From the Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan
Received for publication, January 30, 2001, and in revised form, February 22, 2001
From the School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa 923-1292, Japan
Received for publication, January 30, 2001, and in revised form, February 22, 2001
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ABSTRACT |
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CooA is a heme-containing
transcriptional activator that anaerobically binds to DNA at CO
atmosphere. To obtain information on the conformational transition of
CooA induced by CO binding to the heme, we assigned ring
current-shifted 1H NMR signals of CooA using two
mutants whose axial ligands of the heme were replaced. In the absence
of CO, the NMR spectral pattern of H77Y CooA, in which the axial
histidine (His77) was replaced with tyrosine, was similar
to that of wild-type CooA. In contrast, the spectra of CooA The photosynthetic bacterium Rhodospirillum rubrum has
a heme-based CO sensor, CooA, which activates the expression of
cooMKLXUH and cooFSCTJ operons in response to CO
(1-3). The gene products encoded in these operons constitute a CO
oxidizing system that enables the bacterium to utilize CO as the sole
energy source under CO atmosphere (4, 5). CooA belongs to a family of transcriptional activators that includes the cAMP receptor
protein and the fumarate-nitrate reductase activator protein
(1). The investigation on CooA is therefore important to understand the general activation mechanism of this family of transcriptional activators (1-4).
CooA is an ~50-kDa homodimeric protein, with each monomer
possessing a low spin heme in the ferrous state (6-8, 16). In the
absence of CO, CooA possesses the six-coordinated heme with two
endogenous ligands and is in the inactivated state that does not bind
to DNA (the CO-free state or the low affinity state). The CO-free state
denotes hereafter CooA with the ferrous heme in the absence of CO. In
the presence of CO, one of the axial ligands of the ferrous heme is
displaced with CO, which leads to the capability of CooA to bind to a
specific sequence of DNA (the CO-bound state or the high affinity
state) (7). Thus, the molecular mechanism of the conformational
transition triggered by the CO binding is of essential importance to
understand the function of CooA (7-9).
A significant advance in the understanding of the function of CooA has
been presented in the recent x-ray crystal structure (10). It is
confirmed that the overall fold of the protein is indeed similar to
cAMP receptor protein that forms dimeric structure interfaced by long
helixes (C-helix). The most prominent structural characteristic of CooA
is the ligation of the NH2-terminal Pro2 of one
subunit to the heme iron in the other
subunit.1 The other axial
ligand of the heme is confirmed to be His77 as has been
demonstrated in our previous studies (9, 11). It is further proposed
that the straight C-helix in the low affinity state becomes bent in the
high affinity state upon the CO binding. However, since the x-ray
structure of CooA is presented only for the CO-free state, the
molecular mechanism that triggers the proposed conformational
transition remains to be elucidated.
It was suggested from the resonance Raman spectroscopy that the axial
ligand of the CO-bound form is likely to be His77, and the
release of the other ligand, identified as Pro2 later,
triggers the conformational transition at the DNA binding domain (12).
However, Roberts and his co-workers (13, 14) proposed that CO displaces
His77 instead of Pro2 and the changes in the
His77 ligation triggers the DNA binding. While our recent
investigations using time-resolved resonance Raman and extended x-ray
absorption fine structure spectroscopies have unequivocally
indicated the ligation of His77 in the CO-bound CooA (12,
15), the site-directed mutagenesis of the axial ligands seems
inconsistent with the movement of Pro2 for the trigger of
the DNA binding activity. For example, the deletion of
NH2-terminal residues (CooA To characterize the structural changes induced by the CO binding to the
heme in CooA and clarify the significance of the release of the
NH2-terminal Pro2 ligand, we conducted
1H NMR investigation of CooA in the absence and presence of
CO. As we reported previously, reduced CooA provides
characteristic 1H NMR signals between Procedures for the construction of the expression systems, the
expression and purification for the axial mutants, were reported previously (6-8). NH2-terminal analysis for CooA Proton NMR experiments were performed at 25 °C on a 500-MHz
spectrometer (Brucker, AVANCE DRX500) equipped with an Indy work station (Silicon Graphics) (17). A 1331-pulse sequence was used to
minimize resonances inside the 0-10 ppm region (18). The proton shifts
were referenced to the residual water signal at 4.81 ppm at 25 °C,
which is calibrated against external tetramethylsilane. The wild-type
and mutant CooA proteins were dissolved in 50 mM sodium
phosphate buffers at pH 7.4 containing 10% D2O or at pD 7.4 containing 100% D2O. Protein concentrations were over
300 µM on the heme basis. CooA was converted to the
reduced form by an anaerobic addition of sodium dithionite solution to
a final concentration of about 2 mM. CO-bound CooA was
prepared by flowing CO over the reduced protein for 10 min with mild stirring.
Resonance Raman spectra were measured using the system and the method
reported previously (16).
Assignments of the 1H NMR Peaks for the CO-free
State--
Fig. 1A shows the
1H NMR spectrum for the CO-free state of CooA in
H2O, in which several characteristic and well resolved
resonances are observed in the upfield region (
While upfield shifted NMR signals have been observed for several heme
proteins, the spectral pattern of CooA is unique and indicates the
distinct coordination structure. For example, the ferrous state of
cytochrome c550 M100K mutant (19) indicates signals between
The coordination structure of CooA can consistently explain its NMR
spectrum. Because Pro2 and His77 are the axial
ligands of the CO-free heme (10, 15), the signals b,
d, and e are likely derived from the
nonexchangeable protons of the proline ring of Pro2 and/or
imidazole ring of His77. The nonexchangeable protons of
histidines bound to low spin ferrous hemes, however, are usually
observed at around 1~2 ppm (24). Therefore, given that the signals
b, d, and e originate from the protons
in the axial ligands, they are likely due to the protons from
Pro2. The x-ray structure of the CO-free CooA shows that
the distances from the heme iron to protons attached to
C
The above assignments are supported by the NMR spectra of the axial
ligand mutants, H77Y CooA and CooA
In contrast to CooA
While the assignments for the nonexchangeable signals are convincing,
those of the exchangeable protons are still ambiguous. Since signal
a is the most upfield shifted and is insensitive to the
substitution of His77, the signal should be a proton
located near the heme iron at the Pro2 side. A most
plausible candidate is the NH proton of Pro2. However, the
imino group of Pro2 might be deprotonated when it
coordinates to the heme iron. In addition, the chemical shift and
intensity of a were pH-independent between pH 6 and 11, although generally the pKa of the NH proton of Pro
is ~10.
The assignment of the signal c is also unclear. This signal
disappears by the mutation at His77 and likely belongs to a
residue located in the His77 side. Although
N Assignments of the 1H NMR Peaks for the CO-bound
State--
To obtain an insight into the activation mechanism of CooA,
we measured the NMR spectra of CO-bound wild-type and mutant CooA and
compared them with those in the absence of CO. As shown in Fig.
2A, the CO-bound form of
wild-type CooA shows no NMR signals from the nonexchangeable protons of
Pro2 in the region between
The assignment of Pro2 signals for the CO-free CooA and no
proton signals from the C CO-dependent Activation Mechanism of CooA--
While
the results presented above indicate the movement of Pro2
as the trigger for the activation of CooA, the activities of CooA
We will first examine the CooA
As depicted in Fig. 2, C and F, the CO-bound form
of CooA
We will next consider the H77Y mutant. Noticeably, both of the CO-free
and CO-bound forms of H77Y CooA provide the characteristic three
signals that are similar to those of wild-type CooA in the CO-free form
(Figs. 2B and 1A). For example, the CO-bound
state of H77Y CooA exhibits the triad of the nonexchangeable resonances at
We summarized the coordination structures of wild-type H77Y CooA and
CooAN5, in
which the NH2 termini including the other axial
ligand (Pro2) were deleted, were drastically modulated. We
assigned three signals of wild-type CooA at
4.5,
3.6, and
2.8 ppm
to
1-,
-, and
2-protons of
Pro2, respectively. The Pro2 signals were
undetectable in the upfield region of the spectrum of the CO-bound
state, which confirms that CO displaces Pro2.
Interestingly, the Pro2 signals were observed for CO-bound
H77Y CooA, implying that CO binds to the trans position of
Pro2 in H77Y CooA. The abolished CO-dependent
transcriptional activity of H77Y CooA is therefore the consequence of
Pro2 ligation. These observations are consistent with the
view that the movement of the NH2 terminus triggers
the conformational transition to the DNA binding form.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
N5) fully maintained the activity of CooA (15). Furthermore, the mutation of His77
(H77Y CooA) abolished the CO-induced transcriptional activity of CooA
even though the protein forms CO-bound heme (9, 11). Investigation on
the detailed coordination structures of the heme in these mutants is
necessary to clarify the functional significance of these axial ligands
and their functional differences.
1 and
6 ppm that
show drastic changes upon the CO binding (11). These
ring-current-shifted signals are originating from residues around heme
and therefore should provide us with the detailed information on the
conformational transition triggered by the CO binding (11). However,
due to the unprecedented coordination structure of the heme in CooA, the assignments of the upfield NMR signals have not been presented. We
therefore compared the NMR spectra of CooA in the absence and presence
of CO with those of the axial mutants to assign the upfield NMR
signals. We confirmed that the proximal ligand of the CO-bound CooA is
His77, as we have indicated in the previous studies (12).
Furthermore, the changes in the NMR spectra induced by the axial
mutations provide us with a simple and consistent picture on the
triggering event for the conformational transition of CooA.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
N5 was
carried out by using a protein sequencer (PerkinElmer Life Sciences,
PROCISE 491cLC). Cytochrome f (turnip) was
purchased from Sigma.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
6 to
1 ppm) (11).
These ring-current-shifted signals can be assigned to protons of the amino acid residues nearby the porphyrin ring. As shown in Fig. 1D, three of the signals (b, d, and
e) at
2.8,
3.6, and
4.5 ppm are nonexchangeable. Based
on the intensity of an isolated resonance at 10.3 ppm from the NH of a
single tryptophan at position 110 (spectrum not shown), the intensity
of each signal (b, d, and e)
corresponds to a single proton. Each of the exchangeable signals at
6.1 (a) and
4.0 (c) ppm is also estimated to
possess a single proton intensity.
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Fig. 1.
The upfield region of proton NMR spectra of
CO-free wild-type (WT) and mutant CooA proteins at
25 °C: WT in H2O (A), H77Y CooA
(H77Y) in H2O (B),
CooA N5 (
N5) in
H2O (C), WT in D2O
(D), H77Y CooA in D2O
(E), CooA
N5 in
D2O (F). The concentrations of WT,
H77Y CooA, and CooA
N5 are about 500, 400, and 300 µM
on the heme basis, respectively.
1 and
4 ppm that are assigned to protons of an
axial ligand, Lys100. However, the upfield shifted signals
of CooA were observed in the region below
4 ppm (
4.0,
4.5, and
6.1 ppm). In contrast, the ferrous cytochrome
f,2 in which the
NH2-terminal amino group of Tyr1 is coordinated
to heme (21-23), indicates four nonexchangeable signals at
1.6,
3.3,
6.8, and
8.2 ppm
(20)3. The x-ray structure of
cytochrome f (23) suggests that these signals can be
assigned to C
H of Tyr1,
C
1H of Tyr1, C
H
of Pro2, or C
1H of
Phe4, all of which are located within 5 Å from the heme
iron. While the signal pattern of cytochrome f is different
from that of CooA, the spectrum exemplifies that the coordination of
NH2-terminal amino group can shift signals below
4 ppm.
and C
of Pro2 are less than
4 Å (7) and suggests that signals b, d, and
e are derived from one methyne proton at C
and two methylene protons at the C
position of
Pro2. Furthermore, we detected a significant cross-peak
between the signals b and e in preliminary
two-dimensional COSY measurements (spectra not shown). We therefore
assigned signals b, d, and e to
C
1H,4
C
H, and C
2H of
Pro2, respectively.
N5. We constructed the CooA
N5
mutant, in which four residues,
Pro2-Pro-Arg-Phe5, are deleted from the amino
terminus.5 While the
optical absorption (15) and resonance Raman spectra (spectra not shown)
of CooA
N5 in the CO-free state are very similar to those of
WT6 CooA, the CooA
N5
mutant shows distinct NMR spectra from those of wild-type CooA as shown
in Fig. 1, C and F. Nonexchangeable proton
signals, signals b and d, are completely missing
in the mutant, confirming that these signals are derived from
Pro2.
N5, the spectral changes induced by the
substitution of His77 for Tyr (H77Y CooA) are less drastic,
as shown in Fig. 1, B and E. The spectra of H77Y
CooA7 are almost identical to
those of wild-type CooA, although an exchangeable proton signal at
4.0 ppm (c) disappears and the spectral pattern between
0.5 and
2.0 ppm is perturbed. The minor spectral changes support the
assignment that the signals b, d, and
e originate from the Pro2 side.
H of His77 might be a candidate for the
signal c, this is unlikely since the axial His NH proton of
cytochrome b5 possesses a signal at 1~2 ppm
(24). Another candidate for the signal c would be the
sulfhydryl proton of Cys75, which is one of the axial
ligands for the ferric heme and displaces from the ferrous heme (7, 9,
11, 25). The distance between the SH proton and the heme iron is less
than 4 Å in the CO-free CooA (10), which is consistent with the
prominent upfield shift. The NMR signals for most of sulfhydryl protons
in proteins, however, are not observed due to the rapid exchange with
solvent water (26). If the signal c is due to the SH proton
from Cys75, its exchange rate should be retarded, probably
by a poor accessibility of solvent waters.
2 and
6 ppm. The
exchangeable signal observed at
4.5 ppm is not affected by the
deletion of the NH2 terminus (Fig.
3, C and F).
Therefore, the proton giving the signal at
4.5 ppm should be located
in the His77 side and likely corresponds to the SH proton
of Cys75 that indicates signal c at
4.0 ppm in
the CO-free CooA (Fig. 1, B and E). The small
shift of the peak upon the CO binding might indicate that the
conformational transition of the His77 side is rather
small.
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Fig. 2.
The upfield region of proton NMR spectra of
CO-bound WT and mutant CooA proteins at 25 °C: WT in H2O
(A), H77Y CooA in H2O
(B), CooA N5 in
H2O (C), WT in D2O
(D), H77Y CooA in D2O
(E), CooA
N5 in
D2O (F). The concentrations of WT,
H77Y CooA, and CooA
N5 are about 500, 400, and 300 µM
on the heme basis, respectively.
View larger version (14K):
[in a new window]
Fig. 3.
Coordination structures of the ferrous heme
in CooA elucidated from the NMR and resonance Raman results: CO-free WT
(A), CO-bound WT (B), CO-free H77Y
CooA (C), CO-bound H77Y CooA (D),
CO-free CooA N5 (E), CO-bound
CooA
N5 (F). For H77Y
CooA, only the structures of the predominant species are
presented.7
and C
positions
of Pro2 in the upfield region of the spectrum of the
CO-bound form give us clear evidence that Pro2 displaces
from the heme iron in the CO-bound form of CooA. These results further
support our previous conclusion from the time-resolved resonance Raman
and extended x-ray absorption fine structure spectroscopies (12, 15).
Based on the decrease of the ring-current shift for the proton signals
from Pro2 upon CO binding, the ligand should be displaced
from the original location by at least 4 Å in the CO-bound form (10).
The large displacement of Pro2 induced by the CO binding
suggests the importance of the Pro2 side as the first event
of the conformational transition of CooA.
N5
and H77Y CooA might be interpreted inversely as suggesting the
importance of the His77 side. CooA
N5 fully maintains the
activity of CooA, while H77Y CooA completely abolishes the CO-induced
transcriptional activator activity (9, 11, 15). Examinations of
1H NMR spectra of these mutants in the CO-bound state,
however, give us a clear explanation on their activities based on the
movements of the Pro2 side.
N5 mutant that maintains almost the
same transcriptional activity as wild-type CooA (15). Because the
1H NMR spectra for the CO-free state of CooA
N5 do not
indicate the hyper-fine shifted signals that are characteristic of the five-coordinated ferrous hemes (spectra not shown), the ferrous heme of
CooA
N5 is in a low spin state with two axial ligands. We suggest
that the two ligands are His77 and NH2-terminal
amine, since no other ligands are available around the heme in the
x-ray structure of wild-type CooA (10). The ligation of the truncated
amino terminus is consistent with the 1H NMR
spectrum of CooA
N5 (Fig. 1, C and F) that
indicates nonexchangeable signals at
2.8,
1.4, and
0.8 ppm
assignable to protons of C
H and
-methylene of the
N-terminal Met5. Furthermore, the heme marker lines of the
resonance Raman spectrum appear at frequencies suggesting the
coordination of neutral nitrogen (spectrum not shown), since the
frequencies of
2,
3, and
4 for the CO-free state of CooA
N5 are 1579, 1491, and 1361 cm
1, respectively, which are similar to those
of cytochrome b5 at 1583, 1493, and 1361 cm
1, respectively (27). These results suggest
that the truncated NH2-terminal region is flexible enough
to coordinate to the heme iron.
N5 showed almost the same NMR spectra in the upfield region
as those of wild-type CooA. Furthermore, the resonance Raman spectrum
for the CO-bound CooA
N5 mutant can be superimposed on that of the wild-type CooA, including the heme marker lines and the Fe-C stretching line (spectrum not shown). The frequencies of
2,
3,
4, and
(Fe-C) in the spectrum of
CO-bound CooA
N5 are 1580, 1498, 1370 and 487 cm
1, respectively. Those of CO-bound
wild-type CooA are 1579, 1497, 1369, and 487 cm
1, respectively. These results indicate
that the CO-bound form of CooA
N5 retains the His77-Fe-CO
coordination structure and explain the activity of the mutant that is
similar to that of wild-type CooA (15).
2.6,
3.5, and
4.3 ppm corresponding to the signals for the CO-free state of wild-type CooA at
2.8,
3.6, and
4.5 ppm,
respectively (Fig. 1A). The appearance of the
Pro2 signals implies that CO binds to the trans position to
Pro2 in H77Y CooA, resulting in the formation of the
CO-Fe-Pro2 complex as depicted in Fig. 3D. Since
H77Y CooA is not activated by CO (9, 11), we conclude that the binding
position of CO to the heme iron is crucial for the CooA function.
N5 in the CO-free and CO-bound states in Fig. 3. The binding of
CO to the Pro2 side (Fig. 3, B and F)
can be a trigger to induce the conformational change to the high
affinity form, whereas the binding of CO to the His77 side
and the maintenance of the Pro2 ligation (Fig.
3D) cannot induce the conformational change for the
activation. These results provide us with a simple and consistent picture on the activation mechanism of CooA; the movement of the NH2-terminal loop upon the CO binding for more than 4 Å triggers the conformational transition of the whole protein to the high affinity form. As suggested in the x-ray results (10), the movement of
the NH2-terminal loop might modulate the C-helix, which
forms the dimer interface and locates near the heme, to assume the bent conformation.
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ACKNOWLEDGEMENTS |
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We thank H. Harada (Kyoto University) for his assistance in the NMR measurements. We are grateful to Prof. T. Kitagawa (Institute for Molecular Science) for the permission to use his Raman observation system and Prof. T. Imanaka and Prof. H. Atomi (Kyoto University) for their help in the protein sequence analysis.
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FOOTNOTES |
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* This work was supported by Grants-in-aid 08249102 (to I. M.) and 11116212, 12019222, and 12680631 (to S. A.) for Scientific Research on Priority Areas from Ministry of Education, Culture, Sports, Science, and Technology in Japan.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.
Supported by Research Fellowships of the Japan Society for
the Promotion of Science for Young Scientists.
§ To whom correspondence may be addressed: Tel.: 81-75-753-5921; Fax: 81-75-751-7611; E-mail: morisima@mds.moleng.kyoto-u.ac.jp (for I. M.) or Tel.: 81-761-51-1681; Fax: 81-761-51-1149; E-mail:aono@jaist.ac.jp (for S. A.).
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.C100047200
1 We confirmed that the NH2-terminal methionine (Met1) is removed after the expression of wild-type CooA in the host cell.
2
The reported NMR spectrum for ferrous cytochrome
f only showed the region up to 5 ppm (20). We re-examined
the NMR spectrum of ferrous cytochrome f and found two
additional signals at
6.8 and
8.2 ppm.
3 K. Yamamoto H. Ishikawa, H. Harada, K. Ishimori, and I. Morishima, unpublished results.
4
The -carbon of proline residue has two
protons. We differentiated one of the protons located near the heme
iron as C
1H and other proton as
C
2H.
5
The NH2-terminal analysis for the
CooAN5 mutant showed that methionine is still attached at the
NH2 terminus. Thus, the NH2-terminal residue of CooA
N5 is Met5.
7 The optical absorption and resonance Raman spectroscopies reveal that the CO-free state of H77Y CooA contains both of the six- and five-coordinated hemes, with the six-coordinated heme as the minor component (11, 13, 14). Although broad paramagnetic NMR signals were observed in the downfield region (data not shown), no paramagnetic signals from the five-coordinated heme were detected in the upfield region. The binding of CO to the five-coordinated heme in the CO-free state seems to form the six-coordinated heme, giving the signals shown in Fig. 2, B and E. Therefore, all signals in Figs. 1 and 2 are from the six-coordinated and low spin species of H77Y CooA.
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ABBREVIATIONS |
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The abbreviation used is: WT, wild-type.
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