From the School of Materials Science, Japan Advanced Institute of
Science and Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun,
Ishikawa 923-1292 and RIKEN Harima Institute/Spring-8,
1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan
Received for publication, May 10, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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The CO-sensing transcriptional activator CooA
contains a six-coordinate protoheme as a CO sensor.
Cys75 and His77 are assigned to the fifth
ligand of the ferric and ferrous hemes, respectively. In this study, we
carried out alanine-scanning mutagenesis and EXAFS analyses to
determine the coordination structure of the heme in CooA.
Pro2 is thought to be the sixth ligand of the ferric and
ferrous hemes in CooA, which is consistent with the crystal structure
of ferrous CooA (Lanzilotta, W. N., Schuller, D. J.,
Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and
Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880).
CooA exhibited anomalous redox chemistry, i.e. hysteresis
was observed in electrochemical redox titrations in which the observed
reduction and oxidation midpoint potentials were Hemeproteins are the most popular metalloproteins exhibiting a
wide variety of functions such as oxygen storage/transport, electron
transfer, and redox reactions of various substrates (1, 2). In addition
to these traditional heme proteins, a new class of heme proteins termed
the heme-based sensor proteins have been reported recently in which the
heme acts as a sensor of an effector molecule (3). There are five such
proteins known, soluble guanylate cyclase
(sGC)1 (4-13), FixL
(14-23), DOS (24), HemAT (25), and CooA (26-37). These proteins
contain a heme that acts as a sensing site of NO (sGC), O2
(FixL, DOS, and HemAT), and CO (CooA).
CooA from Rhodospirillum rubrum is a heme-based CO-sensing
transcriptional activator. Although the function of the heme in CooA is
as a sensor of a gaseous effector molecule, similar to other heme-based
sensor proteins, the coordination structure of the heme in CooA is
quite different from that in FixL and sGC. In the case of FixL and sGC,
the heme is five-coordinate with a histidine as a proximal ligand in
the resting state and binds the effector, O2 or NO, at the
distal side trans to the proximal histidine (4-23). On the
other hand, the heme in CooA is six-coordinate in the ferric, ferrous,
and CO-bound forms (26, 28, 29, 34-37). Therefore, CO must replace one
of the axial ligands in the ferrous heme when the heme in CooA binds
its effector, CO (28, 29, 34-36). The exchange of the axial ligand
upon CO binding is functionally relevant for CooA, as described below.
CO is a physiological effector regulating the activity of CooA;
i.e. only the CO-bound form of CooA can be bound to the
target DNA and is active as a transcriptional activator (27, 29, 31-35). Replacement of one of the axial ligands of the ferrous heme
with CO triggers the activation of CooA (28, 29, 34-36). The release
of the axial ligand from the heme upon binding CO causes conformational
changes around the heme and subsequently in the whole molecule (28, 29,
35). These conformational changes induced by the interchange of the
axial ligand are the principal part of the activation of CooA by CO.
Because the heme in CooA plays a central role in sensing CO and in
regulating the transcriptional activator activity of CooA, elucidation
of the coordination structure of the heme in CooA is required to
understand the mechanisms of CO sensing and of the activation of CooA
by CO. Mutagenesis studies, EPR, resonance Raman, and uv/vis
spectroscopies have revealed that the heme is in the six-coordinate and
low-spin state in ferric, ferrous, and CO-bound CooA, and that
Cys75 and His77 are the fifth ligand of the
ferric and ferrous hemes in CooA, respectively (28, 29, 35-38). These
results suggest the possibility that exchange of the axial ligand takes
place between Cys75 and His77 during the change
in the oxidation state of the heme in CooA (29, 35, 36).
Recently, Lanzilotta et al. (39) have reported the crystal
structure of ferrous CooA, which shows that the N-terminal
Pro2 and His77 are the axial ligands of the
ferrous heme in CooA and that the Pro2 of one subunit
provides one ligand to the heme of the other subunit in the CooA
homodimer (39). Although the crystal structure of ferrous CooA has been
reported, the sixth ligand of the ferric heme in CooA remains to be
elucidated. The proximal ligand of CO-bound CooA also remains
controversial. We have proposed that His77 is the proximal
ligand of the CO-bound heme in CooA (28, 29), whereas Vogel et
al. (34) and Dhawan et al. (38) have pointed out the
possibility that CO displaces His77 upon CO binding to the
ferrous heme in CooA.
We report herein the coordination structure of the heme in CooA
revealed by alanine-scanning mutagenesis and EXAFS analyses. In this
study, we also examined the redox properties of the heme in CooA by
means of electrochemical redox titrations.
Site-directed mutagenesis was carried out by using the
Quick-change site-directed mutagenesis kit (Stratagene). The expression and purification of the recombinant CooA were carried out as reported previously (26, 29). A Sephacryl S-100 (Amersham Pharmacia Biotech) gel
filtration column equilibrated with 50 mM Tris-HCl buffer,
pH 8.0 containing 100 mM NaCl was used for the final step of the purification. The purified CooA was concentrated to about 30 µM and about 2 mM by ultrafiltration with a
YM-10 membrane (Amicon, Inc.) for electrochemical redox titration and
EXAFS measurements, respectively.
The gene encoding CooA The PCR product containing the cooA The electrochemical experiments were made using an electrochemical cell
equipped with a quartz optical cell (40 (height) × 10 (width) × 1 mm (thickness)). The optical path length of the optical cell was 1 mm. A working electrode of gold mesh (40 × 9 × 0.7 mm) was immersed in the optical cell. A platinum wire and
Ag2+/AgCl (3 M KCl) electrodes (RE-1B, BSA)
were used as auxiliary and reference electrodes, respectively. The
potential was controlled by a potentiostat (HA-301, Hokuto Denko Co.).
The electronic absorption spectra were measured on a U-3300 Hitachi
uv/vis spectrophotometer.
For electrochemical redox titrations, the following redox mediator dyes
(2 µM each) were added to the sample solution (the values
of E1/2 (versus NHE) of the mediator dyes are
shown in parentheses): phenazine methosulfate (+80 mV, Ref. 40),
gallocyanine (+20 mV, Ref. 40), indigo trisulfonate ( EXAFS spectra at the iron K-edge were measured at liquid nitrogen
temperature using monochromatized synchrotron radiation at the BL 12C
of the Photon Factory in the National Laboratory for High Energy
Physics (Tsukuba, Japan). The EXAFS spectra were measured as
fluorescence excitation spectra using a bent cylinder type focusing
mirror and a silicon (111) double-crystal monochromator. The sample was
placed at an angle of 45 degrees against the incident x-ray beam, and
the fluorescent x-ray intensity perpendicular to the beam was measured
using a solid state 19-element detector (Canberra Industries, Inc.) The
analysis of the EXAFS data was performed using the program XFIT (43) as
previously reported (44-46). The k-windows used for the EXAFS analyses
are shown in figures. The multiple scattering from the outershell atoms
of the porphyrin ring and axial ligand molecules were taken into account. In constrained refinement, the number of parameters was reduced by treating a set of scattering atoms as a unit (47).
Mutagenesis of CooA
Alanine-scanning Mutagenesis--
Mutagenesis and spectroscopic
studies have revealed that the heme in CooA is six-coordinate in the
ferric, ferrous, and CO-bound forms (28-30, 34-36). To date, the
sixth ligand of the ferric and ferrous hemes in CooA is not known,
whereas Cys75 and His77 are assigned to be the
fifth ligand of the ferric and ferrous hemes, respectively (29, 35). We
have reported that His, Met, Cys, and Lys are not candidates for the
axial ligands of the heme in CooA, except for Cys75 and
His77 (29). To identify the unknown ligand of the heme in
CooA, we carried out alanine-scanning mutagenesis on all Arg, Asp, Glu, Asn, Gln, Ser, Thr, and Tyr residues in the heme-binding domain of CooA
in the present work. These side chains could potentially coordinate to
the heme iron, located in the N-terminal region from Met1
to Met131 (27). The mutant CooA proteins constructed in
this work are summarized in Table I. The
individual mutant proteins were partially purified by means of a
Q-Sepharose column and their uv/vis spectra were measured in the
ferric, ferrous, and CO-bound forms. All the mutants thus prepared
exhibited almost the same electronic absorption spectra as did
wild-type CooA (data not shown), indicating that no candidate for the
sixth ligand of the heme in CooA was found by alanine-scanning
mutagenesis.2 These results
suggest that side chains of any amino acid residues, except for
Cys75 and His77, do not coordinate to the heme
iron as an axial ligand in CooA.
Truncated Mutant Lacking Four Residues in the Amino-terminal of
CooA--
To elucidate whether or not the amino group of the
amino-terminal residue is coordinated to the heme in CooA, a truncated mutant (CooA
The transcriptional activator activity of CooA
The above results suggested that the first five residues from the amino
terminus are not responsible for the coordination of the heme in CooA.
However, this conclusion is now known to be incorrect. During the
course of the reviewing process of this paper, Lanzilotta et
al. (39) have reported the crystal structure of the reduced form
of CooA in which His77 and the nitrogen atom from
Pro2 are the axial ligands of the heme (39). Though
Pro2 is deleted in CooA EXAFS Analyses of CooA
To analyze the coordination atoms of the heme iron more directly,
we measured the iron K-edge EXAFS of CooA in the ferric, ferrous, and
CO-bound states. The k3-weighted EXAFS raw data and the
corresponding Fourier transforms are shown in Figs.
2 and 3,
respectively. The following models for the axial ligands of the heme
were used to fit the experimental data: i.e. Cys and Pro,
His and Pro, and His and CO were assumed to be the axial ligands of the
ferric, ferrous, and CO-bound hemes in CooA, respectively. The atomic
coordinates of ferrous CooA (39) were used as a starting model for
refinement of the fitting in the case of ferric and ferrous CooA. The
structural parameters obtained by the fitting are shown in Table
II.
320 mV and
260 mV,
respectively. The redox-controlled ligand exchange of the heme
between Cys75 and His77 is thought to cause the
difference between the reduction and oxidation midpoint potentials.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
N5 was synthesized by polymerase chain
reaction using pKK3CO5 (26) as a template and the following primers:
primer 1, 5'-AGGAGACTCGTATGAACATCGCCAATGTCCTGTTG-3' and primer 2, 5'-TCATTAATCGTCGTCGTCGTCGCGGTC-3'.
N5 gene was
cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen) to
construct pCR-CO
N5. An expression vector of CooA
N5, pKK-CO
N5,
was constructed by inserting the EcoRI fragment containing
the cooA
N5 gene, which was cut from
pCR-CO
N5, into the EcoRI site of pKK223-3 (Amersham Pharmacia Biotech). The activity of CooA
N5 was measured as reported previously (27, 29).
80 mV, Ref. 40),
2-hydroxy-1, 4-naphthoquinone (
120 mV, Ref. 41), anthraquinone
2-sulfonate (
230 mV, Ref. 40), benzyl viologen (
350 mV, Ref. 40),
methyl viologen (
440 mV, Ref. 42), and N,
N'-dimethyl-2,2'-bipyridinium hexafluorophosphate (
720 mV,
Ref. 42). CooA solution containing the mediator dyes was repeatedly
degassed and flushed with argon prior to the measurement and then
~2.5 ml of the sample solution was transferred into the electrochemical cell, which was sealed by a rubber septum, by using a
gas-tight syringe under argon atmosphere. The electrochemical cell was
kept in a thermoelectric cell holder of a U-3300 Hitachi uv/vis
spectrophotometer at 15 °C during the electrochemical titration. The
redox reaction of CooA was followed by recording the absorbance change
in the regions of the Soret and Q bands.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Mutant CooA proteins constructed in this work
N5), in which four residues were deleted from the amino-terminal of CooA, was constructed and its electronic absorption spectra and transcriptional activator activity were measured. The
amino-terminal amino acid sequences of wild-type and CooA
N5, which
were deduced from the DNA sequences, are
1MPPRFNIANV and 1MNIANV,
respectively. The four underlined residues in this sequence were
deleted in CooA
N5. CooA
N5 showed the Soret peak at 422, 424, and
422 nm in the ferric, ferrous, and CO-bound forms, respectively, as
shown in Fig. 1. The spectral features of
CooA
N5 were almost the same as the corresponding ones in the
wild-type though the ferric CooA
N5 showed a slightly blue-shifted
and broad Soret peak with a shoulder at 399 nm compared with wild-type.
The shoulder at 399 nm and a CT-band at 638 nm in the spectrum of
ferric CooA
N5 suggest that a high-spin form of the ferric heme
exists to some extent in ferric CooA
N5.
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Fig. 1.
Electronic absorption spectra of
CooA N5. CooA
N5 was dissolved in 50 mM Tris-HCl buffer, pH 8.0.
N5 was 13.2 and 0.29 units/mg of protein in the presence or absence of CO, respectively.
These values were comparable with those for wild-type CooA; the
activity of wild-type CooA is 15.7 and 0.23 units/mg of protein in the
presence or absence of CO, respectively (27, 29). CooA
N5 was also a
CO-dependent transcriptional activator as is wild-type
CooA. The deletion of four amino acid residues from the amino-terminal
of CooA did not perturb the electronic absorption spectra and
CO-dependent transcriptional activator activity of
CooA
N5. A similar result has been reported recently for the
different N-terminal truncated mutants of CooA (48).
N5, CooA
N5 showed almost the
same uv/vis spectra and activity as those of wild-type CooA. These
results indicate that some compensation for the mutation takes place to maintain CooA
N5 in order to have a six-coordinate heme and to be
active as the CO-dependent transcriptional activator.
However, it is not known at present what is coordinated to the heme in place of Pro2 in the case of CooA
N5.
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Fig. 2.
Iron K-edge k3-weighted EXAFS
curves of the ferric (A), ferrous
(B), and CO-bound CooA (C).
Experimental data are shown by solid lines and the fitting
results by broken lines. The windows used in Fourier filter
are also shown in the figures.
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Fig. 3.
Fourier transformed EXAFS data of the ferric
(A), ferrous (B), and CO-bound CooA
(C). Experimental data are shown by solid
lines and the fitting results by broken lines. The
windows used in Fourier filter are also shown in the figures.
Final refined parameters obtained from restrained refinement of EXAFS
The model of the coordination structure of the heme in CooA elucidated
by EXAFS analyses is shown in Fig. 4.
Cys75 and the nitrogen atom from Pro2 are
thought to be coordinated to the ferric heme in CooA. The distances of
the Fe-SCys and Fe-NPro bonds in the ferric
heme were estimated to be 2.25 and 2.19 Å, respectively. In the
ferrous heme, His77 and the nitrogen atom from
Pro2 are thought to be the axial ligands. The distances of
the Fe-NHis and Fe-NPro bonds in the ferrous
heme were estimated to be 2.02 and 2.16 Å, respectively. Good fitting
results were obtained for CO-bound CooA when His77 was
thought to be retained to be the proximal ligand of the CO-bound heme,
as shown in Figs. 2 and 3. The distances of the Fe-NHis and Fe-CCO bonds in the CO-bound CooA were estimated to be
1.98 and 1.79 Å, respectively. Details of the coordination structure of CooA in each state are discussed in the following sections.
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Coordination Structure of Ferrous CooA-- Lanzilotta et al. (39) have reported the crystal structure of ferrous CooA, which reveals that His77 and Pro2 are the axial ligands of the ferrous heme in CooA (39). As shown in Figs. 2B and 3B, the EXAFS curves can be fitted by using the atomic coordinates of ferrous CooA as a starting model for refinement of the fitting. Compared with the crystallographic result, the EXAFS analysis in this study reveals a slightly short Fe-NHis and a slightly long Fe-NPro bond distances by 0.17 and 0.04 Å, respectively. Although the bond distance of Fe-NPro is slightly longer than that of Fe-NHis, a similar bond distance for Fe-NHis and Fe-NPro does not show that one bond is more labile to be replaced with CO than the other (39). However, EXAFS and time-resolved resonance Raman spectroscopies reveal that His77 remains coordinated to the heme when CO reacts with ferrous CooA, as described below.
Coordination Structure of the Ferrous CO Complex of CooA-- EXAFS analysis of the CO-bound CooA indicated that His77 would be the proximal ligand of the CO-bound CooA, which is consistent with our model reported previously (28, 29), i.e. the mixed coordination of CO and the His77 imidazole, as shown in Fig. 4.
Picosecond time-resolved resonance Raman spectroscopy reveals that a
new intense line because of (Fe-His) at 211 cm
1 is observed immediately after photolysis of CO-bound
CooA (49). The transient 211 cm
1 band is completely
absent in the case of H77Y CooA in which His77 is replaced
by Tyr (49). These results are consistent with the notion that
His77 is the proximal ligand of the CO-bound CooA.
Resonance Raman spectroscopy has revealed the absence of any significant interactions between the bound CO and the distal heme pocket (28). These results indicate that the sixth ligand (Pro2, Ref. 39) replaced by CO moves far away beyond the coordination sphere of the heme.
CO reacts with the six-coordinate ferrous heme under physiological conditions to form CO-bound CooA, by which CooA is activated as the transcriptional activator. In other words, CO replaces one of the axial ligands of the ferrous heme to induce a conformational change required for the activation of CooA (29). The replacement of the axial ligand is functionally relevant for the activation of CooA by CO. It seems reasonable for us to anticipate that a large conformational change, including a concomitant movement of the main chain, is induced by the replacement of Pro2 with CO. Replacement of Pro2 will cause rotation of the C-helices about the dimer interface, as proposed by Lanzilotta et al. (39).
Coordination Structure of Ferric CooA--
The mutagenesis
analyses suggested that side chains of any amino acid residues, except
for Cys75 and His77, do not coordinate to the
heme iron as an axial ligand in CooA. It has been reported that
His77 is not coordinated to the ferric heme in CooA (29,
35, 38). Therefore, a possible candidate for the sixth ligand would be either an exogenous molecule such as H2O or
OH, or the nitrogen atom from Pro2 that is
the sixth ligand of the ferrous heme (39).
Given that an exogenous molecule is coordinated to the heme as the
sixth ligand, it might be H2O or OH. This is
mainly because the EPR spectral feature of the ferric CooA bears good
resemblance to that of cytochrome P450 in the low spin state with the
Cys-Fe3+-H2O/OH
coordination
(29, 37). However, the coordination of H2O or OH
to the ferric heme is unlikely because all the two
axial ligands must be changed upon reduction of the heme, if this is
the case. Furthermore, experiments on ligand binding with imidazole
have shown that the heme center in CooA is remarkably resistant to exogenous ligand binding (35). The electronic absorption spectra of
CooA have been reported to be unchanged between pH 6.5 and 11 (35).
These results apparently indicate that the sixth ligand of the heme in
CooA will be an endogenous ligand, not an exogenous one.
Dawson et al. (50) have reported spectroscopic investigations of ferric cytochrome P450cam ligand complexes. The spectroscopic properties of ferric CooA are similar to those of the nitrogen donor complexes of ferric cytochrome P450cam rather than those of the oxygen donor complexes. Dhawan et al. (38) have reported that magnetic circular dichroism (MCD) spectroscopy reveals the coordination of a neutral nitrogen atom as the sixth ligand to the ferric heme in CooA. As shown in Figs. 2A and 3A, good fitting results were obtained for the EXAFS spectra of ferric CooA when Cys75 and Pro2 were assumed to be the axial ligands of the ferric heme. These results suggest that the nitrogen atom from Pro2 is the most plausible candidate for the sixth ligand of the ferric heme in CooA.
Electrochemical Properties of CooA
Electrochemical Redox Titrations--
The present mutagenesis and
EXAFS experiments showed ligand switching of the heme iron in CooA
coupled with the change in its oxidation state. Then, we examined the
effect of the ligand switching on the redox properties of CooA by
electrochemical techniques. In the presence of redox mediator dyes,
electron transfer proceeded between a gold mesh electrode and CooA. In
electrochemical redox titrations, the electronic absorption spectra of
CooA were measured at each potential after an electric current reached
0.5 µA. Fig. 5A shows a
typical spectral change of CooA in the visible region during reductive
titration of ferric CooA, when the potential was varied from
100 to
600 mV with a step width of 25 or 50 mV. The electronic absorption
spectrum at a potential of
100 mV was identical to that of ferric
CooA as isolated, giving the Soret peak at 423.5 nm. As the applied
potential was shifted negatively, the Soret peak at 423.5 nm decreased
in intensity, whereas a new Soret peak at 424.5 nm increased in
intensity, with a concomitant appearance of the
(558.5 nm) and
(528.0 nm) bands. No spectral change was observed below
600 mV. The
final spectrum was identical to the spectrum of ferrous CooA obtained
by the chemical reduction of the ferric protein with sodium
dithionite.
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After the reductive titration, the potential was shifted positively to reoxidize CooA in the electrochemical oxidative titration. As shown in Fig. 5B, the spectral changes of CooA during the oxidative titration were the reverse of those observed in the reductive titration. At the end point of the oxidative titration, the electronic absorption spectrum of ferric CooA was restored. In the spectral change during the redox titrations, isosbestic points were observed at 411.0, 434.5, 510.0, and 569.5 nm, as shown in Fig. 5. These results show that the redox reaction of CooA proceeded quantitatively and without any side reaction in the electrochemical redox titration system described herein.
Nernst plots to determine the reduction and oxidation midpoint
potentials of wild-type CooA are shown in Fig.
6A. The reduction and
oxidation midpoint potentials of wild-type CooA were calculated from
the plots to be 320 mV and
260 mV, respectively. In the case of
wild-type CooA, the reduction and oxidation midpoint potentials were
not identical; i.e. the oxidation midpoint potential was shifted positively by 60 mV compared with the reduction midpoint potential.
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If a simple redox reaction of the heme in CooA takes place during the
electrochemical redox titrations, the midpoint potentials should be
identical to each other. However, this is not the case for CooA. On the
basis of the coordination structure described in this work, the
reduction and oxidation reactions of the heme iron are accompanied by
the ligand exchange and are thought to proceed according to the scheme
as shown in Scheme 1. A similar scheme
has been reported for analyzing the redox titrations of a mutant
(F82H/C102S) of yeast iso-1-cytochrome c (51) and of cytochrome cd1 from Paracoccus pantotrophus (52). A similar hysteresis in the redox titrations has been observed for cytochrome cd1
from Paracoccus pantotrophus (52). The oxidized
Paracoccus pantotrophus cytochrome cd1 has His/His axial
ligation at the c heme iron and Tyr/His axial ligation at the d1 heme
iron (53, 54). Upon reduction, the ligation of the c heme iron switches to His/Met concomitant with dissociation of the Tyr from the d1 heme
iron (53, 54). Koppenhöfer et al. (52) have reported that the hysteresis in the redox titrations is observed in the case of
Paracoccus pantotrophus cytochrome cd1, because the true equilibrium is not reached for the ligand switching reactions during
the redox titrations.
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In the case of CooA, the change in the oxidation state of the heme iron accompanies the axial ligand switching between Cys75 and His77, which is similar to ligand switching for Paracoccus pantotrophus cytochrome cd1. The reaction intermediates 2 and 4 shown in Scheme 1 seem to be formed during the reduction and oxidation of CooA, respectively, whereas the attempts to observe these intermediates by uv/vis spectroscopy with stopped flow method were unsuccessful so far. The reduction and oxidation of CooA should proceed with the same midpoint potential, if all the reaction steps shown in Scheme 1 are in equilibrium. However, this is not the case for wild-type CooA as shown in Fig. 6A, i.e. the apparent reduction and oxidation midpoint potentials are different from each other. Because the equilibrium is not reached for the ligand switching reactions during the redox titrations, the hysteresis in the redox titrations is thought to be observed also in the case of CooA, as is the case for Paracoccus pantotrophus cytochrome cd1.
To support the above suggestion, we also examined the redox titrations
of the H77G mutant of CooA in which His77, which is one of
the axial ligands of the ferrous heme, is replaced by Gly. The results
of the electrochemical redox titrations for H77G CooA are shown in Fig.
6B. The redox properties of H77G CooA were significantly
different from those of wild-type CooA in which the reduction and
oxidation midpoint potentials (420 mV) were identical to each other.
Because H77G CooA lacks His77, only redox reactions of the
heme iron seem to take place during the redox titrations. The ligand
switching during the redox titrations, the vertical transitions in
Scheme 1, does not take place in the case of H77G CooA, which is the
reason why the hysteresis in the redox titrations was not observed for
H77G CooA.
Redox Properties of CooA Relevant to its Functional and Structural
Properties--
The reduction midpoint potential of ferric CooA (320
mV) is comparable with that of the low-spin form of Pseudomonas
putida d-camphor hydroxylase cytochrome P450cam (E1/2 =
300 mV, Ref. 55), whose axial ligands are a thiolate derived from Cys
and H2O/OH
. This observation is consistent
with the coordination of the Cys75 thiolate to the ferric
heme iron in CooA, because the low reduction potential is thought to be
caused by the strong electron-donating nature of the Cys thiolate (56).
The thiolate coordination is apparently supported by EPR results, in
which the spectral features of ferric CooA are similar to those of the
low-spin form of P450cam (29, 37).
It is also characteristic of CooA that the heme having the histidyl
imidazole as the axial ligand gives 260mV for the oxidation midpoint
potential. Compared with the redox potentials of other heme proteins
having a histidyl imidazole, the value is similar to that of
horseradish peroxidase (
250 mV, Ref. 57) rather than those of
myoglobin (+50 mV, Refs. 58, 59)) and cytochrome b562 (+113 mV, Ref.
60). The difference in the redox potentials has been explained in terms
of the difference in the character of the ligand imidazole group;
i.e. the imidazole ring of peroxidase has an anionic
character, resulting from the strong hydrogen bond of the NH with
carboxylate (61), whereas those in myoglobin and cytochrome b562 have a
neutral character (58, 59, 62). In the case of CooA, deprotonation of
Pro2 that is coordinated to the ferrous heme may be
responsible for the low oxidation potential of CooA.
The low oxidation potential of CooA could be concerned with the
regulation of CooA activation. CooA is required for
CO-dependent expression of the coo operons
encoding a CO-oxidizing system that allows R. rubrum to grow
on CO as the sole energy source (31). CooA is activated only in the
presence of CO under anaerobic conditions to induce the expression of
the coo operons (30, 32, 33). Even in the presence of CO,
however, CooA must not be activated under aerobic conditions because CO
dehydrogenase, a key enzyme for the CO-oxidizing system encoded in the
coo operons, is labile to oxygen (63). The low oxidation
potential of CooA would facilitate the oxidation of the heme to prevent
CooA from being activated in vivo, once oxygen is present in
the cells.
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FOOTNOTES |
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* This work was supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture in Japan 11116212 and 11132219 (to S. A.) and by a grant from the Asahi Glass Foundation (to S. A.). EXAFS data collection at Photon Factory was performed under approval of the Program Advisory Committee (Proposal No. 97G059).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.
§ To whom correspondence should be addressed. Tel.: 81-761-51-1681; Fax: 81-761-51-1149; E-mail: aono@jaist.ac.jp.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M003972200
2 Among the mutants constructed in this work, N6A and D72A CooA showed a slightly blue-shifted Soret peak at 420 nm in the ferric state, whereas the ferrous and CO-bound spectra of these mutants were identical to those of wild-type CooA. However, when Asn6 or Asp72 was replaced by Met, the resulting mutants showed almost the same electronic absorption spectra in the ferric, ferrous, and CO-bound forms as those of wild-type CooA.
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ABBREVIATIONS |
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The abbreviations used are: sGC, soluble guanylate cyclase; EXAFS, extended x-ray absorption fine structure; EPR, electron paramagnetic resonance; NHE, normal hydrogen electrode.
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