YC-1 Facilitates Release of the Proximal His Residue in the NO and CO Complexes of Soluble Guanylate Cyclase*

Ryu MakinoDagger §, Eiji Obayashi, Nana HommaDagger , Yoshitsugu Shiro, and Hiroshi Hori||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha - and beta -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 beta -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.

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 beta -subunit (22), whereas work using a newly discovered antiplatelet reagent suggests that the YC-1-binding site is located on the alpha -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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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 beta -mercaptoethanol (buffer A). Protease inhibitors and beta -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.

    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 min-1 (µmol of cGMP/min/µmol of heme). SDS-PAGE analyses indicated that the enzyme was a heterodimeric protein consisting of the alpha -subunit of 78 kDa and the beta -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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Fig. 1.   Optical spectra of dibromodeuteroheme-reconstituted sGC. Optical spectra of the ferrous and ferrous NO forms of the dibromodeuteroheme-reconstituted sGC at 20 °C in 40 mM TEA buffer, pH 7.5, containing 50 mM NaCl and 5 mM dithiothreitol are shown. The NO complex was prepared by adding about 100 µM NO to the ferrous enzyme under anaerobic conditions.

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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Fig. 2.   Equilibrium dialysis measurements of YC-1 binding. The ferrous sGC (30-40 µM) was incubated with 40-250 µM YC-1 for 6 h at 27 °C. The dissociation constant was calculated by Scatchard analysis. The buffer used was 40 mM TEA, pH 7.5, containing 5% (v/v) ethylene glycol, 4% (v/v) DMF, 50 mM NaCl, and 1 mM of EDTA. When YC-1 dissolved in DMF was added to the sample, the final concentration of DMF was adjusted to 6% (v/v).

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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Fig. 3.   Effects of YC-1 on the activities of the native and dibromodeuteroheme-reconstituted sGC. NO-dependent cyclase activities were measured in the assay mixture containing 1 mM GTP, 4 mM MgCl2, 5 mM dithiothreitol, and 100 µM SNAP in a total volume of 250 µl of 40 mM TEA buffer, pH 7.5 or 8.3, containing 4% (v/v) DMF. When desired, 120 µM YC-1 was added to the mixture. The reaction was started by the addition of SNAP and conducted at 37 °C for 10 min. The enzyme activity was expressed as turnover number (µmol of cGMP formed/min/µmol of heme). In the inset, the structure of dibromodeuteroheme is illustrated. Di-BR sGC in the figure donates dibromodeuteroheme-reconstituted sGC.

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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Fig. 4.   Effects of YC-1 and pH on the ferrous NO complex of native and dibromodeuteroheme-reconstituted sGC. A, optical spectra of the ferrous NO complex of the native sGC. In trace a, the optical spectrum of the NO complex at pH 7.5 in the absence of YC-1 is shown. Trace b shows the spectrum of the NO complex at pH 8.3 in the absence of YC-1, and trace c shows the spectrum at pH 8.3 in the presence of YC-1. B, optical spectra of the ferrous NO complex of dibromodeuteroheme-reconstituted enzyme. Trace a exhibited an optical spectrum at pH 8.3 in the absence of YC-1 and trace b at pH 8.3 in the presence of YC-1. The buffer used was 40 mM TEA buffer, pH 7.5 or 8.3, containing 5% (v/v) ethylene glycol, 2.5% (v/v) DMF, 5 mM dithiothreitol, and 50 mM NaCl. In all samples, 2.5 µl of 10 mM YC-1 dissolved in DMF was added to give a final concentration of 250 µM. Optical absorption spectra of the samples in the EPR tube were recorded by using an optical fiber system. The sample volumes were 100 µl, and the enzyme concentration was 35 µM.

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 (gperp  = 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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Fig. 5.   EPR spectra of the 14N16O complex of native and dibromodeuteroheme-reconstituted sGC. Trace a, EPR spectrum of the 14N16O complex of native sGC in the absence of YC-1 at pH 7.5; trace b, native sGC in the absence of YC-1 at pH 8.3; trace c, native sGC in the presence of YC-1 at pH 8.3; trace d, dibromodeuteroheme-reconstituted sGC in the absence of YC-1 at pH 8.3; trace e, dibromodeuteroheme-reconstituted sGC in the presence of YC-1 at pH 8.3; trace f; native sGC in the presence of 3 mM Ca2+ and 2.5 mM GTP. EPR spectra were taken at 77 K and by 100 kHz field modulation with 0.5 millitesla width. The microwave power was 5 mW. The enzyme concentrations were 35 µM as with heme. In these experiments, 40 scans were averaged. Other experimental conditions are described in the legend of Fig. 4. Di-Br sGC in the figure denotes dibromodeuteroheme-reconstituted sGC.

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 (nu Fe-CO) have been reported (22), but precise analyses of the C-O stretching vibration (nu C-O) and of the effects of YC-1 on the nu 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 nu 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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Fig. 6.   Fourier transform infrared spectra of the CO complex of native sGC. Trace A, Fourier transform infrared spectrum of the CO complex in the absence of YC-1 at 15 °C. Trace B, Fourier transform infrared spectrum of the CO complex in the absence of YC-1 at 25 °C. Trace C, CO complex in the presence of YC-1 at 15 °C. Trace D, CO complex with YC-1 at 25 °C. Trace E, CO complex with YC-1 at 32 °C. In above experiments, the concentrations of sGC and YC-1 are 150 and 280 µM, respectively. In trace F, sGC and YC-1 concentrations are increased to 210 and 350 µM, respectively, and the spectrum was recorded at 32 °C. In the inset, the Fourier transform infrared difference spectrum between with and without YC-1 measured at 32 °C is shown. The buffers used are 40 mM TEA buffer, pH 7.5, containing 3% (v/v) DMF, 5% (v/v) ethylene glycol, and 50 mM NaCl. When YC-1 dissolved in DMF is added to the sample, the final DMF concentration is 6% (v/v) in the all experiments.

To determine the identity of these two CO species, we searched the nu Fe-CO mode by resonance Raman spectroscopy. The nu Fe-CO and the iron-carbon-oxygen bending vibration (delta 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 right-arrow 472 right-arrow 468 right-arrow 466 cm-1), and the latter exhibited a zigzag isotope shift (565 right-arrow 552 right-arrow 565 right-arrow 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 nu Fe-CO and the delta 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 nu Fe-CO mode of the YC-1-bound CO complex. In addition to the nu 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) right-arrow 13C16O (584 cm-1) right-arrow 12C18O (589 cm-1) right-arrow 13C18O (583 cm-1) (Fig. 7, traces b-e). Therefore, this band can be assigned to the delta 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 nu Fe-CO at 488 cm-1, the delta Fe-C-O at 589 cm-1, and the nu C-O at 1972 cm-1. Hereafter, we designate this CO adduct as the major CO adduct.


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Fig. 7.   Effects of YC-1 on resonance Raman frequencies of the iron-CO stretch and the iron-carbon-oxygen bending vibrations. Trace a, the resonance Raman spectrum of the ferrous 12C16O sGC in the absence of YC-1. Trace b, spectrum of the ferrous 12C16O sGC in the presence of YC-1 (100 µM). Trace c, ferrous 13C16O sGC in the presence of YC-1 (100 µM). Trace d, ferrous 12C18O sGC in the presence of YC-1 (100 µM). Trace e, ferrous 13C18O sGC in the presence of YC-1 (100 µM). In the inset, the iron-CO stretching vibration of the ferrous 12C16O or 13C16O sGC in the absence and presence of YC-1 (350 µM) is illustrated. The buffer used is 50 mM TEA buffer, pH 7.5, containing 50 mM NaCl, 5% (v/v) glycerol and 4% (v/v) DMF. All of the samples contained 6 (v/v) DMF including those without YC-1. The resonance Raman spectra were collected using an excitation wavelength of 413.1 nm at ambient temperature.

The 521-cm-1 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 nu 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 nu Fe-CO mode as the minor CO adduct. The shoulder at 1965 cm-1 is a candidate for the nu 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 nu C-O infrared band with the nu Fe-CO Raman band under similar conditions enables us to assign the nu C-O mode of the minor CO adduct. The noticeable difference in the shape between the nu C-O and the nu 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 nu C-O mode can be superposed on the nu Fe-CO mode, indicating that the 1965-cm-1 band should be assigned to the nu C-O mode of the minor CO adduct. Given the mode assignments, the major CO adduct is characterized by the nu Fe-CO and nu C-O modes at 488 and 1972 cm-1, respectively, and the minor one is characterized by the nu Fe-CO at 521 cm-1 and the nu 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 nu C-O and nu 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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Fig. 8.   Plot of nu C-O versus nu Fe-CO frequencies for the CO adducts of sGC and for selected hemes and hemoproteins. The open circles represent data of six-coordinate CO adducts taken from the data cited in Refs. 32, 33, and 35. The closed circles are for the CO-sGC without YC-1 and the major CO adduct of sGC with YC-1. The open squares represent five-coordinate CO adducts (33, 36, 37), and the closed square is for the minor CO adduct of sGC in the presence of YC-1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 sigma -donor ligands and decreases the affinity for exogenous pi -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 sigma -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 ppi * of NO to iron dpi -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 dpi -ppi * 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 alpha - and beta -subunits, whereas the YC-1-binding site is thought to be at the N-terminal side of the beta -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 beta -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 nu 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 beta -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 nu Fe-CO at 488 cm-1, the delta Fe-C-O at 589 cm-1, and the nu C-O at 1972 cm-1. To our knowledge, the delta 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 delta 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 delta Fe-C-O in the major CO adduct by changing from 12CO to 13CO is small. The delta 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 nu Fe-CO frequency is about 535 cm-1. Thus, the separation between nu Fe-CO and delta 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 nu Fe-CO-nu 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 nu 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 alpha - or beta -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 alpha -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 alpha -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 beta -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 beta -subunit so that the reactive azido group is in close contact with those distant cysteine residues of the alpha -subunit. These considerations suggest that the binding site of YC-1 may be located at the dimer interface on the beta -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 nu 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).

    ACKNOWLEDGEMENT

We thank M. Taketsugu for helpful technical assistance.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Furchgott, R. F., and Zawadzki, J. V. (1980) Nature 288, 373-376[Medline] [Order article via Infotrieve]
2. Ignarro, L. J., and Kadowitz, P. J. (1985) Annu. Rev. Pharmacol. Toxicol. 25, 171-191[CrossRef][Medline] [Order article via Infotrieve]
3. Waldman, S. A., and Murad, F. (1987) Pharmacol. Rev. 39, 163-196[Medline] [Order article via Infotrieve]
4. Garthwaite, J., Charles, S. L., and Chess-Williams, R. (1988) Nature 336, 385-388[CrossRef][Medline] [Order article via Infotrieve]
5. Bread, D. S., and Snyder, S. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9030-9033[Abstract]
6. Moncada, S., and Higgs, E. A. (1991) Eur. J. Clin. Invest. 21, 361-374[Medline] [Order article via Infotrieve]
7. Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V., and Snyder, S. H. (1993) Science 259, 381-384[Medline] [Order article via Infotrieve]
8. Stone, J. R., and Marletta, M. A. (1994) Biochemistry 33, 5636-5640[Medline] [Order article via Infotrieve]
9. Kamisaki, Y., Saheki, S., Nakane, M., Palmieri, J. A., Kuno, T., Chang, B. Y., Waldman, S. A., and Murad, F. (1986) J. Biol. Chem. 261, 7236-7241[Abstract/Free Full Text]
10. Humbert, P., Niroomand, F., Fischer, G., Mayer, B., Koesling, D., Hinsch, K.-D., Gauspohl, H., Frank, R., Schultz, G., and Böhme, E. (1990) Eur. J. Biochem. 190, 273-278[Abstract]
11. Ignarro, L. J., Wood, K. S., and Wolin, M. S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2870-2873[Abstract]
12. Wolin, M. S., Wood, K. S., and Ignarro, L. J. (1982) J. Biol. Chem. 257, 13312-13320[Free Full Text]
13. Gerzer, R., Hofmann, F., and Schultz, G. (1981) Eur. J. Biochem. 116, 479-486[Abstract]
14. Ignarro, L. J., Ballot, B., and Wood, K. S. (1984) J. Biol. Chem. 259, 6201-6207[Abstract/Free Full Text]
15. Wedel, B., Humbert, P., Harteneck, C., Foerster, J., Malkewitz, J., Böhme, E., Schultz, G., and Koesling, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2592-2596[Abstract]
16. Zhao, Y., and Marletta, M. A. (1997) Biochemistry 36, 15959-15964[CrossRef][Medline] [Order article via Infotrieve]
17. Deinum, G., Stone, J. R., Babcock, G. T., and Marletta, M. A. (1996) Biochemistry 35, 1540-1547[CrossRef][Medline] [Order article via Infotrieve]
18. Makino, R., Matsuda, H., Obayshi, E., Shiro, Y., Iizuka, T., and Hori, H. (1999) J. Biol. Chem. 274, 7714-7723[Abstract/Free Full Text]
19. Zhao, Y., Brandish, P. E., Ballou, D. P., and Marletta, M. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14753-14758[Abstract/Free Full Text]
20. Wu, C. C., Ko, F. N., Lee, F. Y., and Teng, C, M. (1995) Br. J. Pharmacol. 116, 1973-1978[Abstract]
21. Friebe, A., Schultz, G., and Koesling, D. (1996) EMBO J. 15, 6863-6868[Abstract]
22. Denninger, J. W., Schelvis, J. P. M., Brandise, P. E., Zhao, Y., Babcock, G. T., and Marletta, M. A. (2000) Biochemistry 39, 4191-4198[CrossRef][Medline] [Order article via Infotrieve]
23. Stasch, J.-P., Becker, E. M., Alonso-Alija, C., Apeler, H., Dembowsky, K., Feurer, A., Gerzer, R., Minuth, T., Perzborn, E., Pleibeta , U., Schröder, H., Schroeder, W., Stahl, E., Steinke, W., Straub, A., and Schramm, M. (2001) Nature 410, 212-215[CrossRef][Medline] [Order article via Infotrieve]
24. Kharitonov, V. G., Sharma, V. S., Madge, D., and Koesling, D. (1999) Biochemistry 38, 10699-10706[CrossRef][Medline] [Order article via Infotrieve]
25. Sundberg, L., and Porath, J. (1974) J. Chromatogr. 90, 87-98[CrossRef][Medline] [Order article via Infotrieve]
26. Seybert, D., Moffat, K., Gibson, Q. H., and Chang, C. K. (1977) J. Biol. Chem. 252, 4225-4231[Medline] [Order article via Infotrieve]
27. Yonetani, T. (1961) J. Biol. Chem. 236, 1680-1688[Medline] [Order article via Infotrieve]
28. Paul, K. G., Theorell, H., and Akesson, A. (1953) Acta Chem. Scand. 7, 1284-1287
29. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672
30. Stone, J. R., Sands, R. H., Dunham, W. R., and Marletta, M. A. (1995) Biochem. Biophys. Res. Commun. 207, 572-577[CrossRef][Medline] [Order article via Infotrieve]
31. Kim, S. Y., Deinum, G., Gardner, M. T., Marletta, M. A., and Babcock, G. T. (1996) J. Am. Chem. Soc. 118, 8769-8770[CrossRef]
32. Tsubaki, M., Hiwatashi, A., and Ichikawa, Y. (1987) Biochemistry 26, 4535-4540[Medline] [Order article via Infotrieve]
33. Yu, N.-T., and Kerr, E. A. (1988) in Biological Applications of Raman Spectroscopy (Spiro, T. G., ed), Vol. 3 , pp. 39-95, John Wiley & Sons, Inc., New York
34. Li, X.-Y., and Spiro, T. G. (1988) J. Am. Chem. Soc. 110, 6024-6033
35. Uno, T., Nishimura, Y., Makino, R., Iizuka, T., Ishimura, Y., and Tsuboi, M. (1885) J. Biol. Chem. 260, 2023-2026
36. Ray, B. G., Li, X.-Y., Ibers, A. J., Sessler, J. L., and Spiro, T. G. (1994) J. Am. Chem. Soc. 116, 162-176
37. Vogel, K. V., Spiro, T. G., Shelver, D., Thorsteinsson, M. V., and Roberts, G. P. (1999) Biochemistry 38, 2679-2687[CrossRef][Medline] [Order article via Infotrieve]
38. Chu, G. C., Katakura, K., Tomita, T., Zhang, X., Sun, D., Sato, M., Sasahara, M., Kayama, T., Ikeda-Saito, M., and Yoshida, T. (2000) J. Bio. Chem. 276, 17494-17500[CrossRef]
39. Yamada, H., Makino, R., and Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353[Medline] [Order article via Infotrieve]
40. Makino, R., Iizuka, T., Sakaguchi, K., and Ishimura, Y. (1982) in Oxygenase and Oxygen Metabolism (Nozaki, M. , Yamamoto, S. , Ishimura, Y. , Coon, M. J. , Ernster, L. , and Estabrook, R. W., eds) , pp. 467-477, Academic Press, Inc., New York
41. Walker, F. A., Berioz, D., and Kadish, K. M. (1976) J. Am. Chem. Soc. 76, 3484-3489
42. Yoshimura, T. (1991) Bull. Chem. Soc. Jpn. 64, 2819-2828
43. Shiro, Y., Fujii, M., Isogai, Y., Adachi, S., Iizuka, T., Makino, R., Obayashi, E., Nakahara, K., and Shoun, H. (1995) Biochemistry 34, 9052-9058[Medline] [Order article via Infotrieve]
44. Tsubaki, M., Hiwatashi, A., Ichikawa, Y., and Hori, H. (1987) Biochemistry 26, 4527-4534[Medline] [Order article via Infotrieve]
45. Kon, H., and Kataoka, N. (1969) Biochemistry 12, 4757-4762
46. Wayland, B. B., and Olson, L. W. (1974) J. Am. Chem. Soc. 96, 6037-6041[Medline] [Order article via Infotrieve]
47. Scheidt, W. R., Brinergar, A. C., Ferro, E. B., and Kirner, J. F. (1977) J. Am. Chem. Soc. 99, 7315-7322
48. Kon, H. (1968) J. Biol. Chem. 243, 4350-4357[Abstract/Free Full Text]
49. Walch, A., Nan, H., Chantranunpong, L., and Loew, G. H. (1989) J. Am. Chem. Soc. 111, 2767-2772
50. Migita, C. T., Salerno, J. C., Masters, B. S. S., Martasek, P., McMillan, K., and Ikeda-saito, M (1997) Biochemistry 36, 10987-10992[CrossRef][Medline] [Order article via Infotrieve]
51. Traylor, T. G., and Sharma, V. S. (1992) Biochemistry 31, 2847-2849[Medline] [Order article via Infotrieve]
52. Sharma, V. S., Magde, D., Karitonov, V. G., and Koesling, D. (1999) Biochem. Biophys. Res. Commun. 254, 188-191[CrossRef][Medline] [Order article via Infotrieve]
53. Stone, J. R., and Marletta, M. A. (1998) Chem. Biol. 5, 255-261
54. Zhang, G., Yu, L., Ruoho, A. E., and Hurley, J. H. (1997) Nature 386, 247-253[CrossRef][Medline] [Order article via Infotrieve]
55. Martin, E., Lee, Y.-C., and Murad, F. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12938-12942[Abstract/Free Full Text]


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