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• W2085048960 abstract "We report the characterization by resonance Raman spectroscopy of the oxygenated complex (FeIIO2) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with Nω-hydroxy-l-arginine. The frequencies of the νFe–O and νO–O modes were 530 and 1135 cm–, respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (νFe–O at 517 cm–1 and νO–O at 1123 cm–1) and those of substrate-free saNOS (νFe–O at 517 and νO–O at 1135 cm–1) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953–9962), we propose two models that account for the frequency shift of νFe–O (but not νO–O) upon Nω-hydroxy-l-arginine binding as well as the frequency shift of νO–O (but not νFe–O) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed. We report the characterization by resonance Raman spectroscopy of the oxygenated complex (FeIIO2) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with Nω-hydroxy-l-arginine. The frequencies of the νFe–O and νO–O modes were 530 and 1135 cm–, respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (νFe–O at 517 cm–1 and νO–O at 1123 cm–1) and those of substrate-free saNOS (νFe–O at 517 and νO–O at 1135 cm–1) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953–9962), we propose two models that account for the frequency shift of νFe–O (but not νO–O) upon Nω-hydroxy-l-arginine binding as well as the frequency shift of νO–O (but not νFe–O) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed. Nitric-oxide synthases (NOSs) 2The abbreviations used are: NOSs, nitric-oxide synthases; NOHA, Nω-hydroxy-l-arginine; H4B, tetrahydrobiopterin; saNOS, S. aureus nitric-oxide synthase; nNOS, mammalian neuronal nitric-oxide synthase; bsNOS, B. subtilis nitric-oxide synthase; iNOS, mammalian inducible nitric-oxide synthase; eNOS, mammalian endothelial NOS. 2The abbreviations used are: NOSs, nitric-oxide synthases; NOHA, Nω-hydroxy-l-arginine; H4B, tetrahydrobiopterin; saNOS, S. aureus nitric-oxide synthase; nNOS, mammalian neuronal nitric-oxide synthase; bsNOS, B. subtilis nitric-oxide synthase; iNOS, mammalian inducible nitric-oxide synthase; eNOS, mammalian endothelial NOS. are a family of heme proteins that synthesize NO by catalyzing the two-step oxidation of l-arginine using an O2-dependent mechanism (1Raman C.S. Martásek P. Masters B.S.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. 4. 2000: 293-339Google Scholar, 2Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3190) Google Scholar, 3Andrew P.J. Mayer B. Cardiovasc. Res. 1999; 43: 521-531Crossref PubMed Scopus (568) Google Scholar, 4Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (799) Google Scholar, 5Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). NOSs first hydroxylate l-arginine to Nω-hydroxy-l-arginine (NOHA). This intermediate then becomes a substrate for the second reaction, which produces l-citrulline and NO. To carry out these two reactions, the heme iron at the active site of NOSs must first be reduced to be able to bind molecular oxygen (O2) and to form an oxygenated complex (FeIIO2). The electron is supplied by NADPH via the reductase domain in mammalian NOSs and from an independent reductase for the bacterial NOSs (6Wang Z.Q. Lawson R.J. Buddha M.R. Wei C.C. Crane B.R. Munro A.W. Stuehr D.J. J. Biol. Chem. 2007; 282: 2196-2202Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The FeIIO2 complex must then be activated to form the oxidizing intermediate that oxidizes the substrates (l-arginine and NOHA). The hydroxylation of l-arginine requires an additional external electron initially supplied by tetrahydrobiopterin (H4B) to allow oxygen activation. Oxygen activation is thought to occur via a mechanism similar to that proposed for cytochrome P450 and to involve a Compound I intermediate defined as an oxyferryl (FeIV=O) heme with an associated radical (2Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3190) Google Scholar, 5Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 7Groves J.T. Wang C.C. Curr. Opin. Chem. Biol. 2000; 4: 687-695Crossref PubMed Scopus (178) Google Scholar, 8Rosen G.M. Tsai P. Pou S. Chem. Rev. 2002; 102: 1191-1200Crossref PubMed Scopus (115) Google Scholar, 9Cho K-B. Derat E. Shaik S. J. Am. Chem. Soc. 2007; 129: 3182-3188Crossref PubMed Scopus (55) Google Scholar). Strong evidence suggests that an electron and possibly a proton are supplied from the H4B cofactor to form a peroxy ( FeIIIO2ċ¯) or hydroperoxy (FeIIIOO–H) complex (10Stuehr D.J. Wei C.C. Wang Z. Hille R. Dalton Trans. 2005; 21: 3427-3435Crossref Scopus (45) Google Scholar). A second proton is then recruited, leading to the heterolytic cleavage of the O–O bond and the formation of a water molecule and a Compound I-type intermediate. Unlike the hydroxylation of l-arginine, the conversion of NOHA to NO and citrulline requires only a transiently supplied electron to activate the heme-bound O2. Many mechanisms for the hydroxylation of NOHA have been put forward (8Rosen G.M. Tsai P. Pou S. Chem. Rev. 2002; 102: 1191-1200Crossref PubMed Scopus (115) Google Scholar, 11Huang H. Hah J.M. Silverman R.B. J. Am. Chem. Soc. 2001; 123: 2674-2676Crossref PubMed Scopus (60) Google Scholar, 12Cho K-B. Gauld J.W. J. Am. Chem. Soc. 2004; 126: 10267-10270Crossref PubMed Scopus (22) Google Scholar, 13Cho K-B. Gauld J.W. J. Phys. Chem. B. 2005; 109: 23706-23714Crossref PubMed Scopus (29) Google Scholar). In general, it has been suggested that an activated form of the oxygenated complex (peroxy or hydroperoxy), produced after the transfer of an electron from H4B, can perform an electrophilic attack on the guanidino carbon of NOHA to form a tetrahedral intermediate that undergoes conversion to l-citrulline and NO with a concomitant electron donation back to H4B (14Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). NOSs likely evolved two different mechanisms of oxygen activation to oxidize their two substrates in the first and second catalytic cycles, respectively. Interactions between the oxygen atoms of the FeIIO2 complex and the substrates, together with the electron/proton donor properties of the cofactor, likely play critical roles in determining how NOSs catalyze these reactions. Although H4B has been more extensively characterized, including its involvement as an electron donor in the first and second reactions (10Stuehr D.J. Wei C.C. Wang Z. Hille R. Dalton Trans. 2005; 21: 3427-3435Crossref Scopus (45) Google Scholar, 14Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 16Gorren A.C. Marchal S. Sorlie M. Andersson K.K. Lange R. Mayer B. Biochim. Biophys. Acta. 2006; 1764: 578-585Crossref PubMed Scopus (5) Google Scholar, 17Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Crossref PubMed Scopus (212) Google Scholar, 18Wei C.C. Wang Z.Q. Wang Q. Meade A.L. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2001; 276: 315-319Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) as well as its likely role as a proton donor (19Gorren A.C. Sorlie M. Andersson K.K. Marchal S. Lange R. Mayer B. Methods Enzymol. 2005; 396: 456-466Crossref PubMed Scopus (15) Google Scholar, 20Sorlie M. Gorren A.C. Marchal S. Shimizu T. Lange R. Andersson K.K. Mayer B. J. Biol. Chem. 2003; 278: 48602-48610Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), specific details on the interactions between the hemebound O2 and substrates and the involvement of H4B in modulating these interactions are scarce (21Davydov R. Ledbetter-Rogers A. Martásek P. Larukhin M. Sono M. Dawson J.H. Masters B.S.S. Hoffman B.M. Biochemistry. 2002; 41: 10375-10381Crossref PubMed Scopus (107) Google Scholar, 22Couture M. Stuehr D.J. Rousseau D.L. J. Biol. Chem. 2000; 275: 3201-3205Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We used stopped-flow spectrophotometry and continuous-flow resonance Raman spectroscopy to probe the FeIIO2 complex of Staphylococcus aureus NOS (saNOS) with the substrate l-arginine (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Our results showed that l-arginine binding causes a downshift in the frequency of the νO–O mode but has no effect on the frequency of the νFe–O mode (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A similar downshift of the νO–O frequency has also been reported for the oxygenated complex of mammalian neuronal NOS (nNOS) (24Rousseau D.L. Li D. Couture M. Yeh S-R. J. Inorg. Biochem. 2005; 99: 306-323Crossref PubMed Scopus (94) Google Scholar). Analysis of the kinetics of formation and decay of the FeIIO2 complex revealed that l-arginine stabilizes the complex against autoxidation to the ferric form in saNOS. These findings indicate that hydrogen-bonding interactions involving the heme-bound O2 and l-arginine stabilize the FeIIO2 complex against autoxidation. Hydrogen-bonding interactions also decrease the frequency of the νO–O mode by modulating the amount of π -backbonding from the heme iron (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To complement our first study of the FeIIO2 complex of saNOS in the presence of l-arginine (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we now report an investigation of the interactions of the FeIIO2 complex in the presence of NOHA for saNOS and Bacillus subtilis NOS (bsNOS) by resonance Raman spectroscopy. Specifically, we report the frequencies of the νO–O and νFe–O modes of NOHA-bound saNOS and bsNOS as well as of l-arginine-bound bsNOS. Differences in the frequencies of the νFe–O and νO–O modes were observed in the presence of NOHA with respect to those measured with l-arginine, indicating that the hemebound O2 is involved in substrate-specific interactions in both NOSs. The implications of these results for the mechanisms of oxygen activation by NOSs are discussed. Materials—H4B and NOHA were purchased from Sigma. Argon and 16O2 gas were from Praxair, Inc. (Mississauga, ON, Canada). 18O2 gas (99% purity) was from ICON (Mt. Marion, NY). Enzyme Preparation—saNOS and bsNOS were expressed in Escherichia coli from the cloned genes and purified as described previously (25Chartier F.J.M. Couture M. Biophys. J. 2004; 87: 1939-1950Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26Chartier F.J.M. Couture M. Biochem. J. 2007; 401: 235-245Crossref PubMed Scopus (19) Google Scholar). Samples were maintained in 40 mm HEPES (pH 7.6), 150 mm NaCl, and 1 mm dl-dithiothreitol. Where indicated, NOHA (500 μm) was added to the purified enzymes. For samples containing H4B, a concentration of 500 μm was used. The association of H4B and NOHA with saNOS and bsNOS and that of l-arginine with bsNOS were monitored by measuring the displacement of dl-dithiothreitol bound to the ferric enzymes by optical spectroscopy (25Chartier F.J.M. Couture M. Biophys. J. 2004; 87: 1939-1950Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Stopped-flow Spectroscopy—Stopped-flow experiments were carried out as described previously (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Briefly, anaerobic protein samples (5 μm) were reduced with the minimum amount of sodium dithionite required to reduce the heme. Complete reduction of the sample was verified by optical spectroscopy. Rapid mixing experiments with reduced saNOS and bsNOS and molecular oxygen were carried out at 21 °C. O2 gas (100%) was used to saturate anaerobic buffer with O2. This solution was then used to prepare buffers of the specified oxygen concentrations. The kinetics of formation and decay of the FeIIO2 complex were followed at individual wavelengths in kinetic scanning mode. Kinetic traces were recorded at 5-nm intervals from 380 to 460 nm and from 515 to 610 nm, which generated optical spectra versus time data sets. The kinetic data were analyzed using SPECFIT global analysis software (Spectrum Software Associates, Chapel Hill, NC) with a kinetic model involving four states for the data obtained from saNOS and bsNOS saturated with NOHA: state A corresponded to the initial reduced protein; state B corresponded to the transient FeIIO2 complex; state C corresponded to an intermediate; and state D corresponded to the resting ferric form. For the data obtained from bsNOS saturated with l-arginine, a three-state model was used: state A corresponded to the initial reduced protein; state B corresponded to the transient FeIIO2 complex; and state C corresponded to the resting ferric form. The deconvoluted optical spectra, the fits at all wavelengths, and the time course of the appearance and decay of the four kinetic states were obtained from these analyses. Resonance Raman Spectroscopy—Resonance Raman spectra of the oxygenated intermediates were acquired with a custom-made continuous-flow T-mixer as described previously (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Prior to mixing, the reduced forms of saNOS and bsNOS (80 μm) were prepared by equilibrating the enzymes with pure argon gas for at least 30 min at room temperature and by adding the minimum amount of sodium dithionite required to reduce the heme completely. Optical spectra in the visible region (from 450 to 700 nm), recorded directly from the 10-ml syringe containing the reduced proteins, were recorded to assess the complete reduction of the protein. Pure 16O2 and 18O2 gases were used to prepare oxygenated buffers of known concentrations. The rapid mixer was made anaerobic using a 10 mm sodium dithionite solution, followed by washing with anaerobic buffer to remove the dithionite. Oxygenated buffers (16O2 and 18O2 in two separate mixing experiments) and the reduced proteins were mixed at a 1:1 ratio from 10-ml syringes. The output at 441.6 nm from a helium-cadmium laser (Liconix laser, Melles Griot, Ottawa, Ontario) at ∼10 milliwatts was focused on the sample inside the channel of the quartz flow cell. The resonance Raman spectra of saNOS/NOHA/H4B and saNOS/NOHA samples were acquired 4.5 and 10 ms after mixing, respectively, using previously described equipment (25Chartier F.J.M. Couture M. Biophys. J. 2004; 87: 1939-1950Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Those of bsNOS/l-Arg and bsNOS/NOHA were acquired 22 and 32 ms after mixing, respectively. Several 30-s spectra (12Cho K-B. Gauld J.W. J. Am. Chem. Soc. 2004; 126: 10267-10270Crossref PubMed Scopus (22) Google Scholar, 13Cho K-B. Gauld J.W. J. Phys. Chem. B. 2005; 109: 23706-23714Crossref PubMed Scopus (29) Google Scholar, 14Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) were acquired and averaged. All resonance Raman spectra were obtained at room temperature (25 °C) and were calibrated with the lines of indene. The spectrum of reduced myoglobin was recorded before each experiment to check for small calibration differences on different days. The spectrum of the buffer was also recorded for each experiment to subtract the quartz diffraction signal originating from the detection tube of the continuous-flow mixer. Stopped-flow Spectroscopy—Stopped-flow optical spectroscopy was used to obtain the rates of formation and decay of the FeIIO2 complex of saNOS in the presence of NOHA (without H4B) (Fig. 1). The kinetics of formation and decay of the FeIIO2 complex of saNOS in the presence of NOHA and H4B have already been reported (Table 1) (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Reduced saNOS saturated with NOHA was mixed with O2-saturated buffer. The data were acquired by kinetic scanning in the 380–460 nm (Soret region) (Fig. 1A) and 515–610 nm (supplemental Fig. 1) regions of the absorption spectrum. These kinetic scanning data were first studied by global analysis using a three-state sequential kinetic model (A → B → C). The three-state model did not fit the data properly (supplemental Fig. 2), and inconsistent rates were calculated from the two optical region data sets.TABLE 1Rates of formation and decay of the oxygenated complexes of saNOS at 21 °C and the wavelength maxima of the Soret absorption bands of the oxygenated complexesProteinRate of FeIIO2 formationRate of decay (fraction)aThe rate of decay corresponds to autoxidation when catalysis cannot occur, i.e. with either the substrate or H4B absent. The rate of decay corresponds to catalysis when l-arginine or NOHA is present along with H4B.SoretRef.s-1s-1nmsaNOSbThe experiments were performed using buffer saturated with 10% O2.188039.6 (100%)43023Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarsaNOS/l-ArgbThe experiments were performed using buffer saturated with 10% O2.1065.6 (14%)43023Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar1.0 (86%)saNOS/H4BbThe experiments were performed using buffer saturated with 10% O2.208032.8 (78%)425cThe oxygenated intermediate with the Soret absorption band at 425 nm was converted to the oxygenated intermediate with a Soret absorption band at 430 nm at a rate of 89 s-1.23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar3.6 (22%)430cThe oxygenated intermediate with the Soret absorption band at 425 nm was converted to the oxygenated intermediate with a Soret absorption band at 430 nm at a rate of 89 s-1.saNOS/l-Arg/H4BbThe experiments were performed using buffer saturated with 10% O2.14318.0 (67%)40523Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar5.0 (33%)430saNOS/NOHAdThe experiment was performed using buffer saturated with 100% O2. The rate of conversion of state C to the ferric form was 1.6 s-1 for saNOS and 1.2 s-1 for bsNOS.23034 (100%)425This worksaNOS/NOHA/H4BeThe experiment was performed using buffer saturated with 100% O2. The rate of conversion of the FeIIINO complex to the ferric form was 3.8 s-1.45422.2 (100%)42823Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarbsNOS/l-ArgfThe experiment was performed using buffer saturated with 40% O2.2011.6 (100%)430This workbsNOS/l-ArggThe experiment was performed using air-saturated buffer at 10 °C.600.4 (100%)42728Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (157) Google ScholarbsNOS/NOHAdThe experiment was performed using buffer saturated with 100% O2. The rate of conversion of state C to the ferric form was 1.6 s-1 for saNOS and 1.2 s-1 for bsNOS.944.9 (100%)∼421This worka The rate of decay corresponds to autoxidation when catalysis cannot occur, i.e. with either the substrate or H4B absent. The rate of decay corresponds to catalysis when l-arginine or NOHA is present along with H4B.b The experiments were performed using buffer saturated with 10% O2.c The oxygenated intermediate with the Soret absorption band at 425 nm was converted to the oxygenated intermediate with a Soret absorption band at 430 nm at a rate of 89 s-1.d The experiment was performed using buffer saturated with 100% O2. The rate of conversion of state C to the ferric form was 1.6 s-1 for saNOS and 1.2 s-1 for bsNOS.e The experiment was performed using buffer saturated with 100% O2. The rate of conversion of the FeIIINO complex to the ferric form was 3.8 s-1.f The experiment was performed using buffer saturated with 40% O2.g The experiment was performed using air-saturated buffer at 10 °C. Open table in a new tab A four-state model (A → B → C → D) best fit the data and produced consistent results in both regions of the absorption spectrum (Fig. 1 and supplemental Fig. 1). The kinetic traces recorded at 430 and 450 nm and the corresponding fits to the four-state model are shown in Fig. 1B. These wavelengths show that four states are indeed needed to fit all the transitions observed. The residuals of the fits at all wavelengths were small in amplitude and randomly distributed (Fig. 1C), indicating that a good fit to the four-state model was obtained. State A, with a Soret optical transition centered at 410 nm, corresponded to reduced saNOS (Fig. 1E, solid line) and reacted with molecular oxygen at a rate of 230 s–1 to form the FeIIO2 complex (state B), which had a Soret absorption maximum centered at 425 nm (Fig. 1E, dashed line; and Table 1). State B was identified as an FeIIO2 complex from the isotope shifts induced by 16O2 and 18O2 in the resonance Raman spectra (see below). The FeIIO2 complex subsequently decayed at a rate of 34 s–1 to an intermediate (state C), which had a broad Soret band (Fig. 1E, dotted line). This intermediate may be composed of a mixture of several states. Finally, the enzyme returned at a rate of 1.6 s–1 to the resting ferric state, which had a Soret optical transition at 400 nm (Fig. 1E, dotted/dashed line; and Table 1). In the 515–610 nm region (Fig. 1F), reduced saNOS had a wavelength absorption maximum centered at 552 nm (solid line); the FeIIO2 complex had two maxima near 556 and 570 nm (dashed line); state C had one maximum at 554 nm (dotted line); and the ferric form, which is five-coordinate and high spin, did not have any strong absorption maximum in this optical region (dotted/dashed line). Notably, the absorption bands at 550 and 580 nm observed in the stopped-flow mixing experiments of O2 with reduced saNOS saturated with NOHA and H4B (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), which are characteristic of the FeIIINO complex, were not observed here. Previous studies have shown that an FeIIINO complex is not formed in single turnover experiments from NOHA with pterin-free NOSs (27Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar, 28Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 29Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), thus supporting the crucial role for H4Bin the synthesis of NO from NOHA. Our studies are no exception. We detected NO synthesis as an FeIIINO complex in single turnover experiments with saNOS/NOHA/H4B (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), but not with NOHA alone (Fig. 1). Therefore, even though the rate of decay of the oxygenated complexes of saNOS/NOHA is only moderately increased in the presence of H4B (Table 1), the presence of H4B is nevertheless necessary to observe the FeIIINO complex in the course of the reactions of saNOS/NOHA with O2, thus implying that it is necessary for NO synthesis. Resonance Raman Spectroscopy in the High Frequency Region of the FeIIO2 Complex—To characterize the oxygenated complexes of saNOS with NOHA and H4B and with NOHA alone, we used a custom-made continuous-flow mixer with a dead time of 0.5 ms (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The high frequency region of the resonance Raman spectra was first obtained to determine the oxidation and spin states of the FeIIO2 complex. The spectra were recorded at 4.5 and 10 ms after mixing reduced saNOS/NOHA with the O2-saturated buffer in the presence and absence of H4B, respectively. At these times, the FeIIO2 complexes had reached maximum concentrations of 81 and 72% of the heme available for saNOS/NOHA with (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and without H4B (Fig. 1D), respectively. The spectra showed that the oxidation state marker band ν4 was at 1374 cm–1 and that the coordination and spin state marker band ν3 was at 1501 cm–1 in samples with H4B (Fig. 2, traces A and B) and without H4B(traces C and D). The FeIIO2 complexes of saNOS/NOHA and saNOS/NOHA/H4B were thus ferric and low spin like the oxygenated complexes of other heme proteins (24Rousseau D.L. Li D. Couture M. Yeh S-R. J. Inorg. Biochem. 2005; 99: 306-323Crossref PubMed Scopus (94) Google Scholar, 30Sjodin T. Christian J.F. Macdonald I.D. Davydov R. Unno M. Sligar S.G. Hoffman B.M. Champion P.M. Biochemistry. 2001; 40: 6852-6859Crossref PubMed Scopus (70) Google Scholar). The shoulder on the ν4 mode at 1349 cm–1 and the small intensity ν3 mode at 1468 cm–1 indicated that a small amount of reduced five-coordinate saNOS was present, likely the remains of starting material predicted to correspond to 12 and 11% of the reaction mixtures at 4.5 and 10 ms following the initiation of the reactions based on the kinetic data with NOHA/H4B (23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and NOHA (Fig. 1D), respectively. These assignments are supported by the resonance Raman spectra of saNOS/NOHA obtained later after the initiation of the reactions (Fig. 3). At 56 ms (Fig. 3, trace B) and 81 ms (trace A) after mixing, the ν3 and ν4 lines of reduced saNOS were no longer observed, unlike the 10 ms spectrum (trace C). These results are consistent with stopped-flow data indicating that no reduced form remained at those times (Fig. 1D). Also consistent with stopped-flow data, the spectrum obtained at 81 ms displayed a ν3 line at 1488 cm–1, which corresponded to ferric five-coordinate saNOS and which represented 8% of the heme at that time. Also, at 81 ms, state C had reached its maximum concentration. Our results show that, with a ν4 line at 1372 cm–1 and a ν3 line at 1501 cm–1, this intermediate was mostly six-coordinate and low spin. Isotopic substitution was used to identify an oxygen-sensitive mode in the high frequency region. The 16O2-minus-18O2 difference spectra of saNOS/NOHA/H4B (Fig. 2, trace E) and saNOS/NOHA (trace F) revealed the presence of single isotope-sensitive lines at 1135 cm–1 with 16O2 and 1070 cm–1 with 18O2. These lines corresponded to the νO–O mode, as the isotopic shift of 65 cm–1 was on par with the value expected for an O–O diatomic harmonic oscillator. The frequency was the same as that of substrate-free saNOS (Table 2). These results demonstrate that NOHA and H4B did not change the frequency of the νO–O mode with respect to that of the FeIIO2 complex of substrate-free and pterin-free saNOS at 1135 cm–1 (Table 2). However, the line at 1135 cm–1 was significantly sharper in the spectrum of the FeIIO2 complex with NOHA (width at a half-height of 20 cm–1) than in that of substrate-free saNOS (width at a half-height of 22 cm–1). In the 16O2-minus-18O2 difference spectrum of saNOS/NOHA recorded 81 ms after mixing (Fig. 2, trace G), the intensity of the νO–O line was much lower than at 10 ms (trace F), indicating that only a small amount of the FeIIO2 complex remained after 81 ms, which was expected based on the stopped-flow data (Fig. 1D).TABLE 2Frequencies of the νFe–O and νO–O modes of saNOS, nNOS, iNOS, and P450camProteinνFe–O (18O2)νO–O (18O2)Ref.cm-1cm-1saNOS517 (487)1135 (1071)23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarsaNOS/l-Arg (±H4B)517 (487)1123 (1062), 1135 (1071)23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarsaNOS/H4B517 (487)1123 (1062), 1135 (1071)23Chartier F.J.M. Blais S.P. Couture M. J. Biol. Chem. 2006; 281: 9953-9962Abstract Full Text" @default.
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• W2085048960 title "Substrate-specific Interactions with the Heme-bound Oxygen Molecule of Nitric-oxide Synthase" @default.
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