From the Department of Life Science, College of
Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo
171-8501, Japan, the ¶ Institute of Physical and Chemical
Research, RIKEN Harima Institute, Mikazuki-cho, Sayo, Hyougo 679-5143, Japan, and the
Division of Biophysical Engineering, Graduate
School of Engineering Science, Osaka University, Toyonaka,
Osaka 560-8531, Japan
Received for publication, September 4, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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The benzylindazole compound YC-1 has been shown
to activate soluble guanylate cyclase by increasing the sensitivity
toward NO and CO. Here we report the action of YC-1 on the
coordination of CO- and NO-hemes in the enzyme and correlate the events
with the activation of enzyme catalysis. A single YC-1-binding site on
the heterodimeric enzyme was identified by equilibrium dialysis. To
explore the affect of YC-1 on the NO-heme coordination, the six-coordinate NO complex of the enzyme was stabilized by
dibromodeuteroheme substitution. Using the dibromodeuteroheme enzyme,
YC-1 converted the six-coordinate NO-heme to a five-coordinate NO-heme
with a characteristic EPR signal that differed from that in the absence of YC-1. These results revealed that YC-1 facilitated cleavage of the
proximal His-iron bond and caused geometrical distortion of the
five-coordinate NO-heme. Resonance Raman studies demonstrated the
presence of two iron-CO stretch modes at 488 and 521 cm Soluble guanylate cyclase
(sGC),1 a
protoheme-containing hemoprotein, is a well characterized NO receptor
involved in cell-cell signal transduction pathways associated with
neuronal communication and vasodilation (1-7). sGC purified from rat
and bovine lung are heterodimeric proteins composed of There has been much interest concerning the possible physiological role
of CO in the activation of sGC, but its role as a signaling molecule
remains uncertain because of its poor sGC-stimulating properties. Wu
et al. (20) reported that a benzylindazole compound YC-1
(3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole; structure shown
in Scheme 1) is a NO-independent
activator of platelet sGC. Subsequent work indicated that YC-1
stimulates in vitro cyclase activity of the CO-bound enzyme
to a level comparable with that of NO activation (21). Significant
stimulation of the ferrous and the ferrous NO enzymes by YC-1 was also
noted (21, 22). Despite the important observation of YC-1 sensitization
of the enzyme toward NO and CO, no firm structural information
concerning the binding of this molecule to the enzyme is available. For
instance, some reports indicate that YC-1 binds to the N-terminal
region of the 1 specific to the YC-1-bound CO complex of the
native enzyme. Together with the infrared C-O stretching measurements,
we assigned the 488-cm
1 band to the iron-CO stretch of a
six-coordinate CO-heme and the 521-cm
1 band to the
iron-CO stretch of a five-coordinate CO-heme. These results indicate
that YC-1 stimulates enzyme activity by weakening or cleaving the
proximal His-iron bond in the CO complex as well as the NO complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-subunits (8-10) and catalyze the conversion of GTP to cyclic
3',5'-guanosine monophosphate (cGMP) (8, 11-14). The enzyme is
activated by as much as 200-fold upon NO binding to the heme prosthetic
group (11-14). The enzyme contains a stoichiometric amount of heme
bound to histidine 105 of the N-terminal region of the
-subunit
through a weak His-iron bond (15-17). The C-terminal regions of the
two subunits, which share sequence homology to the catalytic site of
adenylate cyclases, are thought to comprise the catalytic domain. The
weak proximal His-iron bond plays a crucial role in the ability of the
enzyme to form an enzymatically active five-coordinate NO-heme. NO
initially binds to the heme to form an inactive six-coordinate NO
complex, which is then converted to an active five-coordinate NO
complex leading to cleavage of the weak His-iron bond, thereby
resulting in a 200-fold increase in activity above the basal level (18, 19). Although the formation of NO-heme is known to occur in two steps
as described above, details for the activation of the catalytic domain
coupled with NO binding remain elusive.
-subunit (22), whereas work using a newly discovered antiplatelet reagent suggests that the YC-1-binding site is located on
the
-subunit (23).
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Scheme 1.
Chemical structure of YC-1.
The effects of YC-1 on the CO-heme coordination of the enzyme have been examined by the resonance Raman method. The results indicated that although YC-1 altered the CO-heme coordination, the CO-heme of the YC-1-bound enzyme was in a six-coordinate state with a proximal ligand trans to CO (22). Therefore, the YC-1-dependent activation of the CO bound enzyme did not apparently couple to the cleavage of the proximal His-iron bond. However, analyses of CO recombination kinetics suggest that the proximal His-iron bond may weaken or be replaced by a different base upon YC-1 binding (24).
Although there is no evidence that an endogenous YC-1-like molecule
plays a physiological role in regulating sGC activity, an investigation
of its interaction with the enzyme will contribute to understanding
mechanisms of regulation of the catalytic activity. In this study we
attempted to solve the ambiguities regarding the
YC-1-dependent stimulation of the NO and CO complexes of
sGC. To obtain clear evidence of YC-1-dependent changes in
the NO coordination, we have prepared the stable six-coordinate NO-heme
by dibromodeuteroheme substitution. The NO complex of the reconstituted
enzyme contained a significant amount of the six-coordinate NO-heme
when the NO complex was prepared at pH 8.3. Binding of YC-1 resulted in
a complete loss of the six-coordinate NO-heme with the concomitant formation of a five-coordinate NO-heme. This clearly demonstrates that
YC-1 binding facilitates the cleavage of the proximal His-iron bond.
YC-1-dependent scission of the proximal His-iron bond was confirmed by vibrational spectroscopic studies on the native CO-bound enzyme. This is a first observation for the formation of a
five-coordinate CO-heme and provides a molecular mechanism for the
YC-1-dependent CO sensing function of the enzyme.
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EXPERIMENTAL PROCEDURES |
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Red, Blue, and Yellow Agarose Resins-- Agarose resins with dye ligands were prepared by an epoxy coupling method (25). Packed Sepharose 4B (100 g; Amersham Biosciences) was suspended in 150 ml of 1 N NaOH solution containing 500 mg of NaBH4 and 8 ml of 1,4-butanediol-diglycidylether (Sigma-Aldrich). The gel was gently shaken for 5 h at 30 °C and was collected using a glass filter funnel and then washed with 3 liters of H2O. The packed gel was suspended in 150 ml of 0.5 M sodium carbonate buffer, pH 12, and reacted with 1.2 g of Cibacron Brilliant Red 3B-A (Sigma-Aldrich), Cibacron Blue F3G-A (Fluka), or Cibacron Brilliant Yellow 3G-P (Sigma-Aldrich) for 15 h at 42 °C with gentle shaking. The resultant derivatized gels were suspended in 200 ml of sodium carbonate solution containing 0.2 M glycine and gently agitated overnight at 30 °C to completely block any residual epoxy groups.
Enzyme Purification--
Fresh bovine lung (5 kg) was minced and
homogenized using a Waring blender in 12 liters of 50 mM
TEA buffer, pH 7.6, containing 1 mM phenylmethylsulfonyl
fluoride, 1 mM benzamidine, 1 mM EDTA, and 55 mM -mercaptoethanol (buffer A). Protease
inhibitors and
-mercaptoethanol were included in all the buffers
throughout the purification unless stated otherwise. The homogenate was
clarified by centrifugation at 13,500 × g for 20 min,
and the supernatant was then mixed with 1.4 kg of DEAE cellulose A-500
(Seikagaku Kogyo, Tokyo, Japan) equilibrated with buffer A. The slurry
was stirred for 1 h at 4 °C and collected by sedimentation.
Subsequently, the resin was washed two times with buffer A and poured
into a column. The enzyme was eluted with a 3.5-liter linear NaCl
gradient of 0-0.35 M in buffer A. The active fractions
were concentrated and washed with 20 mM MOPS buffer, pH
7.6, using a Minitan concentrator (Millipore) and then was applied to a
Red Sepharose column (5 × 50 cm) equilibrated with the MOPS
buffer described above. The enzyme was eluted by a linear gradient of
0-0.4 M NaCl. The pooled active enzyme, which was
equilibrated with 20 mM MOPS buffer, pH 7.6, was adsorbed
to a Yellow agarose column, and the protein was eluted by a linear
gradient of 0-0.7 mM ATP. The concentrated active
fractions were further purified on a Superdex 200-pg column (2.6 × 60 cm; Amersham Biosciences) followed by a Source Q15 HPLC column
(1.6 × 10 cm; Amersham Biosciences). sGC was purified to apparent
homogeneity using a ceramic hydroxylapatite HPLC column (Bio-Rad),
where the elution was carried out by increasing the phosphate
concentration from 0 to 0.45 M at pH 7.6 in the absence of
EDTA. The overall yield was about 20%. The resultant homogenous enzyme
preparation was stored in liquid nitrogen until use.
Dibromodeuteroheme IX-substituted Enzyme-- The apoenzyme was obtained by a previously described method (18) with the following modifications. The DEAE cellulose column fractions of crude homogenate treated at pH 8.5 were equilibrated with 20 mM MOPS buffer, pH 7.6, and applied to a Blue Sepharose column. When protein was eluted with a linear gradient of 0-0.4 M NaCl, the cyclase activity was recovered as two peaks, one containing the holoenzyme and the other containing the apoenzyme. The apoenzyme was reconstituted with dibromodeuteroheme in 40 mM TEA buffer, pH 7.5, containing the protease inhibitor mixture described earlier supplemented with pepstatin A, leupeptin, and E64, under anaerobic conditions at 20 °C. The remaining purification steps were the same as those used for the native enzyme purification.
Spectral Measurements-- Optical absorption spectra were recorded on a Perkin-Elmer Lambda 18 spectrophotometer. The temperature of the cuvette holder was controlled with thermomodule elements. The buffer used was 40 mM TEA buffer, pH 7.5, containing 50 mM NaCl and 5% (v/v) glycerol or ethylene glycol. The details are given in the figure legends.
EPR spectra were measured on a Varian E-12 X-band EPR spectrometer with 100-kHz field modulation at 77 K. The microwave frequency was calibrated with a microwave frequency counter (Takeda Riken, Model TR 5212), and the magnetic field strength was determined by the nuclear magnetic resonance of water protons. The accuracy of g values is ± 0.005.
NO complexes for EPR measurements were prepared in the buffer containing 5% (v/v) ethylene glycol and 2.5% (v/v) DMF at 20 °C, as follows. The enzyme solution was transferred to a septum capped EPR tube and flushed with oxygen-free N2 gas for 10 min. Then, NO gas previously washed with 1 N NaOH solution was introduced with a gas tight syringe. After incubation at 20 °C for 5 min, the formation of NO complexes was monitored directly by measuring the optical spectrum of the sample. The samples in the EPR tubes were quickly frozen in liquid nitrogen.
Resonance Raman spectra were measured using a JASCO NR-1800 spectrometer equipped with liquid nitrogen cooled CCD detector (Princeton Instruments). Excitation wavelength was the 413.1-nm line from a Krypton ion laser (Coherent, Innova 90). The spectra were collected using a laser power of about 7 mW. To prevent the photodissociation of the bound CO, the laser beam was defocused. The Raman spectrometer was calibrated using indene.
The infrared spectra were measured on a Perkin-Elmer Spectral One FTIR spectrophotometer with a mercury-cadmium-telluride detector. A temperature-controlled cell holder was used. The cell had CaF2 windows with a light path length of 0.1 mm. The ferrous enzyme was used as a reference.
Equilibrium Dialysis-- In equilibrium dialysis, 6% (v/v) DMF was added to the buffer to maintain the required concentration of the poorly water-soluble YC-1. A five-cell rotating equilibrium dialyzer (Spectrum) was used for equilibrium dialysis experiments. Chambers (250 µl) were separated by dialysis membrane with a cut-off of 14 kDa. One chamber was filled with the ferrous enzyme, and the opposite chamber contained the desired amount of YC-1. In some cases, one chamber was filled with the ferrous enzyme and the desired amount of YC-1, and the opposite one contained the buffer solution alone. After introducing the sample (180 µl) into each chamber, the dialysis cells were rotated at a constant rate at 27 °C. The reaction achieved equilibrium within 5 h under these conditions. After dialysis for 6 h at 27 °C under constant rotation, the samples in each chamber were removed by a gas-tight syringe for quantitative analyses of YC-1. An aliquot of the enzyme solution was used to determine the heme concentration and to check the integrity of the enzyme. After dialysis, ~85% of the enzyme was recovered as the active form with the same optical spectra and SDS-PAGE profile as the native enzyme. The samples removed from both chambers were diluted with a 2-fold volume of DMF to prevent the adsorption of YC-1 on the inner surface of sample cup. The concentration of YC-1 was determined by HPLC using a C18 column at a constant flow rate of 1 ml/min of 65% (v/v) methanol. The amount of enzyme-bound YC-1 was calculated from the difference in the concentration between the two chambers with and without enzyme.
Activity Measurements-- The enzyme activity was measured as described previously (18). In brief, the assays were conducted in 50 mM TEA buffer, pH 7.5, supplemented with 5 mM dithiothreitol, 4 mM MgCl2, and 1 mM GTP in a final volume of 235 µl, and 10 µl of the enzyme was added to the mixture. The reaction was started by the addition of 5 µl of SNAP (2 mM) and was conducted for 10 min at 37 °C. After terminating the reaction by addition of 10 µl of 30% (v/v) acetic acid, cGMP was determined by HPLC as described previously (18). For the activation by CO, the assay mixture was saturated with CO gas prior to the addition of the enzyme.
Electrophoresis-- Reducing SDS-PAGE was carried out using an 8% acrylamide running gel. The protein was visualized using either Coomassie Brilliant Blue or silver stain (Daiichi Chemical Co., Tokyo, Japan).
Reagents--
GTP and cGMP were purchased from Wako Pure
Chemical Inc. (Tokyo, Japan). Research grade NO and CO were obtained
from Takachiho Chemical Co. (Tokyo, Japan). YC-1 and SNAP were
purchased from ALEXIS (San Diego, CA). Dibromodeuteroheme was prepared
according to the method of Seybert et al. (26). Other
chemicals, purchased from Nacalai Tesque Co. (Kyoto, Japan), were of
the highest commercial grade and were used without further purification.
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RESULTS |
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Properties of Native and Dibromodeuteroheme-substituted
Enzymes--
The homogenous native enzyme exhibited a
NO-dependent activity of 27-30 µmol/min/mg protein at
37 °C in the presence of Mg2+. This activity
corresponded to a turnover of about 3,800 min1 (µmol of
cGMP/min/µmol of heme). SDS-PAGE analyses indicated that the enzyme
was a heterodimeric protein consisting of the
-subunit of 78 kDa and
the
-subunit of 70 kDa. The enzyme contained 0.95 protoheme
IX/heterodimer, in which the protein and heme were determined by a
modified biuret method (27) and by the pyridine hemochromogen method
(28), respectively. The optical spectra of the ferrous and the ferrous
NO enzymes were identical to previous results (18).
The dibromodeuteroheme-substituted enzyme had a subunit structure
identical to that of the native enzyme (data not shown). The
reconstituted enzyme in the ferrous state exhibited the Soret band at
426 nm and the visible absorption at 553 nm, indicative of a
five-coordinate high spin state (Fig. 1).
The addition of NO yielded the NO complexes with an intense Soret band
at 393 nm with a shoulder around 410 nm. The bands at 393 and 410 nm were assigned to the five- and six-coordinate NO-hemes, respectively, as described later. The weak band at 481 nm was characteristic of the
formation of five-coordinate NO-heme. The absorption band of the
six-coordinate NO-heme at 410 nm was stable for at least 1 h at
20 °C. The addition of YC-1 rapidly converted the six-coordinate NO-heme to the five-coordinate NO-heme (data not shown).
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Stoichiometry of YC-1 Binding--
The stoichiometry of the
binding between YC-1 and the ferrous enzyme was measured by equilibrium
dialysis. The data were analyzed by Scatchard plot (29), in which the
fractional saturation (n = [YC-1]bound/[sGC]total) was plotted against
the fractional saturation divided by the free concentration of YC-1
(n/[YC-1]free). The result obtained by least
square analysis indicates that YC-1 bound/heme is 0.96 with a
dissociation constant of 124 µM at 27 °C (Fig. 2). This approximates to 1 mol of bound
YC-1/mol of heterodimeric enzyme. A different enzyme preparation also
displayed stoichiometry of 0.93 YC-1/heme.
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Effects of pH and YC-1 on Cyclase Activities--
When guanylate
cyclase activities were measured at pH 7.5, YC-1 enhanced the activity
of the native NO-bound enzyme by about 1.1-fold (Fig.
3), confirming previous results (21, 22).
The level of the stimulation by YC-1 is significantly higher at pH 8.3 (1.24-fold) than at pH 7.5. Similar experiments performed using the
dibromodeuteroheme-substituted enzyme provide a clear mechanism of
action for YC-1 (see Fig. 3). In this case, YC-1 stimulates the cyclase
activity by 1.28-fold at pH 7.5 and by 2-fold at pH 8.3, suggesting
that the NO complex of dibromodeuteroheme-substituted enzyme is largely
made up of the catalytically inactive six-coordinate NO-heme. The
activities of the reconstituted enzyme at both pH 7.5 and 8.3 are about
60% of those of the native enzyme even in the presence of YC-1. The
reasons for heme-dependent changes in the activity are
unknown.
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Optical Spectral Characterization of Ferrous NO
Complexes--
Fig. 4 shows the optical
absorption spectra of the NO complex of the native and substituted
enzymes. As demonstrated in Fig. 4, the NO complex of native enzyme at
pH 8.3 comprises a small amount of six-coordinate NO-heme detected as a
shoulder around 420 nm (18). The disappearance of the shoulder at 420 nm and the enhanced 399-nm band after binding of YC-1 indicates
conversion of the six-coordinate NO-heme to the five-coordinate NO-heme
(Fig. 4A, spectrum c). As shown in Fig.
4B, the NO complex of dibromodeuteroheme-substituted enzyme
exhibited two Soret bands at 393 and 410 nm. The 410-nm band was
assigned as the six-coordinate NO complex of the substituted enzyme, as
established by EPR spectroscopy described below. The effects of pH and
YC-1 binding on the optical spectra of the NO complexes were consistent
with the activity measurements, in which the inactive six-coordinate
NO-heme accumulated at pH 8.3. To determine the coordination states of
the NO-hemes, both NO-ligated forms of the native and the reconstituted
enzymes were rapidly frozen and then analyzed by EPR spectroscopy.
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EPR Characterization of Five- and Six-coordinate NO-hemes--
The
14N16O complex of the native enzyme in the
absence of YC-1 exhibits characteristic EPR signals of axially
symmetric five-coordinate NO-heme (g = 2.069 and gz = 2.009) with a triplet 14NO
hyperfine splitting (Fig. 5, trace
a). The spectral features agreed with the previous reports (18,
30). At pH 8.3, a weak EPR signal at g = 1.976 caused
by the six-coordinate NO-heme was observed in addition to EPR signals
of the five-coordinate NO-heme (Fig. 5, trace b). The
addition of YC-1 changed the six-coordinate NO-heme signal to a new
five-coordinate NO-heme signal with rhombic symmetry. The new
five-coordinate NO-heme was characterized by three distinct
g values of gx = 2.103, gy = 2.032, and gz = 2.009 with triplet 14N hyperfine splitting (Fig. 5,
trace c). For the dibromodeuteroheme-substituted enzymes
(Fig. 5, traces d and e), the six-coordinate
NO-heme was detected as an EPR signal at g = 1.976 (trace d). The addition of YC-1 resulted in the loss of the
six-coordinate NO-heme signal and generated an EPR signal with rhombic
symmetry that was essentially identical to the native enzyme (Fig. 5,
trace e). This is the first direct evidence for YC-1-induced
changes to the proximal His-iron bond and the NO coordination of the
five-coordinate NO-heme. Ca2+-GTP also induced the
conversion of the axially symmetric NO-heme signal to the NO-heme
signal with rhombic symmetry as shown in trace f of Fig. 5,
whereas neither Mg2+-ATP nor Ca2+-ATP altered
the g value anisotropy in the NO-heme EPR signal (data not
shown). The increase in g value anisotropy implies that YC-1
and Ca2+-GTP induce a significant geometrical distortion of
the NO-heme coordination.
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Infrared and Resonance Raman Spectra--
YC-1 stimulated the
cyclase activity of the CO-bound enzyme from 0.8 to 27 µmol/min/mg
protein, as reported previously (21, 22). The YC-1-induced changes in
the iron-CO stretching vibration (Fe-CO) have been
reported (22), but precise analyses of the C-O stretching vibration
(
C-O) and of the effects of YC-1 on the
C-O were not carried out (22, 31). The infrared spectra of the 12C16O complex under various conditions
are summarized in Fig. 6. We found a
sharp band at 1987 cm
1 and a broad band centered at 1968 cm
1 both at 15 and 25 °C in the absence of YC-1 (Fig.
6, traces A and B). The bands at 1987 and 1968 cm
1 shifted to 1943 and 1924 cm
1 upon
13C16O substitution, respectively, indicating
that they can be assigned to the
C-O mode (data not
shown). At 15 °C, binding of YC-1 slightly diminished the
1987-cm
1 band and resulted in a small enhancement of the
1972-cm
1 band (Fig. 6, trace C). Raising the
temperature up to 25 or 32 °C markedly intensified the band at 1972 cm
1 with the appearance of a shoulder at 1965 cm
1 (Fig. 6, traces D and E). The
temperature-dependent changes were reversible. The shoulder
at 1965 cm
1 is also seen in the difference spectrum (Fig.
6, inset). No additional CO stretch band was detectable even
when the concentration of sGC and YC-1 was increased (Fig. 6,
trace F). These data demonstrate that there are two types of
CO complex in the presence of YC-1, one with a 1972-cm
1
band and the other with a 1965-cm
1 band. The effect of
YC-1 on the formation of the two CO complexes is more apparent at
elevated temperatures, suggesting an increase in affinity of YC-1 at a
higher temperature.
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To determine the identity of these two CO species, we searched the
Fe-CO mode by resonance Raman spectroscopy. The
Fe-CO and the iron-carbon-oxygen bending vibration
(
Fe-C-O) of the CO complex in the absence of YC-1
have been reported to be 472 and 562 cm
1, respectively
(17). The corresponding Raman bands in our experiments were at 475 and
565 cm
1 (Fig. 7,
trace a). The former Raman band monotonously downshifted (475
472
468
466 cm
1), and the latter
exhibited a zigzag isotope shift (565
552
565
549 cm
1) by increasing the mass of CO (Fig. 7, traces
b-e), indicating that the former and the latter bands were
assigned to the
Fe-CO and the
Fe-C-O,
respectively. YC-1 produced a new Raman band at 488 cm
1
with diminished intensity of the 475- cm
1 band in the
YC-1-free form (Fig. 7, trace b). YC-1 did not alter the
vibrations of the porphyrin macrocycle in the high frequency region
(data not shown). The 488-cm
1 band monotonously
downshifted to 485, 477, and 475 cm
1 by increasing the
mass by 13C16O, 12C18O,
and 13C18O, respectively (Fig. 7, traces
c-e). The Raman band at 488 cm
1
therefore is assigned to the
Fe-CO mode of the
YC-1-bound CO complex. In addition to the
Fe-CO mode at
488 cm
1, YC-1 produced two other isotope-sensitive Raman
bands at 521 and 589 cm
1 (Fig. 7, trace b).
Careful examination revealed that the 589-cm
1 band
exhibits a decrease-increase-decrease frequency shift in the order
12C16O (589 cm
1)
13C16O (584 cm
1)
12C18O (589 cm
1)
13C18O (583 cm
1) (Fig. 7,
traces b-e). Therefore, this band can be
assigned to the
Fe-C-O mode of the CO complex of the
YC-1-bound enzyme. Comparison of the Raman spectrum with the infrared
spectrum (Fig. 6, trace E) leads to the conclusion that one
of the YC-1-bound CO complexes exhibits the
Fe-CO at 488 cm
1, the
Fe-C-O at 589 cm
1,
and the
C-O at 1972 cm
1. Hereafter, we
designate this CO adduct as the major CO adduct.
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The 521-cm1 band of the 12C16O
complex was shifted to 515, 512, and 509 cm
1 by
12C18O, 13C16O, and
13C18O respectively (Fig. 7, traces
b-e). This monotonous frequency shift as the mass of
CO increases assigns this band to the
Fe-CO mode.
Furthermore, this Raman band is not observed in the spectrum of the CO
complex in the absence of YC-1 (Fig. 7, trace a) and is
intensified by increasing the YC-1 concentration (Fig. 7,
inset). These reveal that YC-1 generates another CO adduct
distinct from the major CO adduct. After this, we designate this CO
adduct with the 521-cm
1
Fe-CO mode as the
minor CO adduct. The shoulder at 1965 cm
1 is a candidate
for the
C-O mode of the minor CO adduct, because there
are no other YC-1-sensitive infrared bands in the CO stretching region.
Comparison of the
C-O infrared band with the
Fe-CO Raman band under similar conditions enables us to
assign the
C-O mode of the minor CO adduct. The
noticeable difference in the shape between the
C-O and
the
Fe-CO modes (Figs. 7, inset, and 6, trace F) is because the former mode has a shoulder at 1965 cm
1, whereas the latter exhibits a band at 521 cm
1. Excluding the difference, the
C-O
mode can be superposed on the
Fe-CO mode, indicating
that the 1965-cm
1 band should be assigned to the
C-O mode of the minor CO adduct. Given the mode
assignments, the major CO adduct is characterized by the
Fe-CO and
C-O modes at 488 and 1972 cm
1, respectively, and the minor one is characterized by
the
Fe-CO at 521 cm
1 and the
C-O at 1965 cm
1.
The difference in the CO coordination between the minor and major CO
adducts can be assessed by the well defined correlation curve between
C-O and
Fe-CO (32-36). As shown in Fig.
8, the data points of the YC-1-free CO
complex and the major CO adduct correspond to the correlation curve of
the CO complexes with a neutral histidine trans to CO. In
contrast, the minor CO adduct significantly deviates from the
correlation curve but tends toward data points for a five-coordinate
CO-heme (33, 37, 38). This provides the first clear evidence for the
cleavage of the proximal His-iron bond in the CO complex induced by
YC-1 and accounts for the YC-1-dependent CO responsiveness
of the enzyme.
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DISCUSSION |
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Our recent findings and those of others show that the binding of
NO to the ferrous sGC initially yields a short-lived six-coordinate NO
complex that decays to the five-coordinate NO complex (18, 19). The
six-coordinate NO complex has been shown to be enzymatically inactive
(19). In this study, we have stabilized the six-coordinate NO complex
by heme substitution to examine the action of YC-1 on NO coordination.
Dibromodeuteroheme-substituted enzyme yielded the six-coordinate
NO-heme as a major component at pH 8.3, which was stable even at room
temperature. The electron withdrawing bromo groups decrease the
electron density of the heme-iron, which may explain why the
dibromodeuteroheme-reconstituted enzyme produces a stable
six-coordinate NO-heme (26, 39, 40). The lower electron density on the
heme-iron increases the affinity for exogenous -donor ligands and
decreases the affinity for exogenous
-acceptor ligands such as
oxygen. Therefore, it is feasible that the introduction of electron
withdrawing substituents strengthens the internal proximal His-iron
bond primarily through
-bonding interaction of the imidazole
nitrogen-iron linkage. Indeed, heme derivatives with strong
electron-withdrawing substituents are known to bind imidazole with a
higher affinity (41).
By using the reconstituted enzyme, we solved the question of how YC-1
stimulates the NO-bound enzyme. Optical and EPR spectroscopic studies
indicate that YC-1 facilitates the NO-induced dissociation of the
proximal His ligand and yields a new five-coordinate NO-heme with a
rhombic EPR signal. The EPR signal of the new NO-heme species with YC-1
closely resembles those of P-420-NO complexes or of some heme-model NO
complexes (42-46). In the ferrous NO-heme adducts, bound NO has been
known to adopt a bent iron-nitrogen-oxygen geometry (47). The increase
in the g value anisotropy described for the YC-1 bound form
of the five-coordinate NO complex of sGC indicates an increase in the
delocalization of the unpaired electron residing on the p* of NO to
iron d
-orbital (48). Delocalization of the unpaired electron is
known to be highly sensitive to the geometry of the
iron-nitrogen-oxygen unit (49, 50). On the basis of these
considerations, we propose that YC-1 binding increases the iron-nitrogen-oxygen bond angle, thereby increasing the d
-p
* overlap. The five-coordinate NO-heme with a rhombic EPR signal was also
observed for sGC-NO in the presence of Ca2+-GTP (Fig. 5,
trace f). The formation of the five-coordinate NO complex
with a rhombic EPR signal is specific to GTP among purine nucleotides,
because neither Mg2+-ATP nor Ca2+-ATP causes
detectable changes in the NO-heme signal. This result is puzzling
because GTP-binding sites are located on the C-terminal catalytic
domains of both
- and
-subunits, whereas the YC-1-binding site is
thought to be at the N-terminal side of the
-subunit. However, GTP
may bind to a site other than the catalytic site and thereby serve as
an effector molecule regulating heme reactivity.
Denninger et al. (22) have reported that the heterodimeric
sGC and the enzymatically inactive truncated -subunit formed a
six-coordinate CO-heme but not a five-coordinate CO-heme in the
presence of YC-1. The 521-cm
1 Raman band, which was
assigned to the
Fe-CO of five-coordinate CO-heme in this
work, was obvious in their Raman spectra and was downshifted by
13C18O replacement (Fig. 4 in Ref. 22).
Nevertheless, they have not remarked on this Raman band being
isotope-sensitive. Their finding that the Raman band is absent in the
spectra of the truncated
-subunit implies that the formation of the
five-coordinate CO complex is specific to the heterodimeric sGC. The
detection of a five-coordinate CO-heme presented in this study
represents the first example in a native hemoprotein. The spectroscopic
characteristics match those of the five-coordinate CO-heme reported for
some mutant hemoproteins including the CooA (CO-sensing
transcriptional activator) variant with a H77Y substitution and the
proximal base mutant of bacterial heme oxygenase (37, 38).
The major CO adduct of the YC-1-bound CO complex of sGC was
characterized by the Fe-CO at 488 cm
1, the
Fe-C-O at 589 cm
1, and the
C-O at 1972 cm
1. To our knowledge, the
Fe-C-O frequency at 488 cm
1 of the major
CO adduct is anomalously high for six-coordinate CO- hemes with
proximal neutral imidazole, because the
Fe-C-O frequencies of those CO-hemes are within a limited range of 577-579 cm
1. Moreover, the 5-cm
1 isotope shift of
the
Fe-C-O in the major CO adduct by changing from
12CO to 13CO is small. The
Fe-C-O frequency is similar to that of CO-ligated horseradish and cytochrome c peroxidases at low pH (585-587
cm
1), but in these cases the
Fe-CO
frequency is about 535 cm
1. Thus, the separation between
Fe-CO and
Fe-C-O in the major CO adduct
of sGC is exceptionally large. This characteristic might be explained
by increased bending in the iron-CO unit (34). Whatever the cause of
this anomalous behavior, it is clear that the major CO adduct binds a
neutral ligand at the position trans to CO (Fig. 8). In a
six-coordinate CO-heme with a neutral proximal imidazole, the heme-iron
has been known to tightly bind imidazole by a trans-CO
effect (51). Therefore, release of the imidazole from the
six-coordinate CO-heme seems unlikely. YC-1 may trigger displacement of
the proximal histidine residue by a neutral residue (X)
other than histidine in the formation of the major CO adduct. A neutral
cysteine or methionine are candidate residues for ligand X,
because the ligation of thioether or thiophenol at the position trans to CO provides the same
Fe-CO-
C-O correlation observed for
neutral imidazole adducts (37). To understand the detailed mechanism of
the five-coordinate CO-heme formation, we must await the identification
of the endogenous proximal heme ligand in the major CO adduct.
Some reports demonstrated that YC-1 resulted in the blue shift of the
Soret band of the CO-sGC and increased the binding rate for CO (24,
52), whereas others showed no changes in these measurements (22, 53).
The discrepancy may be attributable to differences in the temperature
employed for the measurements; a significant increase in the CO binding
rate induced by YC-1 was observed at 23 °C but not at 10 °C (24,
53). Our finding for temperature sensitivity in the C-O
band may solve the question.
The results in this paper demonstrate a single binding site for YC-1 on
the heterodimeric sGC. This raises the question as to whether the
binding site for YC-1 is located on the - or
-subunit of sGC. A
newly synthesized pyrazolopyridine derivative BAY41-2272, which shared
an analogous core structure to YC-1, stimulates sGC activity in a
similar manner to YC-1 (23). A photoaffinity labeling analogue of
BAY41-2272 with a reactive azido group identified two cysteine residues
closely located in the
-subunit (23). Competitive binding
experiments demonstrated that the BAY analogue shares a common binding
site with YC-1, suggesting the presence of the YC-1-binding site on the
-subunit. This result appears to be in contrast to a resonance Raman
experimental result, in which the binding site of YC-1 is on the
-subunit (22). However, the azido group of the BAY analogue is
spatially separated from the pyrazolopyridine core by a benzoylic
spacer. Therefore, the pyrazolopyridine core may in fact bind to a site
on the
-subunit so that the reactive azido group is in close contact
with those distant cysteine residues of the
-subunit. These
considerations suggest that the binding site of YC-1 may be located at
the dimer interface on the
-subunit as shown for the binding site of
forskolin, a potent activator of adenylate cyclase (54).
The results from this study are consistent with a view that YC-1
stimulates the CO- and NO-bound sGC by weakening or cleaving the
proximal His-iron bond. The reason why ferrous sGC was stimulated by
YC-1 is not clear, because YC-1 does not cause a detectable shift in
the Fe-His Raman frequency (22). We therefore infer that
the YC-1-dependent stimulation of the ferrous sGC
exclusively occurs through a heme-independent mechanism (55).
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ACKNOWLEDGEMENT |
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We thank M. Taketsugu for helpful technical assistance.
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FOOTNOTES |
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* This work was supported by Special Coordination Funds from the Science and Technology Agency of Japan (to R. M. and Y. S.) and by Grants-in-Aids for Scientific Research on Priority Areas (to R. M., Y. S., and H. H.).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: Dept. of Life Science, and Frontier Project "Life Adaptation Strategies to Environmental Changes," College of Science, Rikkyo (St. Paul's) University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan. E-mail: rmakino@rikkyo.ne.jp.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M209026200
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ABBREVIATIONS |
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The abbreviations used are: sGC, soluble guanylate cyclase; cGMP, cyclic 3',5'-guanosine monophosphate; EPR, electron paramagnetic resonance; TEA, triethanolamine; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; HPLC, high performance liquid chromatography; T, tesla; MOPS, 3-(N-morpholino)propanesulfonic acid; DMF, dimethylformamide.
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