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Department of Biochemistry and Microbiology, Laval University, Quebec City, Quebec, Canada
Correspondence: Address reprint requests to Manon Couture, Assistant Professor, Dept. of Biochemistry and Microbiology, Pavillon Marchand, Room 4165, Laval University, Quebec City, Quebec G1K 7P4, Canada. Tel.: 418-656-2131 ext. 13515; Fax: 418-656-7176; E-mail: manon.couture{at}bcm.ulaval.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Fe-CO at 482 and 497 cm–1 and C-O at 1949 and 1930 cm–1, respectively. Similarly to mammalian NOS, the binding of L-arginine to SANOS caused the formation of a single CO complex with Fe-CO and C-O frequencies at 504 and 1917 cm–1, respectively, indicating that L-arginine induced an electrostatic/steric effect on the CO molecule. The addition of pterins to CO-bound SANOS modified the resonance Raman spectra only when they were added in combination with L-arginine. We found that (6R) 5,6,7,8 tetra-hydro-L-biopterin and tetrahydrofolate were not required for the stability of the reduced protein, which is 5-coordinate, and of the CO complex, which does not change with time to a form with a Soret band at 420 nm that is indicative of a change of the heme proximal coordination. Since SANOS is stable in the absence of added pterin, it suggests that the role of the pterin cofactor in the bacterial NOS may be limited to electron/proton transfer required for catalysis and may not involve maintaining the structural integrity of the protein as is the case for mammalian NOS. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The best characterized NOS are those from mammals that catalyze through two consecutive catalytic cycles the hydroxylation of L-arginine into nitric oxide and L-citrulline (Marletta, 1994). Animal NOS are homodimeric proteins and consist of an amino-terminal domain containing a heme group, a C-terminal reductase domain with FMN and FAD cofactors, and a regulatory region that control the rate of electron transfer between these two domains (Alderton et al., 2001; Marletta, 1994; Raman et al., 2000). The electrons required for the catalytic reactions are provided by NADPH and travel to the heme via the FMN and FAD cofactors of the reductase domain of the enzyme. Essential to the enzyme activity is a pterin cofactor, H4B, without which the enzyme does not produce NO (Adak et al., 2000; Cosentino and Luscher, 1999; Wei et al., 2003). This pterin binds at the dimer interface, close to the heme group. H4B forms extensive Van der Waals interactions and H-bonds with surrounding amino acids and the heme propionates, which helps stabilize the dimeric form of the enzyme (Abu-Soud et al., 1995). In all NOS characterized to date, the heme is attached to the protein moiety through an iron-thiolate coordination bond. The active site of the enzyme is composed of the heme group, the H4B binding site, and the L-arginine binding site near the iron on the opposite side of the iron-thiolate bond.
The first description of a bacterial, L-arginine-dependent NO synthase activity has been from the bacterium Nocardia (Chen and Rosazza, 1994, 1995). Recently, genes encoding proteins similar to animal NOS have been found in other bacteria including Bacillus subtilis (Kunst et al., 1997), Staphylococcus aureus (Kuroda et al., 2001), and Deinococcus radiodurans (White et al., 1999) (Fig. 1). Bacterial NOS are particular in that they lack the reductase domain (Fig. 1) (Bird et al., 2002). Several residues near the amino-terminus are also missing (Fig. 1). In the folded animal NOS, these amino-terminal residues protect the pterin site from exposure to the solvent. Three bacterial NOS, BANOS (Adak et al., 2002a), SANOS (Bird et al., 2002), and DANOS (Adak et al., 2002b), have been expressed as recombinant proteins in Escherichia coli. BANOS was found to synthesize NO in single-turnover experiments when supplied with NG-hydroxy-L-arginine and H4B (Adak et al., 2002a), and both BANOS and DANOS were shown to synthesize nitrite under equilibrium conditions using L-arginine or NG-hydroxy-L-arginine as substrates (Adak et al., 2002a,b). However, the turnover number was found to be very small possibly due to the use of the reductase domain of a mammalian NOS instead of the cognate reductase. It must be stressed that although the bacterial NOS are able to carry out NO synthesis in vitro, no evidence exists for such a reaction in vivo.
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) and that of SANOS in complex with the NOS inhibitor SEITU (S-ethylisotiourea) were recently published (Bird et al., 2002). The structure showed that in SANOS, the pterin binding site is more exposed to the solvent than that of mammalian NOS because several of the N-terminal residues that protect the pterin from solvent exposure in mammalian NOS are missing. Nonetheless, the protein exists as a dimer in solution and the crystal structure revealed that the dimer is stabilized by alternative interactions (Bird et al., 2002). The pterin binding site is larger in SANOS than in mammalian NOS and is occupied by a NAD+ molecule in the crystals. It appears that some bacteria, such as S. aureus, in which NOS genes have been found, may not be able to synthesize H4B because they lack the sepiapterin reductase, the last enzyme of the biosynthetic pathway for the synthesis of H4B (Adak et al,. 2002b; Raman et al., 2000). However, it is possible that B. subtilis may be able to synthesize H4B (Adak et al., 2002a). The nature of the natural cofactor of the bacterial NOS remains to be determined. To characterize the environment of the active site of bacterial NOS, we purified the recombinant SANOS and probed it with resonance Raman spectroscopy, which is a very powerful technique that provides a wealth of information about the structure and electronic properties of the heme group and of the heme axial ligands. We show that the binding of L-arginine to SANOS induces changes to the heme group similar to those observed in mammalian NOS. However, the binding of H4B and THF have little structural/electronic effects on the protein and we show that these are not required for stability of the ferrous and the CO complex. Our results suggest that, in contrast to mammalian NOS, the pterin cofactor of the bacterial NOS may not have a role in the maintenance of the structural integrity of the protein although it would be required for the catalytic activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Protein expression and purification
The SANOS gene was amplified by the PCR method from S. aureus genomic DNA (American Type and Culture Collection (ATCC) 35556) and cloned in the pet30B expression vector (Novagen, Madison, WI) in frame with an N-terminal tag composed of six histidine residues. The cloned gene was sequenced at a local facility and found to be identical to the sequence deposited at the National Center for Biotechnology Information (NCBI) (NC_002745). The recombinant protein was expressed in E. coli BL21(DE3) and was purified by affinity chromatography over a Ni2+-Sepharose column (Amersham Biosciences, Baie d'Urfé, Canada). Briefly, E. coli cells grown in Terrific Broth medium (Sambrook et al., 1989) supplemented with 10 µg/ml of kanamycin were recovered after an overnight induction by 1 mM isopropyl-ß-D-thiogalactopyranoside that was added when the optical density of the culture had reached 0.8. The cells were disrupted using a French pressure cell and the cell lysate was centrifuged at 10000 x g for 30 min at 4°C. Ammonium sulfate precipitation was then carried out on the soluble proteins fraction. Proteins that precipitated between 35% and 50% ammonium sulfate saturation were harvested by centrifugation, resuspended in purification buffer (40 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM PMSF) and loaded on a 40-ml Ni2+-sepharose column. The column was washed successively with 10 column volumes of purification buffer containing 20 mM, 50 mM, and 100 mM imidazole, respectively. SANOS was eluted in purification buffer containing 400 mM imidazole. The purified protein was dialyzed at 4°C against 40 mM Hepes, pH 7.5, buffer containing 1 mM DTT, 150 mM NaCl and 10% glycerol and was kept at –80°C. The purified protein migrated with an apparent molecular weight of 42 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight in solution, determined by gel filtration over a Superdex HR200 column (Amersham Biosciences, Baie d'Urfe, Canada), was 72 kDa indicating that SANOS is a dimer in solution (The calculated molecular mass of the SANOS monomer including heme and the His-tag is 45.9 kDa). The buffer used for gel filtration chromatography was 40 mM Hepes, pH 8, 150 mM NaCl, and 1 mM DTT. The heme b content was quantified by the pyridine hemochrome method (Appleby, 1978).
Affinity for L-arginine
The affinity of SANOS for L-arginine was determined by the spectral changes caused by L-arginine binding to the ferric enzyme (McMillan and Masters, 1993).
Sample preparation for spectroscopy
The buffer used was 40 mM Hepes, pH 7.6, containing 1 mM DTT and 40 mM NaCl. To obtain spectrum of the ferric protein in the absence of substrate, DTT was not included in the buffer as this compound forms a low-spin complex with the heme. Where indicated, 1 mM L-arginine, 600 µM THF, and 1.2 mM H4B were added to the protein samples.
Optical spectroscopy
Optical spectra were recorded with a Cary 3 spectrophotometer with samples placed in a 1-cm path-length anaerobic cuvette (Hellma, Müllheim, Germany). The Fe2+ sample was prepared by equilibration of the sample solution with argon followed by the addition of a small volume of a freshly prepared anaerobic solution of sodium dithionite. The CO complexes were prepared by incubating the protein samples with argon, adding a known volume of CO gas, and reducing the heme with a small amount of freshly prepared sodium dithionite. The protein concentration was 5 µM.
Resonance Raman spectroscopy
To obtain resonance Raman spectra, the 441.6 nm line of an He/Cd laser (Liconix laser, Melles-Griot, Canada) was used to probe CO complexes, and the 406 and 413 lines of a Kr-ion laser (Innova 302 Kr laser, Coherent, Santa Clara, CA) were used to probe ferric and ferrous forms of SANOS. The laser beam was focused to an 
55 µM spot on a custom-made sample cuvette, which was kept rotating at 1000 rpm to avoid local heating of the sample. The scattered light was collected at a 90° angle with an F#1 lens and refocused with an F#9.8 lens on the entrance slit of a 0.75-m spectrograph (Acton Research, Acton, MA) equipped with an 1800 lines/mm diffraction grating. A Notch filter, designed to block light at 441.6 nm (MK Photonics, Albuquerque, NM) or at 406 and 413 nm (Kaiser Optical, Ann Arbor, MI) was placed in front of the entrance slit of the spectrometer to prevent Rayleigh scattered light from entering the spectrometer. The width of the spectrometer slit was set at 100 microns. The diffracted light was detected at the spectrometer exit with a liquid nitrogen-cooled CCD camera (Spec10:100B, Roper Scientific, Trenton, NJ). Cosmic rays were automatically removed from spectra by a software routine of WinSpec (Roper Scientific). Typically, several 1-min spectra were recorded with a low excitation power, 1.4–4.8 mW, at room temperature and averaged with the Grams software (ThermoGalactic, Salem, NH). The spectra were calibrated with the lines of indene in the 200–1700 cm–1 region and with the lines of acetone and potassium ferrocyanide in the 1600–2100 cm–1 region. To verify the stability of the samples, optical spectra of the samples in the Raman cuvette were recorded before and after resonance Raman spectra were obtained. Typically, samples containing 30–40 µM protein, based on the heme content, were used to acquire the resonance Raman spectra. To obtain the resonance Raman spectra of ferrous SANOS in the perpendicular and parallel orientations, a polarizer (Newport, Irvine, CA) in combination with a polarization scrambler was placed in front of the entrance slit of the spectrometer. The efficiency of this setup was verified by measuring the polarization ratio of the 460 cm–1 line of CCl4.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The addition of H4B or THF to SANOS changed the optical spectrum of the ferric enzyme and we observed an increase of the intensity of the band at 398 nm and a diminution of the intensity at 418 nm (not shown). This behavior is similar to that of mammalian NOS, which display a shift from a mixture of 5-coordinate and 6-coordinate forms to a 5-coordinate, high-spin form with a Soret band near 397 nm when saturated with H4B (Ghosh et al., 1997). These spectral changes caused by the addition of H4B or THF alone to SANOS indicate that these molecules bind to the protein and that they induce a shift in the heme coordination state similar to that of the mammalian NOS.
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). Addition of CO to the ferrous protein shifted the Soret band to 445 nm with a minor contribution near 423 nm (Fig. 2). For the CO complex of NOS and P450, a Soret band near 450 nm indicates that the heme forms an iron-thiolate bond on the proximal side whereas a Soret band near 420 nm is indicative of a protein in an inactive state called P420 (Wang et al., 1995; Wells et al., 1992). For P450cam, the proximal ligand of the P420 form was shown to be a histidine residue (Wells et al., 1992). No change in the optical spectrum of the CO complex of SANOS was observed upon addition of L-arginine, confirming that the majority of the protein is already in a native-like conformation with a cysteine residue as the proximal ligand to the heme. In contrast to the situation observed for the mammalian NOS (Wang et al., 1995), the CO complex of SANOS is very stable in the absence of pterin and substrate and no significant increase in the content of the P420 form was observed over the course of a 24-h incubation at room temperature (not shown). Under the same conditions, nNOS and iNOS convert to the P420 form with time (Wang et al., 1995).
Resonance Raman spectra of ferric SANOS
The high-frequency region of the resonance Raman spectra of heme proteins (1300–1700 cm–1) contains in-plane vibrational modes of the porphyrin ring that are sensitive to the oxidation, coordination, and spin state of the heme iron (Hu et al., 1996; Spiro and Li, 1988; Wang et al., 1996). In particular, the 4 mode is sensitive to the electron density of the porphyrin ring and the 2 and 3 modes are sensitive to the coordination and spin state of the heme iron (Spiro and Li, 1988; Wang et al., 1996). The assignment of the coordination and spin state of ferric SANOS was obtained from the resonance Raman spectra of the substrate-free and L-arginine-bound enzyme recorded in the high-frequency region (Fig. 3). The frequencies of the 4 line at 1373 cm–1 for the substrate-free (Fig. 3 B) and at 1369 cm–1 for the L-arginine-bound protein (Fig. 3 C), respectively, are typical of oxidized heme proteins. Two 3 lines, at 1489 and 1503 cm–1, respectively, were identified in the spectrum of the substrate-free SANOS (Fig. 3 B), indicating a mixture of 5-coordinate and high-spin (1489 cm–1) and 6-coordinate and low-spin (1503 cm–1) ferric hemes. Upon the addition of L-arginine, a single 3 line is observed at 1488 cm–1 (Fig. 3 C), indicating that the heme is totally 5-coordinate and high-spin. In the latter spectrum, the identification of the 2 line at 1563 cm–1 confirms this assignment. The assignment of the 2 line of the substrate-free protein was not possible because this region of the spectrum is complicated by the presence of additional heme modes (Fig. 3 B).
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10 mode that is usually observed near 1638 cm–1 in the spectra of 6-coordinate and low-spin ferric heme proteins. For SANOS, the 10 mode is observed at 1639 cm–1 (Fig. 3 B). As reported for eNOS (Schelvis et al., 2002) and nNOS (Wang et al., 1995), the 10 mode of the 5-coordinate and high-spin form of SANOS convolutes with the C-C vinyl stretching mode at 1624 cm–1. A single 10/vinyl stretching mode was identified in the presence (1624 cm–1) or absence (1626 cm–1) of L-arginine (Fig. 3, B and C). The nearly identical frequencies indicate that the binding of L-arginine does not induce a significant change in the orientation and/or environment of the vinyl groups. A similar behavior has been reported for the mammalian eNOS (Schelvis et al., 2002).
Resonance Raman spectra of ferrous SANOS
The high-frequency region of the resonance Raman spectrum of the ferrous form of substrate-free and pterin-free SANOS is showed in Fig. 3 D. The 4 and 3 lines are found at 1349 cm–1 and 1467 cm–1, respectively, showing that the heme is 5-coordinate and high-spin. The low-frequency of the 4 mode is similar to the frequencies of the 4 modes of P450 (Champion et al., 1978) and nNOS (Wang et al., 1995) and is indicative of an iron-thiolate coordination. Other heme proteins with histidine-iron coordination have a 4 line at higher frequency, near 1355 cm–1. The heme coordination and spin state of the ferrous SANOS did not change upon L-arginine binding (results not shown), and the heme thus remained 5-coordinate and high-spin. In contrast, ferrous nNOS is mostly 6-coordinate and low-spin in the absence of pterin and 5-coordinate and high-spin in the presence of H4B (Wang et al., 1995). The 10/vinyl region of ferrous SANOS, with lines at 1602 and 1619 cm–1 (Fig. 3 D), is similar to that of nNOS/H4B with lines at 1600 and 1617 cm–1 (Table 1). The ferrous, H4B-depleted nNOS, representing the P420 form, displays a single 10/vinyl mode at 1615 cm–1 (Table 1) (Wang et al., 1995). The similarity of the ferrous spectrum of SANOS with that of nNOS/H4B is a further indication that very little inactive P420 form is present in our protein preparation.
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), is present in the reduced spectrum of SANOS (Fig. 3 D). As discussed above, it is unlikely that this line corresponds to the 4 line of a P420 form; moreover, the 4 line of such a P420 form would be expected near 1361 cm–1 based on the frequency observed for the ferrous P420 form of P450 (Wells et al., 1992) and nNOS (Wang et al., 1995). The 1369 cm–1 line also does not arise from the 4 line of oxidized heme. First, the optical spectrum of the ferrous sample used to obtain the Raman spectrum was verified to rule out the possibility that the sample oxidized during data acquisition. Second, the 4 line of the substrate-free ferric enzyme is actually at a higher frequency, 1373 cm–1 (Fig. 3 B). Measurement of the polarization sensitivity of ferrous SANOS revealed that the 1369 cm–1 line is not polarized, with a polarization ratio of 0.99, in contrast to the 4 line at 1349 cm–1 that displays a polarization ratio of 0.28 (Fig. 3, D and E). This result establishes that the 1369 cm–1 arises from a heme mode other than 4, and therefore we conclude that ferrous SANOS is 5-coordinate and high-spin in the presence or absence of L-arginine and pterins with no other detectable form.
Resonance Raman spectra of the CO complexes
CO is widely used to probe the active site of heme proteins because it produces a very stable heme complex and because it is a sensitive probe of electronic and steric interactions within the heme pocket (Aono et al., 2002; Couture et al., 2001; Fan et al., 1997; Igarashi et al., 2003; Mukai et al., 2002; Phillips et al., 1999; Uchida et al., 1998; Wang et al., 1997). In the high-frequency region of the resonance Raman spectrum of the CO complex (Fig. 3 A), the 4 line and the 3 line are identified at 1370 and 1495 cm–1, respectively, revealing that the complex is 6-coordinate and low-spin. No significant photodissociation of the heme-bound CO molecule occurred during spectral acquisition as indicated by the absence of the 4 line (1349 cm–1) and of the 3 line (1467 cm–1) of the 5-coordinate, high-spin ferrous form. In contrast, these two lines are present in the resonance Raman spectrum of the CO complex acquired at high laser power (50 mW; results not shown).
CO isotopes were used to identify the Fe-CO and 
Fe-C-O modes in the low-frequency region of the resonance Raman spectra. The spectra of the substrate-free enzyme were recorded first. Fig. 4 shows an isotope-sensitive line located approximately at 489 cm–1 that shifts monotonously in going from 12C16O (Fig. 4 D), to 13C16O (Fig. 4 C), to 12C18O (Fig. 4 B), and to 13C18O (Fig. 4 A). This behavior is expected for the Fe-CO stretching mode of a CO complex and has been observed for many heme proteins (Wang et al., 1997; Yu et al., 1984). The isotope sensitive line at 489 cm–1, which is quite large (with a width at half-height of 35 cm–1), was further investigated by spectral deconvolution. For the 12C16O spectrum, two lines, at 482 and 497 cm–1, respectively, with approximately the same area under the curve, were deconvoluted (not shown). In contrast, the 13C18O spectrum revealed two major lines at 470 cm–1 and 481 cm–1 and a smaller intensity line at 498 cm–1 (Fig. 4, inset). Since the line at 498 cm–1 is not shifting with the different CO isotopes, the latter was assigned to a heme mode. We conclude that the 497 cm–1 line deconvoluted from the 12C16O spectrum is composed of a small-intensity heme mode at 498 cm–1 and of a larger-intensity line at 497 cm–1 that we assign to a Fe-CO mode. SANOS thus displays two Fe-CO modes located at 482 cm–1 and 497 cm–1, respectively, with 12C16O. These lines shift to 470 cm–1 and 481 cm–1, respectively, with 13C18O. The magnitude of the 12C16O – 13C18O isotope shift of the 497 cm–1 mode, 16 cm–1, corresponds to the value expected for a system behaving as a diatomic oscillator and the magnitude of the isotope shift of the 482 cm–1 line, 12 cm–1, is smaller than the expected shift but is similar to that observed in nNOS (Wang et al., 1997) (Table 2).
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Fe-C-O bending mode of SANOS as it displayed the typical zigzag pattern of the Fe-C-O bending mode of heme proteins (Yu et al., 1984).
To identify the C-O stretching mode of substrate-free SANOS, the resonance Raman spectra were recorded in the 1600–2100 cm–1 region (Fig. 5, A–C). Two lines, at 1949 cm–1 and 1930 cm–1, respectively, that shifted to 1857 cm–1 and 1843 cm–1, respectively, with 13C18O, were assigned to two C-O stretching modes as they fall in the region observed for the C-O stretching modes of other heme proteins (Table 2). The magnitude of the isotope shifts, 92 cm–1 and 87 cm–1, respectively, corresponds well to the theoretical value expected for a diatomic oscillator (91 cm–1). Based on the reversed relationship displayed by the frequencies of the Fe-CO and C-O modes (Li and Spiro, 1988; Ray et al., 1994; Vogel et al., 2000), we propose the existence of two CO complexes of SANOS: one is characterized by the 1930 cm–1 C-O mode and the Fe-CO mode at 497 cm–1 whereas the second is characterized by the 1949 cm–1 C-O mode and the Fe-CO mode at 482 cm–1. As shown in Fig. 7, a plot of the Fe-CO versus C-O frequencies shows that the modes for SANOS, open circles 1 and 2, fall in the region of heme proteins having thiolate coordination.
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Fe-CO mode of substrate-bound nNOS (Table 2) and is therefore assigned to the Fe-CO mode of L-arginine-bound SANOS. The isotope shift of 8 cm–1 is smaller than expected but is similar to that of nNOS (Table 2). Thus the two Fe-CO modes detected in the absence of L-arginine are replaced in the L-arginine-bound SANOS by a single Fe-CO mode at higher frequency. The bending mode of the CO complex of L-arginine-bound SANOS is also found at a higher frequency, 567 cm–1 (Fig. 6 D), than that of the CO complex of L-arginine-free SANOS, 560 cm–1 (Fig. 4 D, Table 2).
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C-O region of the CO spectrum of L-arginine-bound SANOS, a single isotope-sensitive line was detected at 1917 cm–1 with 12C16O and at 1830 cm–1 with 13C18O (Fig. 5). This line falls in the region where the C-O modes of other heme proteins occur (Table 2) and it displayed an isotopic shift, 87 cm–1, close to the theoretical value expected for a diatomic oscillator, 90 cm–1. It is therefore assigned to the C-O stretching mode of the CO complex of L-arginine-bound SANOS. Thus, the two C-O stretching modes observed at 1930 and 1949 cm–1, respectively, with the L-arginine-free enzyme are replaced by a single C-O mode at 1917 cm–1 upon L-arginine binding (Table 2).
Inspection of the resonance Raman spectra of the L-arginine-bound enzyme (Fig. 6 D) and the L-arginine-free enzyme (Fig. 4 D) indicated that binding of L-arginine caused several changes to the resonance Raman spectrum of the CO complex. These differences are best seen in the difference spectrum calculated from the spectrum of the L-arginine-bound enzyme minus that of the substrate-free enzyme (Fig. 6 F). Among these, the most significant changes are new maxima at 691 cm–1 and 734 cm–1, an increase of the intensity of lines near 747 and 801 cm–1, as well as a narrowing of the Fe-CO region that is revealed by the decrease in intensity near 484 cm–1. Based on the assignment of heme modes of myoglobin (Hu et al., 1996), the line at 747 cm–1 may correspond to the 15 pyrrole breathing mode whereas the line at 801 cm–1 may correspond to 6, a heme ruffling mode. These changes at 747 and 801 cm–1 reveal that the heme itself undergoes structural and electronic modifications after the binding of L-arginine. In contrast, the binding of H4B alone does not perturb the spectrum of the CO complex as shown by the difference spectrum calculated between the H4B-saturated enzyme and the H4B-free protein (Fig. 6 H) that displayed only a small imbalance of the intense line at 676 cm–1. THF alone did not change the spectrum either (results not shown). However, the addition of H4B (Fig. 6 A) or THF (Fig. 6 B) in combination with L-arginine revealed changes in the intensity and position of several heme modes. This is best observed in the difference spectrum calculated from the spectrum of the L-arginine/H4B sample minus the spectrum of the L-arginine saturated sample (Fig. 6 G). For instance, there is an increase of the intensity in the Fe-CO mode at 499 cm–1, of the bending mode at 563 cm–1, and other small changes to heme modes in the 700–800 cm–1 region.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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), in S. aureus (Kuroda et al., 2001), and in D. radiodurans (White et al., 1999). We presented here the first detailed characterization of the heme environment of a bacterial NOS in solution.
Heme coordination and spin state of ferric and ferrous SANOS
For the purpose of comparison with the mammalian NOS, the effects of pterin and L-arginine binding on the heme coordination state of ferric and ferrous SANOS were determined. Ferric SANOS, as purified, represents a mixture of 6-coordinate, low-spin and 5-coordinate, high-spin species (Fig. 3, Table 1). Six-coordinate and low-spin ferric forms are observed for P450cam, nNOS, and eNOS (Table 1). For the P450cam enzyme, the low-spin species is attributed to a form with a water molecule that is bound to the iron on the distal side of the heme (Poulos et al., 1986). Upon binding of substrate (L-arginine for NOS and camphor for P450cam) or cofactor (H4B in NOS), this water molecule is displaced to give rise to a 5-coordinate and high-spin species (Table 1). Similarly, ferric SANOS is converted into a 5-coordinate and high-spin species upon binding L-arginine or H4B as shown in the resonance Raman spectra where a single 3 line is detected at 1488 cm–1 (Table 1). This change of coordination and spin state suggests the existence of a binding site for L-arginine and for a pterin cofactor and that their occupancy by a substrate or a pterin molecule, respectively, prevents the coordination of a water molecule to the iron.
It is noteworthy that ferrous SANOS is 5-coordinate and high-spin, even in the absence of L-arginine and pterin. This is shown by the low frequency of the 4 line (1349 cm–1) that is typical of other thiolate-coordinated, 5-coordinate reduced heme proteins, and by the frequency of the 3 line at 1467 cm–1 that is typical of 5-coordinate and high-spin ferrous hemes. Also of interest is the finding that in contrast to mammalian nNOS and iNOS characterized in the absence of the pterin cofactor (Wang et al., 1995), there is no evidence of a 6-coordinate, low-spin form of ferrous SANOS (Fig. 3, D and E).
The comparison of the CO complexes of SANOS with respect to those of the mammalian NOS
CO is widely used to probe the heme environment of heme proteins. It is particularly sensitive to polar interactions due to the possible electronic delocalization on the Fe-C-O unit (Phillips et al., 1999). It is also sensitive to steric interactions that cause the bending or the tilting of the Fe-C-O unit. We have determined the frequencies of the Fe-CO, C-O, and Fe-C-O modes of SANOS. We use these to deduce the properties of the heme pocket and compare them to those of mammalian NOS and P450. First, in the absence of exogenous substrate, we detect two Fe-CO modes at 482 and 497 cm–1, respectively, and two C-O modes at 1949 and 1930 cm–1, respectively. This indicates that in SANOS, the CO molecule can adopt two conformations. In nNOS, two conformations of the Fe-C-O unit were observed and assigned to an open conformation, Fe-CO at 487 cm–1 and C-O at 1949 cm–1, in which the CO molecule is not interacting with neighboring groups, and a closed conformation, with Fe-CO at 501 cm–1 and C-O at 1930 cm–1, in which a positively charged group interacts with the heme-bound CO molecule (Fan et al., 1997). Similarly, for SANOS, the two conformations can be described as an open conformation where the CO molecule does not interact strongly with neighbor groups, with Fe-CO at 482 cm–1 and C-O at 1949 cm–1, and a conformation in which a nearby group, positively charged, is interacting with the CO molecule, that give rise to a Fe-CO mode at 497 cm–1 and a C-O mode at 1930 cm–1. However, upon inspection of the SANOS crystal structure, it does not appear that a positively charged group is present in the vicinity of the heme on the distal side (Bird et al., 2002). It may be that the structure of the ferrous protein in complex with CO differs from that of the ferric enzyme, which is the form crystallized, and that a conformational change occurs upon heme reduction or ligand binding. When plotted on the Fe-CO versus C-O correlation graph, the values of the Fe-CO and C-O modes of SANOS and SANOS/Arg fall in the region observed for heme proteins having thiolate coordination (Fig. 7, 1–3). This confirms that the fifth axial ligand of the enzyme in the CO complex is a cysteine residue.
In ferric SANOS, the change in coordination and spin state induced upon binding of L-arginine occurs in the absence of exogenously added pterin, which suggests that SANOS forms a strong complex with L-arginine. This is supported by the finding that the affinity for L-arginine is high, 4.4 ± 0.2 µM (Chartier and Couture, unpublished results), close to the values observed for nNOS (Ghosh et al., 1997). The structure of the heme pocket of SANOS is affected by the binding of L-arginine as observed in the low-frequency region of the resonance Raman spectrum of the CO complex, which displays modifications to several heme modes. Similar to nNOS (Wang et al., 1997) and iNOS (Fan et al., 1997; Li et al., 2004), we observe an increase in the intensity of the modes at 692, 751, and 801 cm–1, respectively, upon L-arginine binding. Recently, Li et al. showed that several modes in the low-frequency region of iNOS in complex with CO and NO are modulated by L-arginine and H4B binding (Li et al., 2004). These changes were attributed to distortion of the heme imposed by the binding of L-arginine and H4B (Li et al., 2004). The similarity of the resonance Raman spectra of L-arginine-bound SANOS and that of the mammalian NOS indicate that L-arginine binds in a similar manner in these proteins. The correlation between the modifications to the heme structure, imposed by L-arginine binding, and the catalytic properties of NOS are not known but they may be relevant to the various rates of heme reduction displayed by NOS (Santolini et al., 2001).
In addition to changes to heme modes in the low-frequency region, L-arginine binding caused the replacement of the two Fe-CO and C-O modes detected with the L-arginine free enzyme (Table 2) by a single Fe-CO mode at 504 cm–1. The frequency of the Fe-CO mode of the L-arginine-bound SANOS complex is similar to that of nNOS/Arg, Fe-CO at 503 cm–1, but the C-O frequency differs significantly, 1917 cm–1 versus 1929 cm–1, respectively (Table 2). This is more easily observed on the Fe-CO versus C-O correlation graph (Fig. 7), where the values for the L-arginine-bound SANOS complex (open circle 3) are on the correlation line whereas those of nNOS/Arg are above the correlation line (solid circle 6). This observation may be due to a stronger Fe-thiolate bond in SANOS in comparison to the mammalian NOS. Determination of the frequency of the Fe-Cys mode should reveal whether the strength of the Fe-thiolate bond differs in SANOS as compared to that of eNOS (Schelvis et al., 2002), the only mammalian NOS for which this value has been determined.
The bending mode of the Fe-CO complex is a sensitive indicator of the heme proximal ligand. It is found in the 556–567 cm–1 range in P450 and NOS (Table 2), whereas it is in the 577–579 cm–1 range in Mb, human Hb, and cytochrome oxidase (Ray et al., 1994). The Fe-C-O bending mode of SANOS is detected, with and without substrate, in the range expected from a heme protein with a Fe-thiolate bond (Table 2). Distortion from linearity of the Fe-C-O unit seems to activate the bending mode in some heme proteins. However, distal polar interactions may be even more important, as Ray et al. observed that the intensity of the Fe-C-O bending mode is increased by polar interactions in the distal heme pocket and is correlated with significant back-bonding (Ray et al., 1994). That the bending mode is already intense in the absence of substrate in SANOS suggests either that the Fe-C-O unit is distorted from linearity in the substrate-free protein or that a heme pocket group is involved in polar interactions with the CO. Upon L-arginine binding, the bending mode of SANOS becomes more intense and shifts to higher frequency. This shift to higher frequencies is similar to that observed with nNOS and iNOS upon L-arginine binding (Table 2) and it indicates that steric/polar interactions of the CO molecule with L-arginine are similar in these NOS. That strong polar/steric interactions between CO and L-arginine occur in SANOS is also supported by the smaller than expected isotopic shift of the Fe-CO mode in the presence of L-arginine (8 cm–1, Table 2), as compared to the isotopic shifts in the absence of L-arginine (16 and 12 cm–1, Table 2). This indicates that the CO complex of L-arginine-bound SANOS is not well described by the simple model composed of a two-body oscillator.
Stability of pterin-free SANOS and relation to function
We have shown that ferrous SANOS is totally 5-coordinate and high-spin in the absence of substrate and pterin. We also showed that the CO complex of SANOS is unusually stable in comparison to the CO complex of H4B-depleted nNOS (Wang et al., 1995) since it shows little change of the absorption spectrum over a 24-h period of incubation at room temperature; the Soret band remained at 445 nm. The band at 445 nm is indicative of the presence of a native structure with thiolate coordination. From the analysis of the primary amino acid sequence and the crystal structure of SANOS (Bird et al., 2002), one might have expected that the protein may have been unstable in the absence of pterin since several residues defining the pterin binding site in mammalian NOS are missing in SANOS, leaving the pterin site more solvent-exposed. This is clearly not the case. Hence, SANOS must have evolved to maintain the structural integrity of the reduced protein in the absence of added cofactor as both the ferrous and ferrous-CO complex are stable with time under these conditions.
That SANOS is stable in the ferrous state without added pterin does not mean that the protein does not require a pterin for its function. With BANOS, it was shown that NO synthesis in single turnover experiments with NG-hydroxy-L-arginine as the substrate was dependent on the presence of H4B (Adak et al., 2002a). With DANOS, nitrite formation was stimulated by H4B in an in vitro assay performed with the reductase domain of nNOS (Adak et al., 2002a). SANOS (F. Chartier and M. Couture, unpublished results), BANOS (Adak et al., 2002a), and DANOS (Adak et al., 2002b) display a slow catalytic rate as determined from the rate of nitrite synthesis under turnover conditions in the presence of the reductase domain of a mammalian NOS. This slow catalytic activity may be related to the use of a pterin that is not the native pterin of bacterial NOS. However, the slow catalytic rates may also result from the use of the reductase domain of a mammalian NOS instead of the native reductase present in the bacteria harboring a NOS gene. Although several N-terminal residues defining the pterin binding site are missing in SANOS and the other bacterial NOS, the crystal structures of SANOS (Bird et al., 2002) and BANOS (Pant et al., 2002) reveal the presence of a cavity corresponding to the H4B binding site of mammalian NOS. The presence of this cavity and the enhancement of catalytic activity by H4B suggest that a pterin-like molecule likely binds at this site in the bacterial NOS in vivo. A gene encoding a putative sepiapterin reductase was identified in B. subtilis genome by sequence comparison (Kunst et al., 1997). Sequence data bank search with sepiapterin reductase sequences are not conclusive about the presence of a similar gene in S. aureus (F. Chartier and M. Couture, unpublished results). Little is known about the pathways for pterin biosynthesis in bacteria harboring a NOS gene, and clearly, more studies are required to determine the nature of the native cofactor of bacterial NOS.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Submitted on March 8, 2004; accepted for publication May 26, 2004.
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REFERENCES |
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Adak, S., K. S. Aulak, and D. J. Stuehr. 2002a. Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis. J. Biol. Chem. 277:16167–16171.
Adak, S., A. M. Bilwes, K. Panda, D. Hosfield, K. S. Aulak, J. F. McDonald, J. A. Tainer, E. D. Getzoff, B. R. Crane, and D. J. Stuehr. 2002b. Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans. Proc. Natl. Acad. Sci. USA. 99:107–112.
Adak, S., Q. Wang, and D. J. Stuehr. 2000. Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric-oxide synthase. Implications for mechanism. J. Biol. Chem. 275:33554–33561.
Alderton, W. K., C. E. Cooper, and R. G. Knowles. 2001. Nitric oxide synthases: structure, function and inhibition. Biochem. J. 357:593–615.[CrossRef][Medline]
Aono, S., T. Kato, M. Matsuki, H. Nakajima, T. Ohta, T. Uchida, and T. Kitagawa. 2002. Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis. J. Biol. Chem. 277:13528–13538.
Appleby, C. A. 1978. Purification of Rhizobium cytochromes P-450. Methods Enzymol. 52:157–166.[Medline]
Bird, L. E., J. Ren, J. Zhang, N. Foxwell, A. R. Hawkins, I. G. Charles, and D. K. Stammers. 2002. Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus. Structure (Camb). 10:1687–1696.[Medline]
Champion, P. M., I. C. Gunsalus, and G. C. Wagner. 1978. Resonance Raman investigations of cytochrome P450cam from Pseudomonas putida. J. Am. Chem. Soc. 100:3743–3751.[CrossRef]
Chen, Y., and J. P. Rosazza. 1994. A bacterial nitric oxide synthase from a Nocardia species. Biochem. Biophys. Res. Commun. 203:1251–1258.[CrossRef][Medline]
Chen, Y., and J. P. N. Rosazza. 1995. Purification and characterization of nitric oxide synthase (NOSNOC) from Nocardia species. J. Bacteriol. 177:5122–5128.
Cosentino, F., and T. F. Luscher. 1999. Tetrahydrobiopterin and endothelial nitric oxide synthase activity. Cardiovasc. Res. 43:274–278.
Couture, M., T. Burmester, T. Hankeln, and D. L. Rousseau. 2001. The heme environment of mouse neuroglobin. Evidence for the presence of two conformations of the heme pocket. J. Biol. Chem. 276:36377–36382.
Das, T. K., J. M. Friedman, A. P. Kloek, D. E. Goldberg, and D. L. Rousseau. 2000. Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin. Biochemistry. 39:837–842.[CrossRef][Medline]
Fan, B., J. Wang, D. J. Stuehr, and D. L. Rousseau. 1997. NO synthase isozymes have distinct substrate binding sites. Biochemistry. 36:12660–12665.[CrossRef][Medline]
Fuchsman, W. H., and C. A. Appleby. 1979. CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin. Biochemistry. 18:1309–1321.[CrossRef][Medline]
Ghosh, D. K., C. Wu, E. Pitters, M. Moloney, E. R. Werner, B. Mayer, and D. J. Stuehr. 1997. Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction. Biochemistry. 36:10609–10619.[CrossRef][Medline]
Hu, S., and J. R. Kincaid. 1991. Resonance Raman characterization of nitric oxide adducts of cytochrome P450cam: the effect of substrate structure on the iron-ligand vibrations. J. Am. Chem. Soc. 113:2843–2850.[CrossRef]
Hu, S., K. M. Smith, and T. G. Spiro. 1996. Assignment of protoheme resonance Raman spectrum by heme labeling in myoglobin. J. Am. Chem. Soc. 118:12638–12646.[CrossRef]
Igarashi, J., A. Sato, T. Kitagawa, I. Sagami, and T. Shimizu. 2003. CO binding study of mouse heme-regulated eIF-2alpha kinase: kinetics and resonance Raman spectra. Biochim. Biophys. Acta. 1650:99–104.[Medline]
Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G., Bertero, P. Bessieres, A. Bolotin, S. Borchert, et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 390:249–256.[CrossRef][Medline]
Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, and others. 2001. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet. 357:1225–40.[CrossRef][Medline]
Li, D., D. J. Stuehr, S. R. Yeh, and D. L. Rousseau. 2004. Heme distortion modulated by ligand-protein interactions in inducible nitric oxide synthase. J. Biol. Chem. 279:26489–26499.
Li, X.-Y., and T. G. Spiro. 1988. Is bound CO linear or bent in heme proteins? Evidence from resonance Raman and infrared spectroscopic data. J. Am. Chem. Soc. 110:6024–6033.[CrossRef]
Marletta, M. A. 1994. Nitric oxide synthase: aspects concerning structure and catalysis. Cell. 78:927–930.[CrossRef][Medline]
McMillan, K., and B. S. Masters. 1993. Optical difference spectrophotometry as a probe of rat brain nitric oxide synthase heme-substrate interaction. Biochemistry. 32:9875–9880.[CrossRef][Medline]
Mukai, M., P. Y. Savard, H. Ouellet, M. Guertin, and S. R. Yeh. 2002. Unique ligand-protein interactions in a new truncated hemoglobin from Mycobacterium tuberculosis. Biochemistry. 41:3897–3905.[CrossRef][Medline]
Pant, K., A. M. Bilwes, S. Adak, D. J. Stuehr, and B. R. Crane. 2002. Structure of a nitric oxide synthase heme protein from Bacillus subtilis. Biochemistry. 41:11071–11079.[CrossRef][Medline]
Phillips, G. N. J., M. L. Teodoro, T. Li, B. Smith, and J. S. Olson. 1999. Bound CO is a molecular probe of electrostatic potential in the distal pocket of myoglobin. J. Phys. Chem. B. 103:8817–8829.
Poulos, T. L., B. C. Finzel, and A. J. Howard. 1986. Crystal structure of substrate-free Pseudomonas putida cytochrome P-450. Biochemistry. 25:5314–5322.[CrossRef][Medline]
Raman, C. S., P. Martasek, and B. S. S. Masters. 2000. Structural themes determining function in nitric oxide synthases. In The Porphyrin Handbook. K. M. Kadish, K. M. Smith, and R. Guilard, editors. Academic Press, New York. 293–339 p.
Ramsden, J., and T. G. Spiro. 1989. Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin. Biochemistry. 28:3125–3128.[CrossRef][Medline]
Ray, G. B., X.-Y. Li, J. A. Ibers, J. L. Sessler, and T. G. Spiro. 1994. How far can proteins bend the FeCO unit? Distal polar and steric effects in heme proteins and models. J. Am. Chem. Soc. 116:162–176.[CrossRef]
Rodriguez-Crespo, I., P. Moenne-Loccoz, T. M. Loehr, and P. R. Ortiz de Montellano. 1997. Endothelial nitric oxide synthase: modulations of the distal heme site produced by progressive N-terminal deletions. Biochemistry. 36:8530–8538.[CrossRef][Medline]
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Santolini, J., A. L. Meade, and D. J. Stuehr. 2001. Differences in three kinetic parameters underpin the unique catalytic profiles of nitric-oxide synthases I, II, and III. J. Biol. Ch
-helices and the ß-sheets of SANOS are shown below the alignment (Bird et al., 2002
