of a
cleft in the molecule denoted by the arrow in the Fig. 8.
While numerous small differences exist between the
rsC552 and rC557
structures, the only residues in the rC557
monomer that have greatly different positions relative to
rsC552 are Ala-12 and Cys-11. This can be
understood if the disulfide bond between the protomers of
rC557 serves to pull this region of the molecule out of the conformation it would take in
rsC552.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-meso axis leads to
significant perturbation of the heme methyl shift pattern (Ref. 48; see
Refs. 34-41). Interestingly, we observe resonances attributed to a
minor form of rC552(C11A) with similar shifts as
rC557 (resonances marked with
asterisks in Fig. 2B); these may arise from a
minority conformer in which the heme is flipped in this mutant.
-bonding
p-orbital of the
-N atom of the liganding imidazole ring
(31, 32). The average value of these angles in both
rsC552 and rC557 is
~95-100°. From Fig. 6 of Shokhirev and Walker (32), the predicted
order of the methyl shifts is C3 > C8 > C5 > C1. This order corresponds to our
assignments in rsC552 (see Fig. 3A).
Further, rotating the heme about its
,
-axis should not change the
orientation of the magnetic axes with respect to the protein; however,
the positions of the methyl groups with respect to the magnetic axes
will change, and a different order of methyl group shifts is predicted.
Unfortunately, probably because of microheterogeneity in the
rC557 structure, we have not been able to assign
the three-intensity lines shown in Fig. 3C.6
Given the great similarity of the axial ligand orientations in rsC552 and rC557,
assigning the methyl resonances in rC557 would provide a valuable test of the theory, but these must await further experimentation.
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ACKNOWLEDGEMENTS |
---|
J. A. F. thanks Drs. Shelagh Ferguson-Miller and Denise Miller of Michigan State University for valuable discussions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM35342 (to J. A. F.) and GM48495 (to D. E. M.). Portions of this work are based upon research conducted at the Stanford Synchrotron Radiation Laboratory, which is funded by the Department of Energy, Office of Basic Energy Sciences. The Biotechnology Program is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program and the Department of Energy, Office of Biological and Environmental Research.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.
The atomic coordinates and the structure factors (code 1FOC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** To whom correspondence and reprint requests should be addressed. Tel.: 858-534-4424; Fax: 858-534-0936; E-mail: jfee@ucsd.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008421200
2 We acknowledge the contribution of Dr. Denise A. Mills, Michigan State University, who discovered that rC557 is differentially adsorbed to hydroxyapatite, allowing its complete separation from rC552; details will be published elsewhere.
3 Cytochrome rC552(C11A) was pure by electrospray mass spectrometry exhibiting 8+ and 9+ ions at 1855 m/z and 1648 m/z, respectively, which correspond to the expected molecular mass of 14,289 ± 2 Da (617 Da from heme plus 14,213 from apo protein having a Cys to Ala mutation).
4 This splitting becomes more pronounced at cryogenic temperatures (J. Van der Kooi, unpublished observations).
5 K. M. Patel and K. L. Bren, unpublished results.
6 Two-dimensional NOESY spectra of cytochrome rC557 have been collected at 30 and 50 °C. A small number of weak cross-peaks involving heme resonances are observed at 30 °C, but the large line width of the 39.4-ppm resonance (attributed to conformational exchange) precludes detection of NOESY cross peaks to it. While resonance line widths decrease dramatically at 50 °C, NOESY spectra collected at 50 °C fail to reveal any resolved NOESY cross-peaks.
7 The discussion of cytochrome c folding on in Ref. 42 is immediately relevant to our observations regarding a protein molecule containing an inverted heme.
8 Rasnik, I., Sharp, K. A., Fee, J. A., and Vanderkooi, J. M. (2000) J. Phys. Chem., in press.
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ABBREVIATIONS |
---|
The abbreviations used are: Ccm and ccm, cytochrome c maturation proteins and genes, respectively; rC552, major species of recombinant cytochrome c obtained from the expression of the truncated Thermus cycA gene in E. coli; rC557, dimeric species of cytochrome c obtained from the expression of the truncated Thermus cycA gene in E. coli; rsC552, recombinant cytochrome c obtained from the expression of the chimeric T. versutus/T. thermophilus cycA gene in E. coli cells also bearing plasmid pEC86; NOESY, nuclear Overhauser effect spectroscopy.
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