Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase
2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês
10.1074/jbc.m000050200
ISSN1083-351X
AutoresHusam M. Abu‐Soud, Koji Ichimori, Anthony Presta, Dennis J. Stuehr,
Tópico(s)Neuroscience of respiration and sleep
ResumoWe studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0.13 to 86 s−1 upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg orN ω-hydroxy-l-arginine (0.005 and 0.014 s−1, respectively, at 10 °C). O2binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved withN ω-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparentKm for O2to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0.13 to 86 s−1 upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg orN ω-hydroxy-l-arginine (0.005 and 0.014 s−1, respectively, at 10 °C). O2binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved withN ω-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparentKm for O2to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. nitric-oxide synthase rat neuronal NO synthase bovine endothelial NO synthase calmodulin mouse inducible NO synthase (6R)-5,6,7,8-tetrahydro-l-biopterin N ω-hydroxy-l-arginine 3-diol nitric oxide Nitric-oxide synthases (NOSs)1 catalyze a stepwise oxidation of l-arginine (Arg) to citrulline and nitric oxide (NO) (1.Marletta M.A. Hurshman A.R. Rusche K.M. Curr. Opin. Chem. Biol. 1998; 2: 656-663Crossref PubMed Scopus (199) Google Scholar, 2.Hemmens B. Mayer B. Titheradge M.A. Methods in Molecular Biology. 100. Humana Press, Totowa, NJ1997: 1-32Google Scholar, 3.Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). In mammals, three NOSs are expressed that differ in their primary sequence, post-translational modifications, cellular location, and tissue expression (4.Michel T. Feron O. J. Clin. Invest. 1997; 100: 2146-2152Crossref PubMed Scopus (841) Google Scholar, 5.Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 6.MacMicking J. Xie Q.-W. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3430) Google Scholar), consistent with their participating in a range of physiologic and pathologic systems. Two NOSs (neuronal, nNOS or NOS-I; and endothelial, eNOS or NOS-III) are constitutively expressed and participate in signal cascades by synthesizing NO in response to Ca2+-dependent CaM binding. A third NOS (cytokine-inducible, iNOS or NOS-II) is primarily regulated by transcriptional mechanisms, binds CaM irrespective of the Ca2+ concentration to be always active, and functions as both a regulator and effector of the immune response. Although NO synthesis activities of the NOS isoforms differ considerably, how and why this occurs is unclear. A comparison of published steady-state rates shows that eNOS is about four to eight times slower than either nNOS or iNOS (7.Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (167) Google Scholar, 8.Martasek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar, 9.List B.M. Klosch B. Volker C. Gorren A.C.F. Sessa W.C. Werner E.R. Kukovetz W.R. Schmidt K. Mayer B. Biochem. J. 1997; 323: 159-165Crossref PubMed Scopus (138) Google Scholar, 10.Moali C. Boucher J.L. Sari A.M. Stuehr D.J. Mansuy D. Biochemistry. 1998; 37: 10453-10460Crossref PubMed Scopus (176) Google Scholar, 11.Ghosh S. Gachhui R. Crooks C. Wu C. Lisanti M.P. Stuehr D.J. J. Biol. Chem. 1998; 273: 22267-22271Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 12.Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar, 13.Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 14.Rodriguez-Crespo I. Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 11462-11467Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Because NO synthesis is actually the result of many steps, it is imperative to identify which steps limit the activity of a particular NOS isoform. Work with NOS chimeras containing swapped reductase domains has suggested heme reduction could be responsible for the low activity of eNOS (30.Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, it seems that NOS catalysis is comprised of two parts (15.Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (799) Google Scholar,16.Adak S. Crooks C. Wang Q. Crane B.R. Tainer J.A. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 1999; 274: 26907-26911Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar): an active component that includes all steps involved in NO generation and an inactive component that includes NO binding to the NOS heme and subsequent dissociation or oxidation of the heme-NO complex to regenerate active enzyme. For iNOS and nNOS, evidence suggests that their activities are significantly decreased in a number of settings because a majority of enzyme partitions into an NO-bound inactive form (17.Hurshman A.R. Marletta M.A. Biochemistry. 1995; 34: 5627-5634Crossref PubMed Scopus (109) Google Scholar, 18.Abu-Soud H.M. Wang J. Rousseau D.L. Fukuto J. Ignarro L.J. Stuehr D.J. J. Biol. Chem. 1995; 270: 22997-23006Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). This also increases theirKm for O2, which has physiologic consequences (19.Abu-Soud H.M. Rousseau D.L. Stuehr D.J. J. Biol. Chem. 1996; 271: 32515-32518Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 20.Dweik R.A. Laskowski D. Abu-Soud H.M. Kaneko F.T. Hutte R. Stuehr D.J. Erzurum S.C. J. Clin. Invest. 1998; 101: 660-666Crossref PubMed Scopus (259) Google Scholar). In nNOS, a residue that controls enzyme partitioning between the active and NO bound forms has recently been identified (16.Adak S. Crooks C. Wang Q. Crane B.R. Tainer J.A. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 1999; 274: 26907-26911Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In contrast, the extent by which heme-NO complex formation may limit eNOS NO synthesis is still unknown. To address this issue, we analyzed several steps involved in NO synthesis by eNOS and its propensity to partition between active and inactive forms during catalysis. NO gas was purchased from Matheson Gas Products, Inc., and O2 gas was purchased from the Ohio Gas Company and used without further purification.N ω-Hydroxy-l-arginine was a generous gift from Dr. Bruce King of Wake Forest University. All other materials were obtained from Sigma or from sources reported previously (11.Ghosh S. Gachhui R. Crooks C. Wu C. Lisanti M.P. Stuehr D.J. J. Biol. Chem. 1998; 273: 22267-22271Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13.Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16.Adak S. Crooks C. Wang Q. Crane B.R. Tainer J.A. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 1999; 274: 26907-26911Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18.Abu-Soud H.M. Wang J. Rousseau D.L. Fukuto J. Ignarro L.J. Stuehr D.J. J. Biol. Chem. 1995; 270: 22997-23006Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Bovine eNOS with a six-histidine tag at its N terminus was expressed in Escherichia coliBL21(DE3) using the pCWori expression vector and purified in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (H4B) by ammonium sulfate precipitation and sequential nickel-nitrilotriacetic acid and 2′,5′-ADP affinity chromatography as described previously (11.Ghosh S. Gachhui R. Crooks C. Wu C. Lisanti M.P. Stuehr D.J. J. Biol. Chem. 1998; 273: 22267-22271Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The purified enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. The eNOS was quantitated based on its P450 heme content which was determined using an extinction coefficient of 74 mm−1 cm−1 for its dithionite-reduced, CO bound form (11.Ghosh S. Gachhui R. Crooks C. Wu C. Lisanti M.P. Stuehr D.J. J. Biol. Chem. 1998; 273: 22267-22271Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). NO concentration was monitored using an ISO-NO mark II NO meter equipped with a ISO-NOP 200 sensor (World Precision Instruments, Inc., Sarasota, FL). Applied voltage was +865 mV. Measurements were made in a water-jacketed and stirred 4-ml cell at 15 °C. Electrode calibration involved the consecutive addition of 1 μl of saturated NO solution to 3 ml of argon-deoxygenated Hepes buffer (40 mm, pH 7.4) in a rubber-sealed cell at 15 °C. The NO solution was made in argon-deoxygenated buffer, and its NO concentration was determined with oxyhemoglobin. The electrode current was proportional to NO concentration up to 10 μm, and sensitivity was typically 410 nm NO/nA at 15 °C. Reactions (1.5-ml total volume) were run in the stirred 4-ml cell at 15 °C and contained 40 mm Hepes buffer, pH 7.4, 0.2 mm NOHA or Arg, 0.3 mm dithiothreitol, 4 μm H4B, 1 mm Ca2+, 0.6 mm EDTA, 2.5 μm CaM, 0.1 m NaCl, and 1.5 μmeNOS. NO synthesis was started by adding 50 μm NADPH. NO concentration was monitored with the NO electrode. Concurrent citrulline production was followed by removing 10-μl aliquots from the reaction at various time points and quenching with diluted HCl. Citrulline was measured after derivatization with orthopthaldialdehyde using a fluorometric high pressure liquid chromatography detection method as described previously (21.Presta A.P. Siddhanta U. Wu C. Sennequier N. Huang L. Abu-Soud H.M. Erzurum S. Stuehr D.J. Biochemistry. 1998; 37: 298-310Crossref PubMed Scopus (149) Google Scholar). Concurrent NADPH oxidation was measured at 340 nm in replica reactions that were run in cuvettes under the same conditions except the total volume was 0.7 ml. Concentrated eNOS was placed in septum-sealed cuvettes and diluted with various ratios of N2-, air-, or O2-saturated buffer solutions that contained 40 mm BisTris propane, pH 7.4, 1 mm Arg, 15 μg CaM, 1.2 mm Ca2+, 0.9 mm EDTA, 1 mm dithiothreitol, 5–10 μm oxyhemoglobin, and 4 μm each of FAD, FMN, and H4B (final volume 1 ml). Final eNOS concentrations for the NADPH oxidation and NO synthesis rate measurements were 300 and 80 nm, respectively. The initial O2 concentration in each reaction was calculated based on the solution mixing ratio and the O2 concentration of air- or O2-saturated buffer at 25 °C (0.26 and 1.26 mm, respectively). Reactions were run at 25 °C and initiated by injecting 10 μl of NADPH solution to give 100 μm final concentration. The rate of NO synthesis was determined by monitoring the NO-mediated conversion of oxyhemoglobin to methemoglobin at 401 nm (11.Ghosh S. Gachhui R. Crooks C. Wu C. Lisanti M.P. Stuehr D.J. J. Biol. Chem. 1998; 273: 22267-22271Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), whereas the initial rate of NADPH oxidation was determined at 340 nm in the presence or absence of Arg and in the absence of oxyhemoglobin.Km values for O2 were estimated from double reciprocal plots of the data. Optical spectra were recorded on a Hitachi 3010 UV-visible spectrophotometer at 15 °C. Anaerobic spectra were recorded using septum-sealed quartz cuvettes that could be attached through a quick-fit joint to a vacuum system. eNOS samples were made anaerobic by repeated cycles of evacuation and equilibrated with catalyst-deoxygenated nitrogen. Cuvettes were maintained under nitrogen or NO atmosphere during spectral measurements. Kinetic measurements were carried out using a Hi-Tech stopped-flow apparatus (model SF-51) equipped for anaerobic work. Rates of NO and O2 binding to ferrous eNOS at different ligand concentrations were obtained as described previously (22.Abu-Soud H.M. Wu C. Ghosh D.K. Stuehr D.J. Biochemistry. 1998; 37: 3777-3786Crossref PubMed Scopus (111) Google Scholar). Experiments were carried out at 10 °C and initiated by rapidly mixing an anaerobic buffered solution that contained 2 μm ferrous eNOS (generated in a cuvette by titrating in an anaerobic dithionite solution), 10 μm H4B, 1 mm dithiothreitol, and 0.15 m NaCl with a buffered solution that contained 0.15 m NaCl and different concentrations of O2or NO. Dithionite reduction was omitted when studying ferric eNOS. All NO and O2 binding rates were measured in the presence or absence of Arg. In some cases, O2 binding to ferrous eNOS was monitored using a rapid scanning diode array detector (Hi-Tech MG-6000) designed to collect 96 complete spectra in a specific time frame. The diode array detector was calibrated relative to five reference absorbance wavelengths of holmium oxide filter (HY-I) at 362, 420, 446, 460, and 536 nm. Spectra were collected after rapidly mixing anaerobic ferrous eNOS with air-equilibrated buffer at 10 °C. In all cases, 6–10 replica scans were collected and utilized to derive mean kinetic values. Solution compositions were as described above. Rates of NADPH-dependent flavin and heme reduction were measured at 10 °C under similar stopped-flow conditions as described above, except that the anaerobic ferric eNOS solution also contained 0.9 mm EDTA with or without 1.2 mmCa2+ and 3 μm CaM and was mixed with an anaerobic buffered solution that contained 0.2 mm NADPH and 0.15 m NaCl. Flavin reduction was monitored at 485 nm, and heme reduction was monitored at 400 nm. Signal-to-noise ratios were improved by averaging 6–10 individual traces. The time courses were fit using a nonlinear least-squares method provided by the instrument manufacturer. We first characterized UV-visible transitions that occur during reduction of eNOS flavin and heme centers by NADPH. As shown in the upper panel of Fig. 1, an anaerobic sample of ferric eNOS containing H4B displayed a broad Soret absorbance peak centered near 400 nm with flavin absorbance peaks ranging from 445 to 550 nm, consistent with earlier reports (8.Martasek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar, 9.List B.M. Klosch B. Volker C. Gorren A.C.F. Sessa W.C. Werner E.R. Kukovetz W.R. Schmidt K. Mayer B. Biochem. J. 1997; 323: 159-165Crossref PubMed Scopus (138) Google Scholar, 23.Presta A. Liu J. Sessa W.C. Stuehr D.J. Nitric Oxide Biol. Chem. 1997; 1: 74-87Crossref PubMed Scopus (53) Google Scholar). Adding NADPH in the absence of bound CaM caused flavin reduction as judged by the disappearance of the flavin visible absorption bands. No heme reduction occurred as judged by our observing only a slight decrease in heme Soret absorbance, which could be attributed to flavin reduction in this region of the spectrum, and no change in ferric heme absorbance at 640 nm. Adding Ca2+ to promote CaM binding led to partial heme reduction, as judged by an intermediate decrease and red shift in Soret absorbance. Upon adding Arg to the sample, heme reduction became complete. The lower panel of Fig. 1 shows spectra from a similar experiment that initially contained ferric eNOS saturated with both H4B and Arg. Under this condition, Ca2+-promoted CaM binding led to complete heme reduction as judged by the decrease in Soret absorbance and its shift to 414 nm. Heme reduction was blocked when N-nitro-l-arginine methyl ester replaced Arg in the experiment (data not shown), as also occurs in eNOS isolated from mammalian cells (23.Presta A. Liu J. Sessa W.C. Stuehr D.J. Nitric Oxide Biol. Chem. 1997; 1: 74-87Crossref PubMed Scopus (53) Google Scholar). These results confirm that CaM triggers heme reduction in H4B-bound eNOS, indicate that Arg facilitates heme reduction, and provide wavelengths to monitor the kinetics of flavin and heme reduction. We studied the kinetics of flavin and heme reduction using stopped-flow spectroscopy under anaerobic conditions at 10 °C. Based on spectra in Fig. 1 and our previous work with nNOS (24.Gachhui R. Abu-Soud H.M. Ghosh D.K. Presta A. Blazing M.A. Mayer B. George S.E. Stuehr D.J. J. Biol. Chem. 1998; 273: 5451-5454Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), we followed flavin and heme reduction at 485 and 400 nm, respectively. Reduction of flavins was studied by rapidly mixing CaM-free or CaM-bound eNOS with a solution containing excess NADPH. Fig. 2shows flavin reduction in CAM-free eNOS was very slow and required about 25 s to reach completion (left panel), whereas flavin reduction in CaM-bound eNOS was fast and reached completion in less that 0.1 s (right panel). In both cases, the spectral change was essentially monophasic and fit well to a single exponential function with rate constants of 0.13 and 85 s−1, respectively. Replica experiments were run to monitor heme reduction in eNOS at 400 nm. In CaM-free eNOS, the spectral change at 400 nm was monophasic with a rate of 0.15 s−1, essentially identical to that observed at 485 nm and consistent with only flavin reduction occurring in this circumstance. In contrast, for CaM-bound eNOS that contained Arg and H4B the absorbance change was biphasic. The first phase was attributed to flavin reduction with a rate constant identical to that obtained at 485 nm, whereas the slow phase was attributed to heme reduction. As shown in Fig. 3, heme reduction in the presence of Arg had a rate constant of 0.005 s−1 at 10 °C and was about three times faster (0.014 s−1) with NOHA. The slow phase attributed to heme reduction at 400 nm was absent when eNOS contained the heme reduction inhibitorN-nitro-l-arginine methyl ester (data not shown). Adding Arg, NOHA, or N-nitro-l-arginine methyl ester did not alter the rate of flavin reduction (data not shown). We next characterized O2binding to ferrous eNOS. We used rapid-scanning stopped-flow spectroscopy to identify species that form upon mixing a prereduced, anaerobic solution of H4B-saturated eNOS with air-equilibrated buffer. As shown in Fig. 4, the starting spectrum recorded after 3 ms is characteristic of ferrous eNOS, which displays a Soret absorbance peak at 414 nm and almost no absorbance at 630 nm. This was followed by buildup of a transient species whose spectrum was characterized by absorbance peaks at 427, 560, and a shoulder at 600 nm, identical to the ferrous-dioxy complex of nNOS obtained under similar temperature and buffer conditions (25.Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 26.Sato H. Sagami I. Daff S. Shimizu T. Biochem. Biophys. Res. Commun. 1998; 253: 845-849Crossref PubMed Scopus (37) Google Scholar). This transient species decayed to form ferric eNOS, as judged by a shift in Soret absorbance to 396 nm and buildup of visible absorbance centered near 630 nm. The eNOS ferrous-dioxy complex formed and decayed at sufficiently different rates such that the kinetics of both steps could be studied using single wavelength stopped-flow methods. We therefore examined the rates of ferrous-dioxy formation and decay as a function of O2 concentration. We studied H4B-bound eNOS in the presence and absence of Arg or NOHA and monitored the change in absorbance at 430 nm (Fig. 4). At all O2 concentration tested, there was a monophasic increase in absorbance attributed to buildup of the ferrous-dioxy complex, followed by a slower, essentially monophasic decrease attributed to its conversion to ferric eNOS (data not shown). As shown in Fig. 5, plots ofk obs versus O2concentration were linear in all cases, with a positive intercept indicating that the reaction is reversible and follows a one step mechanism. The kinetic parameters for O2 binding were estimated from the slope and y intercept of each plot and are listed in Table I, along with the rates of ferrous-dioxy complex decay, which were independent of O2 concentration under each circumstance (data not shown).Figure 5Rate of ferrous-dioxy complex formationversus O2 concentration and effect of substrate. eNOS ferrous-dioxy complex formation at 10 °C was followed at 430 nm to determine an observed rate at each indicated O2 concentration. Experiments contained H4B-bound eNOS either in the absence of substrate (○) or in the presence of 1 mm Arg (●) or 1 mm NOHA (■). The lines are a least squares fit for each data set.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IKinetics of O2 and NO binding to H4B-saturated eNOS determined by stopped-flow spectroscopySpeciesLigandk onk offk decaym −1 s −1s −1s −1Ferrous eNOSO2 No substrate3.1 × 105383.0 +Arg3.4 × 105282.8 +NOHA2.6 × 105182.0Ferric eNOSNO No substrate6.1 × 10593 +Arg8.2 × 10570 +NOHA2.8 × 105100Ferrous eNOSNO No substrate1.1 × 10645 +Arg1.1 × 10670 +NOHA8.9 × 10550Standard deviations fell within 15% of the reported mean in all cases. Open table in a new tab Standard deviations fell within 15% of the reported mean in all cases. As shown in Fig.6, adding excess NO to H4B-bound ferric or ferrous eNOS formed stable 6-coordinate nitrosyl complexes under anaerobic conditions in the absence of Arg. The ferric-NO complex displayed a Soret absorbance peak at 440 nm and two absorbance bands centered at 549 and 580 nm, whereas the ferrous-NO complex had a Soret peak at 436 nm and a broad visible band centered at 580 nm. These spectral features are essentially identical to NO complexes of iNOS, nNOS, and eNOS (17.Hurshman A.R. Marletta M.A. Biochemistry. 1995; 34: 5627-5634Crossref PubMed Scopus (109) Google Scholar, 18.Abu-Soud H.M. Wang J. Rousseau D.L. Fukuto J. Ignarro L.J. Stuehr D.J. J. Biol. Chem. 1995; 270: 22997-23006Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27.Negrerie M. Berka V. Vos M.H. Liebl U. Lambry J.-C. Martin J.-L. J. Biol. Chem. 1999; 274: 24694-24702Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Kinetics of NO binding were studied at 10 °C in the presence or absence of Arg or NOHA. Reaction of NO solutions of different concentration with ferric or ferrous eNOS was monitored at 440 or 436 nm, respectively. For all six conditions tested, plots of k obs versus NO concentration were linear with positive intercept at the yaxis (Fig. 7), indicating that NO binding is reversible and follows a simple one step mechanism. Kinetic constants for NO binding estimated from these plots are listed in TableI.Figure 7Rate of heme-NO complex formationversus NO concentration and effect of substrate.The rate of eNOS ferrous-NO (A) or ferric-NO (B) complex formation was followed at 10 °C and at 436 and 440 nm, respectively, at each indicated NO concentration. Experiments contained H4B-bound eNOS either in the absence of substrate (○) or in the presence of 1 mm NOHA (■) or 1 mm Arg (●). The lines are a least squares fit for each data set.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because iNOS and nNOS both form heme-NO complexes during NO synthesis (17.Hurshman A.R. Marletta M.A. Biochemistry. 1995; 34: 5627-5634Crossref PubMed Scopus (109) Google Scholar, 18.Abu-Soud H.M. Wang J. Rousseau D.L. Fukuto J. Ignarro L.J. Stuehr D.J. J. Biol. Chem. 1995; 270: 22997-23006Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), we examined if eNOS would also do so. Fig. 8, upper panel, contains spectra of eNOS recorded prior to, during, and after NO synthesis with Arg as the substrate. NADPH was limiting in the reaction. The spectra clearly show that the majority of eNOS molecules contained reduced flavins and oxidized (ferric) heme during the steady state, with very little or no NO complex present. This is similar to the state in which eNOS exists when it oxidized NADPH in the absence of substrate (upper panel inset). We then examined if a heme-NO complex would form during NO synthesis from NOHA, which for eNOS supports a higher rate of NO synthesis compared with Arg (23.Presta A. Liu J. Sessa W.C. Stuehr D.J. Nitric Oxide Biol. Chem. 1997; 1: 74-87Crossref PubMed Scopus (53) Google Scholar). Again, spectra were recorded prior to, during, and after NO synthesis. As shown in thelower panel of Fig. 8, the spectrum recorded during NO synthesis from NOHA had less ferric heme character (absorbance at 400 nm) as compared with Arg and displayed a shoulder above 420 nm. As theinset shows, the shoulder is actually a gain in absorbance centered near 430 nm. Thus, some heme-NO complex built up during NO synthesis from NOHA. We next examined the kinetics of heme-NO complex formation and decay during NO synthesis from NOHA using the stopped-flow method. The reaction was initiated by rapid mixing an NADPH solution with a solution of CaM-bound eNOS that contained H4B and NOHA. The upper panel of Fig. 9 follows buildup and decay of the heme-NO complex at 436 nm, along with concurrent NADPH oxidation at 340 nm. Buildup of the eNOS-nitrosyl complex was relatively slow, approached a steady state, and then decayed at a rate of 0.01 s−1 after the NADPH was consumed. The rate of NADPH oxidation was slowed by about a factor of 2 or 3 upon buildup of the heme-NO complex. The absorbance change at 436 nm during the first 100 s of the reaction (Fig. 9, lower panel) best fit to a single exponential function and gave an observed rate of 0.065 s−1. This absorbance increase at 436 nm was actually preceded by a more rapid absorbance decrease (lower panel inset), which best fit to a single exponential function with an apparent rate constant of 94 s−1 and can be attributed to flavin reduction. This initial drop also explains why the trace at 436 nm in the upper panel appears not to return to its initial level after the reaction terminated and flavins become oxidized. We next utilized an electrode to monitor NO concentrations during NO synthesis from Arg or NOHA to see how these levels correlate with rates of NADPH oxidation and citrulline formation. Experiments were carried out by immersing a NO-selective electrode in a reaction vial that contained eNOS and all the necessary substrates and cofactors. Aliquots were removed for citrulline analysis at timed intervals after initiating the reaction with NADPH, and replica experiments were run in a cuvette to monitor concurrent NADPH oxidation by eNOS under each condition. As shown in the upper panel of Fig. 10, the NO concentration rose after initiating NO synthesis from Arg, achieved a maximum of 61 nm after 1 min, fell as NADPH continued to be consumed, and then fell more rapidly after all NADPH was oxidized. The rate of NADPH consumption was approximately linear during the reaction and increased only slightly when the NO scavenger oxyhemoglobin was present. When NOHA was used in place of Arg (Fig. 10, middle panel), the NO concentration rose to a much higher level during the reaction (840 nm) and then gradually fell as in the Arg reaction. NADPH consumption by eNOS was slowed about 3 times as the NO concentration built up. This effect was NO-dependent, because NADPH oxidation in a replica reaction that c
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